METHODS FOR DETERMINING COMPLEMENT-FIXING ANTIBODIES

Abstract

The invention relates to methods for determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in a sample from a subject. Further, the invention is related to methods for the concomitant determination of the presence and/or amount of complement-fixing antibodies reactive to different flaviviruses in a sample from a subject. Moreover, the invention is related to in vitro methods for diagnosing a flavivirus infection in a subject. In addition, the present invention also provides kits for carrying out the methods.

Claims

1. A method for determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in a sample from a subject comprising the steps of: Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; Step 2: contacting an amount of complement component 1q (C1q) with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow binding of the C1q to a heavy chain constant region of the complement-fixing antibodies; Step 3: contacting an amount of a reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the reporter antibody to the C1q, wherein the reporter antibody binds to the C1q with a variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and Step 4: detecting a signal from the reporter antibody bound to the C1q in step 3, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

2. The method according to claim 1, comprising the further steps of: Step 5: determining the presence and/or amount of the reporter antibody from the signal of step 4; and Step 6: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 5.

3. The method according to claim 1, comprising the steps of: Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; Step 2: contacting an amount of C1q with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow binding of the C1q to the heavy chain constant region of the complement-fixing antibodies; Step 3.1: contacting an amount of a pre-reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the pre-reporter antibody to the C1q, wherein the pre-reporter antibody binds to the C1q with a variable region of the pre-reporter antibody; Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C1q in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to a heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and Step 4: detecting a signal from the reporter antibody bound to the pre-reporter antibody in step 3.2, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

4. The method according to claim 3, wherein the method further comprises the steps of: Step 5: determining the presence and/or amount of the reporter antibody from the signal of step 4; and Step 6: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 5.

5. The method according to claim 1, to wherein the antigen is selected from the group consisting of virus like particle (VLP), non-structural protein 1, envelope protein, pre-membrane protein, membrane protein, capsid protein, non-structural protein 2A, non-structural protein 2B, non-structural protein 3, non-structural protein 4A, non-structural protein 4B, and non-structural protein 5 and any derivative thereof.

6. The method according to claim 1, wherein the detectable label is a fluorescence label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof.

7. A method for the concomitant determination of the presence and/or amount of complement-fixing antibodies reactive to different flaviviruses in a sample from a subject comprising the steps of: Step 1: contacting an amount of at least two microsphere complexes with the sample, wherein a first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and a second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen, wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein an emission signal of the first detectable label differs from an emission signal of the second detectable label, Step 2: contacting an amount of C1q with the complement-fixing antibodies bound to the first and/or the second flavivirus antigen in step 1 to allow binding of the C1q to a heavy chain constant region of the complement-fixing antibodies, Step 3: contacting an amount of a reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the reporter antibody to the C1q, wherein the reporter antibody binds to the C1q with the variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and simultaneously detecting a signal from the reporter antibody bound to the C1q in step 3 of the at least one microsphere upon irradiation with a second light source, Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative of the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

8. The method according to claim 7, wherein the method further comprises the steps of: Step 7: determining the presence and/or amount of the reporter antibody from the summarized signal in step 6, and Step 8: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 7.

9. The method according to claim 7, comprising the steps of: Step 1: contacting an amount of at least two microsphere complexes with the sample, wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen, wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, Step 2: contacting an amount of C1q with the complement-fixing antibodies bound to the first and/or the second flavivirus antigen in step 1 to allow binding of the C1q to a heavy chain constant region of the complement-fixing antibodies, Step 3.1: contacting an amount of a pre-reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the pre-reporter antibody to the C1q, wherein the pre-reporter antibody binds to the C1q with a variable region of the pre-reporter antibody, Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C1q in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to a heavy chain constant region of the pre-reporter antibody with a variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and simultaneously detecting a signal from the reporter antibody bound to the C1q in step 3 of the at least one microsphere upon irradiation with a second light source, Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

10. The method according to claim 9, wherein the method further comprises the steps of: Step 7: determining the presence and/or amount of reporter antibody from the summarized signal in step 6, and Step 8: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of reporter antibody determined in step 7.

11. The method according to claim 7, wherein the antigen is selected from the group consisting of virus like particle (VLP), non-structural protein 1, envelope protein, pre-membrane protein, membrane protein, capsid protein, non-structural protein 2A, non-structural protein 2B, non-structural protein 3, non-structural protein 4A, non-structural protein 4B, and non-structural protein 5 and any derivative thereof, preferably the antigen is VLP.

12. The method according to claim 7, wherein the third detectable label is a fluorescence label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof.

13. The method of claim 1, wherein the flavivirus is selected from the group consisting of dengue virus, zika virus, West Nile virus, Japanese encephalitis virus, Tick-Borne encephalitis virus, Yellow Fever virus, Murray Valley encephalitis virus, and St. Louis encephalitis virus, preferably dengue virus

14. The method of claim 1, wherein the antigen is selected from the group consisting of virus like particle (VLP), non-structural protein 1, envelope protein, pre-membrane protein, membrane protein, capsid protein, non-structural protein 2A, non-structural protein 2B, non-structural protein 3, non-structural protein 4A, non-structural protein 4B, and non-structural protein 5 and any derivative thereof, preferably the antigen is VLP

15. The method of claim 1, wherein in step 1, an amount of a first microsphere complex comprising a first microsphere coupled to a dengue 1 virus VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a dengue 2 virus VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a dengue 3 virus VLP, and an amount of a fourth microsphere complex comprising a fourth microsphere coupled to a dengue 4 virus VLP is contacted with the sample.

16. The method of claim 1, wherein the C1q is purified.

17.-21. (canceled)

22. The method according to claim 1, wherein the sample is heat-inactivated.

23. The method according to claim 1, wherein the subject is selected from the group consisting of mouse, primate, non-human primate, human, rabbit, cat, rat, horse, and sheep, preferably the subject is human.

24. A kit for detecting flavivirus-reactive complement-fixing antibodies in a sample comprising: an amount of at least one microsphere complex comprising a microsphere coupled to a flavivirus antigen, an amount of C1q, an amount of a reporter antibody, and optionally, an amount of a pre-reporter antibody, wherein the reporter antibody binds to the C1q with a variable region of the reporter antibody, and wherein the reporter antibody is attached to a detectable label and wherein the pre-reporter antibody binds to the C1q with a variable region of the pre-reporter antibody and the reporter antibody binds to a heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody.

25. The kit according to claim 24, comprising an amount of at least two microspheres complexes, wherein a first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and a second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen, wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label.

26.-32. (canceled)

33. A method for determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in a sample from a subject comprising the steps of: Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow the formation of a complement component fragment C3d (C3d); Step 3: contacting an amount of a reporter antibody with the C3d formed in step 2 to allow binding of the reporter antibody to the C3d, wherein the reporter antibody binds to the C3d with a variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and Step 4: detecting a signal from the reporter antibody bound to the C3d in step 3, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

34. The method according to claim 33, comprising the steps of: Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow the formation of C3d; Step 3.1: contacting an amount of a pre-reporter antibody with the C3d formed in step 2 to allow binding of the pre-reporter antibody to the C3d, wherein the pre-reporter antibody binds to the C3d with a variable region of the pre-reporter antibody; Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C3d in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to a heavy chain constant region of the pre-reporter antibody with a variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and Step 4: detecting a signal from the reporter antibody bound to the pre-reporter antibody in step 3.2, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

35. The method of claim 33, comprising the further steps of: Step 5: determining the presence and/or amount of the reporter antibody from the signal of step 4; and Step 6: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 5.

36.-37. (canceled)

38. A method for the concomitant determination of the presence and/or amount of complement-fixing antibodies reactive to different flaviviruses in a sample from a subject comprising the steps of: Step 1: contacting an amount of at least two microsphere complexes with the sample, wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen, wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow the formation of C3d, Step 3: contacting an amount of a reporter antibody with the C3d formed in step 2 to allow binding of the reporter antibody to the C3d, wherein the reporter antibody binds to the C3d with a variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and simultaneously detecting a signal from the reporter antibody bound to the C3d in step 3 of the at least one microsphere upon irradiation with a second light source, Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

39. The method according to claim 38, comprising the steps of: Step 1: contacting an amount of at least two microsphere complexes with the sample, wherein a first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and a second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen, wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the first and/or the second flavivirus antigen in step 1 to allow the formation of C3d, Step 3.1: contacting an amount of a pre-reporter antibody with the C3d formed in step 2 to allow the formation of C3d and to allow binding of the pre-reporter antibody to the C3d, wherein the pre-reporter antibody binds to the C3d with a variable region of the pre-reporter antibody, Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C3d in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to a heavy chain constant region of the pre-reporter antibody with a variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and simultaneously detecting a signal from the reporter antibody bound to the C3d in step 3 of the at least one microsphere upon irradiation with a second light source, Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized detected signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

40. The method according to claim 38, wherein the method further comprises the steps of: Step 7: determining the presence and/or amount of the reporter antibody from the summarized signal in step 6, and Step 8: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 7.

41.-51. (canceled)

52. A kit for detecting flavivirus-reactive complement-fixing antibodies in a sample comprising: an amount of at least one microsphere complex comprising a microsphere coupled to a flavivirus antigen, an amount of a complement competent serum, an amount of a reporter antibody, and optionally, an amount of a pre-reporter antibody, wherein the reporter antibody is attached to a detectable label and wherein the reporter antibody binds to the C3d with a variable region of the reporter antibody, wherein the pre-reporter antibody binds to the C3d with a variable region of the pre-reporter antibody and the reporter antibody binds to a heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody.

53. The kit according to claim 52, comprising an amount of at least two microspheres complexes including a first microsphere complex and a second microsphere complex, wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen, wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label.

54.-59. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0157] FIG. 1 Optimization of coupling pH exemplarily shown for DENV2 VLP (A) and DENV2 NS1 (B). Mean fluorescence intensity (MFI) is shown for the negative control sample that did not contain DENV-reactive complement-fixing Abs, as well as the high control sample, comprising a high amount of DENV-reactive complement-fixing Abs in dependence of the coupling pH and sample dilution. For instance, a 10-fold sample dilution refers to a logo (sample dilution) of 1.

[0158] FIG. 2 Schematic overview of the DENV-quadruplex complement-fixing assay. The DENV-quadruplex complement-fixing assay according to the present invention comprises application of microspheres coupled to dengue 1 virus (DV1), dengue 2 virus (DV2), dengue 3 virus (DV3), and dengue 4 virus (DV4) virus like particles (VLPs). The DV1, DV2, DV3, and DV4 VLP-coupled microspheres are applied as a mixture allowing detection of complement-fixing Abs reactive to the different DENV serotypes in one single experiment. The VLP-coupled microspheres are incubated with a sample comprising complement-fixing Abs to allow binding of these Abs to the VLPs. Afterwards, purified human C1q is added to the mixture to bind complement-fixing Abs from the sample bound to the VLPs. Bound human C1q is then further detected using an anti-human C1q pre-reporter antibody and an anti-sheep IgG phycoerythrin-coupled reporter antibody generating a fluorescence signal upon irradiation with a light source.

[0159] FIGS. 3 and 4 Validation of the DENV-quadruplex complement-fixing assay set-up. In order to validate the DENV-quadruplex complement-fixing assay set-up, a reference sample, a negative control sample, as well as control samples comprising a high, medium, and low concentration of DENV-reactive complement-fixing Abs were analyzed. Mean fluorescence intensity (MFI) depending on the sample dilution (presented as log.sub.10 sample dilution) and on the DENV serotype i.e. DENV1 (FIG. 3A), DENV2 (FIG. 3B), DENV3 (FIG. 4A) and DENV4 (FIG. 4B) was fitted according to a sigmoidal curve. For instance, a 10-fold sample dilution refers to a logo (sample dilution) of 1.

[0160] FIGS. 5 and 6 Validation of the DENV-quadruplex complement-fixing assay set-up (linearity). The obtained complement-fixing antibody concentration was plotted against the expected concentration for each DENV VLP, i.e. DENV1 VLP (FIG. 5A), DENV2 VLP (FIG. 5B), DENV3 VLP (FIG. 6A) and DENV4 VLP (FIG. 6B) and was fitted by a linear regression.

[0161] FIGS. 7 and 8 Validation of the DENV-quadruplex complement-fixing assay set-up. Mean fluorescence signal (MFI) is exemplarily presented for DENV2 VLP in dependence of the sample dilution, as well as sample type. High control sample (FIG. 7A), medium control sample (FIG. 7B), as well as low control sample (FIG. 8), comprising a high, medium, and low concentration of DENV-reactive complement-fixing Abs were examined in five replicates together with the reference sample. For instance, a 10-fold sample dilution refers to a logo (sample dilution) of 1.

[0162] FIGS. 9 and 10 Validation of the DENV-quadruplex complement-fixing assay set-up. Complement-fixing antibody titers in EU/mL (arbitrary unit) are presented dependent on the operator, sample type (high, medium, and low control sample, comprising high, medium, and low amounts of DENV-reactive complement-fixing Abs), as well as DENV VLP. Results for DENV1 VLP are shown in FIG. 9A, for DENV2 VLP in FIG. 9B, for DENV3 VLP in FIG. 10A, and for DENV4 VLP in FIG. 10B.

[0163] FIGS. 11 and 12 Validation of the DENV-quadruplex complement-fixing assay set-up (intra-assay precision). The percent coefficient of variation (% CV) was calculated for the replicates dependent on the operator, sample type (high, medium, and low control sample, comprising high, medium, and low amounts of DENV-reactive complement-fixing Abs), as well as DENV VLP. Results for DENV1 VLP are shown in FIG. 11A, for DENV2 VLP in FIG. 11B, for DENV3 VLP in FIG. 12A, and for DENV4 VLP in FIG. 12B.

[0164] FIG. 13 Validation of the DENV-quadruplex complement-fixing assay set-up (inter-assay precision). The percent coefficient of variation (% CV) was calculated for the replicates carried out by the two operators dependent on the sample type (high, medium, and low control sample, comprising high, medium, and low amounts of DENV-reactive complement-fixing Abs), as well as DENV VLP.

[0165] FIG. 14 Analysis of human serum samples with the DENV-quadruplex complement-fixing assay set-up. Mean fluorescence intensity (MFI) resulting from analysis of DENV-1 VLP-coupled microspheres in the quadruplex assay set-up depending on the sample dilution (presented as logo sample dilution) was fitted according to a sigmoidal curve. Besides the data concerning human serum samples 1-5, the results from analysis of a reference sample are further included. For instance, a 10-fold sample dilution refers to a logo (sample dilution) of 1.

[0166] FIGS. 15 to 18 Analysis of samples from human subjects seropositive and seronegative for DENV. The DENV-quadruplex complement fixing-assay was applied to evaluate complement-fixing Abs in seronegative (FIGS. 15 and 17) and seropositive (FIGS. 16 and 18) subjects from two clinical studies (#1 and #2) prior (day 0 in FIGS. 15 and 16, or day 1 in FIGS. 17 and 18, respectively) and post vaccination with a tetravalent DENV vaccine, comprising live attenuated viruses of DENV1, DENV2, DENV3, and DENV4. Data are presented as median values with error bars (upper panels) and box plots (lower panels). As day 0 in FIGS. 15 and 16 refers to samples taken prior to vaccination, day 0 can be referred to as the starting day of the study. As day 1 in FIGS. 17 and 18 refers to samples taken prior to vaccination, day 1 can be referred to as the starting day of the study.

[0167] FIG. 19 Receiver Operator Curve (ROC) to evaluate accuracy of the DENV-quadruplex complement fixing-assay for diagnosis. The sensitivity, meaning the percentage of true positive samples i.e. samples from seropositive subjects with an MNT50 titer of 10 being also classified as positive in the complement assay is plotted on the y-axis and 1-specificity, meaning the percentage of true negative samples i.e. samples from seronegative subjects with an MNT50 titer of <10 being also classified as negative in the complement assay is plotted on the x-axis for several thresholds (or complement-fixing antibody titers).

[0168] FIGS. 20 and 21 Analysis of human samples by a 10-plex complement-fixing assay set-up using ZIKV VLP, DENV1-4 VLPs, ZIKV NS1, and DENV1-4 NS1 as antigens coupled to the microspheres. Mean fluorescence intensity (MFI) is presented for all VLPs (FIG. 20) and NS1 (FIG. 21). Complement-fixing Abs were determined in samples collected in the Dominican Republic in 2016 to 2017 from subjects suffering from febrile illness consistent with DENV and ZIKV infections (panel #1), as well as in samples collected in Colombia either before or after 2016 from subjects 8-40 days post febrile illness consistent with DENV and ZIKV infection (panels #2 and #3). Of note, zika seroprevalence in Colombia reached around 50% after 2016. In addition, a negative control lacking flavivirus-reactive Abs and a blank was included.

[0169] FIG. 22 Evaluation of non-human primate samples by the DENV-quadruplex complement-fixing assay set-up. (A) A phylogenetic tree was prepared based on the envelope amino acid sequence of the DENV serotype strains used to infect non-human primates. (B) A total of 16 animals were infected with each one DENV serotype (indicated above the graphics) and complement-fixing Ab titers were determined against each serotype for 1 year after infection (at days 0, 69, and 341) and presented for each animal. The samples on day 0 are taken prior to vaccination.

[0170] FIG. 23 Evaluation of non-human primate samples by the DENV-quadruplex complement-fixing assay set-up. A total of 16 animals were infected with each one DENV serotype (indicated above the graphics) and complement-fixing Ab titers were determined against each serotype for 1 year after infection (at days 0, 69, and 341). Complement-fixing Ab titers are averaged for all animals infected with the same serotype. The samples on day 0 are taken prior to vaccination.

[0171] FIG. 24 ZIKV NS1 complement-fixing Abs evaluated by the 10-plex assay set-up. (A) A total of 15 non-human primates were infected with each one DENV serotype (indicated next to the animal numbers), followed by ZIKV infection 1 year later. ZIKV NS1-reactive complement-fixing Ab titers in mean fluorescence intensity (MFI) were determined with the 10-plex complement-fixing assay. (B) ZIKV NS1-reactive complement-fixing Ab titers in mean fluorescence intensity (MFI) were determined with the 10-plex complement-fixing assay in human samples. Human samples analyzed were negative controls lacking ZIKV-reactive complement-fixing Abs, WNV positive samples lacking ZIKV-reactive complement-fixing Abs but containing WNV-reactive complement-fixing Abs, as well as ZIKV positive samples containing ZIKV-reactive complement-fixing Abs.

[0172] FIG. 25 Complement fixing antibody titers measured by the DENV-quadruplex complement-fixing assay in non-human primates that were vaccinated with either YFV, ZIKV, WNV, TBEV, or JEV vaccine (indicated above the graphics). The titers are shown for all four DENV VLPs (DENV1-4) for each animal. Titers were determined at days 1, 57, and 169. PIZV=Purified inactivated Zika vaccine.

[0173] FIG. 26 Neutralizing and complement-fixing antibody levels against all four dengue virus serotypes in subjects living in dengue endemic areas. Microneutralization (MNT50) antibody (A) and complement-fixing antibody (B) levels are shown per analyzed sample for each dengue serotype. In total, 53 samples were analyzed. Horizontal lines represent the threshold for considering a subject as seropositive (?10 for MNT50 and ?3 EU/mL for complement-fixing antibodies).

[0174] FIG. 27 Correlation analysis between neutralizing (MNT50) and complement-fixing antibodies against all four dengue virus (DENV) serotypes in subjects living in dengue endemic areas. Correlation analysis was performed using Log.sub.10-transformed MNT50 and complement-fixing antibody titers and the correlation coefficient (R.sup.2) was calculated for each DENV serotype. Vertical and horizontal lines represent LLOQ of the MNT50 (10) and complement-fixing antibody assays (3 EU/mL), respectively.

[0175] FIG. 28 Total IgG titers against all four dengue virus serotypes in subjects living in dengue endemic areas. The horizontal dotted line represents the assay LLOQ (200 RU/mL; RU=relative units).

[0176] FIG. 29 Correlation analysis between total IgG titers and complement-fixing antibody titers against all four dengue virus serotypes in subjects living in endemic areas. Correlation analysis was performed using Log.sub.10-transformed total IgG titers and complement-fixing antibody titers and the correlation coefficient (R.sup.2) was calculated for each DENV serotype. Vertical and horizontal lines represent LLOQ of the total IgG binding (200 RU/mL) and complement-fixing antibody assays (3 EU/mL), respectively.

[0177] FIG. 30 Schematic overview of C3d production by the classical complement activation pathway (A) and C3d deposition assay set-up (B). For C3d production, C1q binds to the immunocomplex and changes its conformation, which in turn activates the enzymes C1r and C1s. These enzymes cleave C4 and the larger fraction (C4b) remains covalently bound to C1q(rs)2. Then, C2 is cleaved and the larger fraction (C2a) remains bound to the C4b forming an enzyme C3 convertase (C4b2a). The convertase cleaves C3 and the larger fraction (C3b) binds to C4b2a, as well as the surroundings. C3b is then finally further cleaved into C3d, which is detected in the C3d deposition assay set-up.

[0178] FIG. 31-34 Complement fixing antibody titers measured by the C1q-based DENV-quadruplex complement-fixing assay set-up (FIGS. 31 and 32) and the C3d deposition DENV-quadruplex complement-fixing assay set-up (C3dfixing antibody titers; FIGS. 33 and 34) in a panel of 9 serum samples collected from DENV seropositive healthy adults living in dengue-endemic areas in Colombia from 2015-2016. In addition, high, medium, and low control samples comprising high, medium, and low number of DENV-reactive complement-fixing Abs were included.

[0179] FIG. 35 Correlation between C1q-based complement-fixing antibody titers and C3d-based complement-fixing antibody titers as determined in FIG. 31-34 (R.sup.2=correlation coefficient; CI=confidence interval).

DETAILED DESCRIPTION

[0180] In the following sections, various exemplary compositions and methods are described in order to detail various embodiments. It will be obvious to one skilled in the art that practicing the various embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times and other specific details may be modified through routine experimentation.

Microsphere Complex

[0181] The microsphere complex for use in the methods and kits of the present application comprises a microsphere coupled to a flavivirus antigen.

Microsphere

[0182] The microsphere useful for the invention ranges in the size from about 0.01 to about 100 ?m in diameter, more preferably from about 1 to about 20 ?m, and most preferably a microsphere has a diameter from about 5 to about 7 ?m. In a preferred embodiment the microsphere has a diameter of about 6.5 ?m. The size of a microsphere can be determined in practically any flow cytometry apparatus by so-called forward or small-angle scatter light.

[0183] The microsphere may be constructed of any material to which molecules like VLPs or NS1 may be attached to. For example, acceptable materials for the construction of microspheres include but are not limited to: polystyrene, polyacrylic acid, polyacrylonitrile, polyacrylamide, polyacrolein, polybutadiene, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, or combinations thereof. In a preferred embodiment of the present invention, microspheres are constructed of polystyrene.

[0184] The microsphere may comprise surface affinity groups for attachment of molecules. Said affinity groups may be, but are not limited to, Ni 2+(for immobilization of His-tagged molecules), Protein A, Protein G, Protein L, anti-human IgG Ab, anti-rabbit IgG Ab, anti-mouse IgG Ab, anti-goat IgG Ab, anti-FLAG Ab, streptavidin, avidin, and glutathione.

[0185] The microsphere may comprise functional groups on the surface useful for attachment of molecules, such as the antigens of the present invention. Said functional groups may be, but are not limited to, carboxylates, esters, alcohols, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides, maleimides, or halides. In a preferred embodiment the microsphere comprises carboxylates on the surface. Molecules like antigens can be covalently coupled to the microspheres using chemical techniques described herein. In a preferred embodiment molecules like antigens (i.e. VLPs or NS1) can be coupled to the microsphere by carbodiimide coupling using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS). Thereby, the EDC is reacting to an unstable o-acylisourea ester with a carboxylate on the surface of the microspheres. The unstable o-acylisourea ester readily reacts with Sulfo-NHS to form a semi-stable amine reactive NHS-ester. The NHS-ester finally reacts with an amine group provided by an antigen, thereby forming a stable amide bond.

[0186] As amine-containing compounds other than those provided by the antigen, glycerol, urea, imidazole, azide, and some detergents may interfere with the carbodiimide coupling, they should be removed from the antigen preparation with a suitable buffer exchange method. For instance, a suitable buffer for carbodiimide coupling is 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer. The pH value for coupling may be between about 5 and about 9. Coupling of the antigen to the microsphere may be carried out by incubation for about 2 hours.

[0187] The microsphere may be magnetic. In a preferred embodiment the microsphere may be superparamagnetic. Magnetic microspheres can be easily captured by a magnetic plate separator for instance to wash the microspheres.

[0188] A magnetic plate separator can be used for separating the microspheres within the 96-well plate from the solution within the wells of the 96-well plate by magnetic capture and refers to a construction for holding a 96-well plate. A magnetic plate separator enables the user to quickly decant the supernatant within the wells and washing of the wells, while fixing the microspheres at the bottom of the 96-well plate by magnetic capture. Application of a magnetic plate separator reduces the risk that microspheres are getting lost during washing procedures.

[0189] The microsphere may comprise a detectable label by which the microsphere can be identified with the help of a detection system. Identification of a microsphere likewise allows identification of the antigen which is coupled to the microsphere.

[0190] Concerning the detection of such labels with suitable detection systems, reference is also made to the section Detection system.

[0191] In preferred embodiments the detectable label is at least one fluorescent dye. The fluorescent dye may be for instance selected from the group consisting of squaraine, phthalocyanine, naphthalocyanine, and any derivative thereof. For instance, a derivative of a fluorescent dye may be the dye further comprising a methyl group at any position.

[0192] In some embodiments the microsphere comprises one fluorescent dye. The microsphere can be identified by the emission signal of the one fluorescent dye upon irradiation with a suitable light source.

[0193] In other embodiments different microspheres comprise different concentrations of the same fluorescent dye. In such embodiments, the microsphere can be identified by the intensity of the emission signal of the one fluorescent dye upon irradiation with a suitable light source.

[0194] In some embodiments the microsphere comprises two or more fluorescent dyes. The microsphere can be identified by the emission signal of the two or more fluorescent dyes upon irradiation with a suitable light source.

[0195] In some embodiments different microspheres comprise different concentrations of the two or more fluorescent dyes. In such embodiments, the microsphere can be identified by the intensity of the emission signal of the two or more fluorescent dyes (indicative for the ratio of the two or more fluorescent dyes) upon irradiation with a suitable light source.

[0196] In certain embodiments where the microsphere comprises two or more fluorescent dyes, the emission signal of the two or more fluorescent dyes is resulting from an overlay of the emission signal of the single fluorescent dyes. The intensity of the emission signal is therefore indicative for the ratio of the two or more fluorescent dyes.

[0197] The at least one fluorescent dye can be excited with any suitable light source as for instance a laser or a light emitting diode (LED) using a suitable detection system.

[0198] The at least one fluorescent dye can be covalently attached onto the surface of the microsphere, or can be internally incorporated during microsphere production (i.e. polystyrene polymerization), or the microsphere can be dyed after production by placing the microsphere in a suitable solution comprising the at least one fluorescent dye. A suitable solution comprising the at least one fluorescent dye is for instance an organic solution.

[0199] In one embodiment, one microsphere may comprise two fluorescent dyes having an emission signal maximum at 675 nm, another microsphere may comprise two different fluorescent dyes having an emission signal maximum at 700 nm.

[0200] In another embodiment, one microsphere may comprise a fluorescent dye at one specific concentration, another microsphere may comprise the same fluorescent dye at another specific concentration, wherein the emission signal maxima of the two microspheres occur at the same wavelength but with different emission signal intensities.

[0201] In certain embodiments different microspheres comprising different concentrations of fluorescent dyes can be excited by the same light source (e.g. the one or more fluorescent dyes at specific concentrations in the different microspheres are excitable by the same wavelength). In specific embodiments the different microspheres are excitable with a wavelength within the range from about 600 to about 650 nm, more preferably with a wavelength of about 615 nm to about 640 nm, and even more preferably with a wavelength of about 620 to about 635 nm. In one embodiment the different microspheres are excitable with a wavelength of about 635 nm. An advantage of such a set-up is, that only one light source is needed for distinguishing all microspheres present within a microsphere mixture and thereby further simplifying a set-up in which multiple antigens can be analyzed in one single experiment.

[0202] The microspheres may also be identified by their size, if different microspheres are of a different size using a suitable detection system. The size of the microspheres ranges from 0.01 to 100 ?m in diameter, more preferably from about 1 to about 10 ?m in diameter. For instance, one microsphere may be about 6 ?m in diameter, another microsphere may be about 6.5 ?m in diameter.

[0203] The microsphere may also be identified by a specific shape of the microsphere, if different microspheres are of a different shape using a suitable detection system.

[0204] To allow the simultaneous detection of complement-fixing antibodies reactive to different antigens in one single experiment, microspheres with a different size or a different detectable label or a different shape are coupled to different antigens and mixed. Microspheres coupled to the same antigen have the same size or the same detectable label or the same shape. Although the microspheres are mixed each microsphere can be identified by the specific size or detectable label or shape of the microsphere. Thereby, the antigen coupled to the microsphere can be simultaneously identified.

[0205] Microspheres may be one out of the list consisting of MagPlex? microspheres, MicroPlex? microspheres, LumAvidin? microspheres, MagPlex?-Avidin microspheres, and SeroMAP? microspheres produced by the Luminex Corporation (Austin, Texas). The type of microsphere which can be used depends on the detection system applied (reference is also made to the section Detection system).

[0206] In a preferred embodiment the microspheres are the MagPlex? microspheres, which are superparamagnetic polystyrene microspheres with surface carboxyl groups and a diameter of about 6.5 ?m produced by Luminex Corporation (Austin, Texas). MagPlex? microspheres comprise two or more fluorescent dyes at a specific concentration allowing each microsphere to be identified by a detection system as for instance a MAGPIX? instrument as produced by the Luminex Corporation (Austin, Texas). Microspheres of different MagPlex? microsphere catalog numbers (Luminex Corporation, Austin, Texas) comprise the two or more fluorescent dyes at different concentrations. The MagPlex? microspheres can be excited by the same excitation wavelength and therefore only one light source is required for microsphere identification.

[0207] In specific embodiments the excitation wavelength is from about 600 to about 650 nm, preferably from about 615 to about 640 nm, more preferably from about 620 to about 635 nm. For instance, the excitation wavelength is about 635 nm.

Flavivirus Antigen

[0208] The flavivirus antigen to which the microsphere is coupled to can be a whole virion surrogate, such as virus like particle (VLP). Virus like particles comprise one or more of the structural proteins of the flavivirus from which they are derived i.e. prM, M, and E protein. The flavivirus VLP may be for instance a ZIKV VLP or a DENV VLP. The DENV VLP may be of any serotype (DENV1-4).

[0209] Flavivirus VLPs of the present invention may be derived from any flavivirus strain, including any DENV or ZIKV strain. Flavivirus VLPs may be produced in any suitable cell such as a mammalian cell or an insect cell (such as cells from Aedes albopictus (C6/36) or Spodoptera frugiperda (Sf9). In some embodiments, the cells used for VLP production are human cells. In specific embodiments, the cells used for VLP production are HEK293 cells.

[0210] In one embodiment, the flavivirus antigen is a DENV1 VLP derived from DENV1 strain Puerto Rico/US/BID-V853/1998 (GenBank accession No. EU482592.1; SEQ ID NO: 1 and 5). DENV1 VLP may be produced in HEK293 cells. In more specific embodiments the DENV1 VLP comprises structural proteins from DENV1 strain Puerto Rico/US/BID-V853/1998 (GenBank accession No. EU482592.1; SEQ ID NO: 1 and 5). In even more specific embodiments the DENV1 VLP comprises the E protein, M protein, and prM protein which are at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to those encoded by DENV1 strain Puerto Rico/US/BID-V853/1998 (GenBank accession No. EU482592.1; SEQ ID NO: 1 and 5).

[0211] In one embodiment, the flavivirus antigen is a DENV2 VLP derived from DENV2 strain Thailand/16681/84 (EMBL-EBI accession No: U87411.1; SEQ ID NO: 2 and 6). DENV2 VLP may be produced in HEK293 cells. In more specific embodiments the DENV2 VLP comprises structural proteins from DENV2 strain Thailand/16681/84 (EMBL-EBI accession No: U87411.1; SEQ ID NO: 2 and 6). In even more specific embodiments the DENV2 VLP comprises the E protein, M protein, and prM protein which are at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to those encoded by DENV2 strain Thailand/16681/84 (EMBL-EBI accession No: U87411.1; SEQ ID NO: 2 and 6).

[0212] In one embodiment, the flavivirus antigen is a DENV3 VLP derived from DENV3 strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (GenBank accession No. AY099336.1; SEQ ID NO: 3 and 7). DENV3 VLP may be produced in HEK293 cells. In more specific embodiments the DENV3 VLP comprises structural proteins from DENV3 strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (Gen Bank accession No. AY099336.1; SEQ ID NO: 3 and 7). In even more specific embodiments the DENV3 VLP comprises the E protein, M protein, and prM protein which are at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to those encoded by DENV3 strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (GenBank accession No. AY099336.1; SEQ ID NO: 3 and 7).

[0213] In one embodiment, the flavivirus antigen is a DENV4 VLP derived from DENV4 strain Dominica/814669/1981 (EMBL-EBI accession No: AF326825.1; SEQ ID NO: 4 and 8). DENV4 VLP may be produced in HEK293 cells. In more specific embodiments the DENV4 VLP comprises structural proteins from DENV4 strain Dominica/814669/1981 (EMBL-EBI accession No: AF326825.1; SEQ ID NO: 4 and 8). In even more specific embodiments the DENV4 VLP comprises the E protein, M protein, and prM protein which are at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to those encoded by DENV4 strain Dominica/814669/1981 (EMBL-EBI accession No: AF326825.1; SEQ ID NO: 4 and 8).

[0214] In specific embodiments, for production of the DENV1-4 VLPs, the C-terminal 20% of DENV E protein were replaced by the corresponding Japanese encephalitis virus (JEV) SA-14 sequence (EMBL-EBI accession No: M55506.1, E protein amino acids 399-497 (DENV1 VLP), 397-495 (DENV2 VLP), 399-492 (DENV3 VLP), 400-495 (DENV4 VLP). The replaced sequence corresponds to the transmembrane and intraparticle portion of the protein.

[0215] In one embodiment, the flavivirus antigen is a ZIKV VLP derived from ZIKV strain Suriname/Z1106033 (GenBank Accession No. KU312312.1 and ALX35659.1; SEQ ID NO: 9 and 10). ZIKV VLP may be produced in HEK293 cells. In more specific embodiments the ZIKV VLP comprises structural proteins from ZIKV strain Suriname/Z1106033 (GenBank Accession No. KU312312.1 and ALX35659.1; SEQ ID NO: 9 and 10). In even more specific embodiments the ZIKV VLP comprises the E protein, M protein, and prM protein which are at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to those encoded by ZIKV strain Suriname/Z1106033 (GenBank Accession No. KU312312.1 and ALX35659.1; SEQ ID NO: 9 and 10).

[0216] The flavivirus antigen to which the microsphere is coupled to can be a single protein, such as a structural or non-structural protein as for instance non-structural protein 1 (NS1). Further proteins suitable for coupling to the microsphere are prM, M, E, C, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, or fragments or variants thereof.

[0217] The flavivirus antigen to which the microsphere is coupled to can be any derivative of a structural and non-structural protein such as a protein domain or a part of a protein, such as the domain III of the envelope protein (E).

[0218] The protein or protein domain or part of a protein can be produced recombinantly (recombinant protein) in any suitable cells such as mammalian cells, insect cells, or bacterial cells.

[0219] In one embodiment, the flavivirus antigen is a DENV1 NS1 derived from DENV1 strain Nauru/Western Pacific/1974 (SEQ ID NO: 11 and 12; GenBank accession No. AY145121). DENV1 NS1 may be produced in HEK293 cells. In more specific embodiments the DENV1 NS1 is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to the NS1 encoded by DENV1 strain Nauru/Western Pacific/1974 (SEQ ID NO: 11 and 12; GenBank accession No. AY145121). In even more specific embodiments the sequence of the DENV1 NS1 the microspheres are coupled to is shown in SEQ ID NO: 13.

[0220] In one embodiment, the flavivirus antigen is a DENV2 NS1 derived from DENV2 strain Thailand/16681/84 (EMBL-EBI accession No: U87411.1; SEQ ID NO: 2 and 6). DENV2 NS1 may be produced in HEK293 cells. In more specific embodiments the DENV2 NS1 is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to the NS1 encoded by DENV2 strain Thailand/16681/84 (EMBL-EBI accession No: U87411.1; SEQ ID NO: 2 and 6). In even more specific embodiments the sequence of the DENV2 NS1 the microspheres are coupled to is shown in SEQ ID NO: 14.

[0221] In one embodiment, the flavivirus antigen is a DENV3 NS1 derived from DENV3 strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (GenBank accession No. AY099336.1; SEQ ID NO: 3 and 7). DENV3 NS1 may be produced in HEK293 cells. In more specific embodiments the DENV3 NS1 is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to the NS1 encoded by DENV3 strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (GenBank accession No. AY099336.1; SEQ ID NO: 3 and 7). In even more specific embodiments the sequence of the DENV3 NS1 the microspheres are coupled to is shown in SEQ ID NO: 15.

[0222] In one embodiment, the flavivirus antigen is a DENV4 NS1 derived from DENV4 strain Dominica/814669/1981 (EMBL-EBI accession No: AF326825.1; SEQ ID NO: 4 and 8). DENV4 NS1 may be produced in HEK293 cells. In more specific embodiments the DENV4 NS1 is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to those encoded by DENV4 strain Dominica/814669/1981 (EMBL-EBI accession No: AF326825.1; SEQ ID NO: 4 and 8). In even more specific embodiments the sequence of the DENV4 NS1 the microspheres are coupled to is shown in SEQ ID NO: 16.

[0223] In one embodiment, the flavivirus antigen is a ZIKV NS1 derived from ZIKV strain Suriname/Z1106033 (GenBank Accession No. KU312312.1 and ALX35659.1; SEQ ID NO: 9 and 10). ZIKV NS1 may be produced in HEK293 cells. In more specific embodiments the ZIKV NS1 is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to those encoded by ZIKV strain Suriname/Z1106033 (GenBank Accession No. KU312312.1 and ALX35659.1; SEQ ID NO: 9 and 10).

Reporter and Pre-Reporter Antibody

[0224] The reporter and pre-reporter Ab for use in the methods and kits of the present invention may be any Abs suitable. The reporter and pre-reporter Ab may be recombinant Abs, monoclonal Abs, or polyclonal Abs.

[0225] According to certain embodiments of the invention the reporter and the pre-reporter antibody are immunoglobulin (Ig) molecules, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.

[0226] The reporter and pre-reporter Ab may be derived from any origin. According to certain embodiments of the invention the reporter and the pre-reporter Ab are derived from a non-human origin such as sheep, mouse, rabbit, goat, or donkey.

[0227] In a first aspect, when the assay is based on the detection of binding of C1q to flavivirus antigen/complement-fixing antibody complexes, the reporter Ab is capable of binding to the C1q with the variable region of the reporter antibody. Preferably, the reporter Ab is specific for C1q, more preferably for human C1q. Antibodies specific for C1q are commercially available. Alternatively, antibodies specific for C1q can be produced by methods known in the art such as immunization of laboratory animals and selection of C1q-specific hybridoma.

[0228] In a second aspect, when the assay is based on the detection of the formation of C3d due to the formation of flavivirus antigen/complement-fixing antibody complexes in the presence of complement competent serum, the reporter Ab is capable of binding to the formed C3d. Preferably, the reporter Ab is specific for C3d, more preferably for human C3d. Antibodies specific for C3d are commercially available. Alternatively, antibodies specific for C3d can be produced by methods known in the art such as immunization of laboratory animals and selection of C3d-specific hybridoma.

[0229] Within the embodiments of the present invention, the reporter Ab is attached to a detectable label. In preferred embodiments, the reporter Ab is attached to the detectable label by the heavy chain constant region of the reporter Ab.

[0230] In embodiments wherein the reporter Ab is used together with a pre-reporter Ab in the methods of the present invention, the reporter Ab is capable of binding to the heavy chain constant region of the pre-reporter Ab with the variable region of the reporter Ab. In a third aspect, when applied together with a pre-reporter Ab, the reporter Ab is capable of binding to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody. The reporter Ab is attached to a detectable label, preferably with the heavy chain constant region of the reporter antibody.

[0231] According to one embodiment of the invention the detectable label to which the reporter antibody is attached to is a compound or moiety that comprises one or more appropriate chemical substances or enzymes, which directly or indirectly generate a detectable compound or signal in a chemical, physical or enzymatic reaction. Labeling can be achieved by methods well known in the art (see, for example, Lottspeich, F., and Zorbas H., Springer Spektrum 2012, Bioanalytik).

[0232] According to one embodiment of the invention the detectable label is selected from the group consisting of fluorescent labels, magnetic labels, enzyme labels, colored labels, chromogenic labels, luminescent labels, radioactive labels, haptens, biotin, metal complexes, metals, and colloidal gold. All these types of labels are well established in the art.

[0233] According to one embodiment of the invention the label is selected from such which provide the emission of fluorescence or phosphorescence upon irradiation or excitation or the emission of X-rays when using a radioactive label.

[0234] According to one embodiment of the invention the label is an enzyme label, which include but are not limited to alkaline phosphatase, horseradish peroxidase (HRP), ?-galactosidase, and ?-lactamase. Enzyme labels catalyze the formation of chromogenic reaction products.

[0235] In specific embodiments the detectable labels are fluorescent labels. Numerous fluorescent labels are well established in the art and commercially available from different suppliers (see, for example, The HandbookA Guide to Fluorescent Probes and Labeling Technologies, 10th ed. (2006), Molecular Probes, Invitrogen Corporation, Carlsbad, CA, USA). Examples of fluorescent labels include but are not limited to xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin (PE), cyanine, coumarin, and any derivative thereof.

[0236] According to preferred embodiments of the invention, the fluorescent label is PE.

[0237] Concerning the detection of such labels with suitable detection systems, reference is also made to the section Detection system.

[0238] In the embodiments wherein the detectable label to which the reporter Ab is attached to is a fluorescent label the fluorescent label can be irradiated/excited with any suitable light source present within a detection system. The light source may be a laser or a LED. In the case the fluorescent label is PE, the excitation wavelength of the light source is in the range of about 505 to about 535 nm, for instance about 511 nm.

Detection System

[0239] According to the invention the detection system refers to any system which is suitable for determining values indicative for the presence and/or amount of reporter antibody.

[0240] According to the invention the detection system may also be able to determine values indicative for the presence and/or amount of a specific microsphere.

[0241] The selection of a suitable detection system depends on several parameters such as the type of detectable labels used for detection or the kind of analysis performed. Various optical and non-optical detection systems are well established in the art. A general description of detection systems that can be used with the method can be found, e.g., in Lottspeich, F., and Zorbas H., Springer Spektrum 2012, Bioanalytik.

[0242] According to one embodiment of the invention, the detection system is an optical detection system. In some embodiments, performing the method involves simple detection systems, which may be based on the measurement of parameters such as fluorescence, optical absorption, resonance transfer, and the like.

[0243] According to one embodiment of the invention the detection system measures fluorescence. Such systems measure the capacity of particular molecules to emit their own light when excited by light of a particular wavelength resulting in a characteristic absorption and emission behavior. In particular, quantitative detection of fluorescence signals is performed by means of modified methods of fluorescence microscopy (for review see, e.g., Lichtman, J. W., and Conchello, J. A. (2005) Nature Methods 2, 910-919; Zimmermann, T. (2005) Adv. Biochem. Eng. Biotechnol. 95, 245-265). Thereby, the signals resulting from light absorption and light emission, respectively, are separated by one or more filters and/or dichroites and imaged on suitable detectors. Data analysis is performed by means of digital image processing. Image processing may be achieved with several software packages well known in the art (such as Mathematica Digital Image Processing, EIKONA, or Image-PRO). Another suitable software for such purposes is the Iconoclust software (Clondiag Chip Technologies GmbH, Jena, Germany). Suitable detection systems may be based on classical methods for measuring a fluorescent signal such as epifluorescence or darkfield fluorescence microscopy (reviewed, e.g., in: Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Publishing Corp., NY). Another optical detection system that may be used is confocal fluorescence microscopy, wherein the object is illuminated in the focal plane of the lens by a point light source. Importantly, the point light source, object and point light detector are located on optically conjugated planes. Examples of such confocal systems are described in detail, for example, in Diaspro, A. (2002) Confocal and 2-photon-microscopy: Foundations, Applications and Advances, Wiley-Liss, Hobroken, NJ. The fluorescence-optical system is usually a fluorescence microscope without an autofocus, for example a fluorescence microscope having a fixed focus. Further fluorescence detection methods that may also be used include inter alia total internal fluorescence microscopy (see, e.g., Axelrod, D. (1999) Surface fluorescence microscopy with evanescent illumination, in: Lacey, A. (ed.) Light Microscopy in Biology, Oxford University Press, New York, 399-423), fluorescence lifetime imaging microscopy (see, for example, Dowling, K. et al. (1999) J. Mod. Optics 46, 199-209), fluorescence resonance energy transfer (FRET; see, for example, Periasamy, A. (2001) J. Biomed. Optics 6, 287-291), bioluminescence resonance energy transfer (BRET; see, e.g., Wilson, T., and Hastings, J. W. (1998) Annu. Rev. Cell Dev. Biol. 14, 197-230), and fluorescence correlation spectroscopy (see, e.g., Hess, S. T. et al. (2002) Biochemistry 41, 697-705). In specific embodiments, detection is performed using FRET or BRET, which are based on the respective formation of fluorescence or bioluminescence quencher pairs. The use of FRET is also described, e.g., in Liu, B. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 589-593; and Szollosi, J. et al. (2002) J. Biotechnol. 82, 251-266. The use of BRET is detailed, for example, in Prinz, A. et al. (2006) Chembiochem. 7, 1007-1012; and Xu, Y. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 151-156.

[0244] In one embodiment the detection system comprises a first light source, e.g. an argon laser or a light emitting diode (LED), which has an excitation wavelength in the range of about 400 to about 700 nm and a second light source, e.g. an argon laser or a LED, which has an excitation wavelength in the range of about 300 to about 700 nm and a suitable detection component as for instance a photodiode such as an avalanche photodiode (APD) in combination with a photomultiplier or a charge-coupled device (CCD) sensor. The first light source may be used for the identification of the detectable label of a microsphere, wherein the detectable label may be one or more fluorescent dyes at a specific concentration. The second light source may be used for excitation of the detectable label of a reporter antibody.

[0245] In a preferred embodiment the first light source, e.g. the argon laser or LED has an excitation wavelength in the range of about 600 to about 650 nm and the second light source, e.g. the argon laser or LED has an excitation wavelength in the range of about 500 to about 600 nm. In a more preferred embodiment the first light source, e.g. the argon laser or LED has an excitation wavelength in the range of about 615 to about 640 nm and the second light source, e.g. the argon laser or LED has an excitation wavelength in the range of about 505 to about 540 nm. In an even more preferred embodiment the first light source, e.g. the argon laser or LED has an excitation wavelength in the range of about 620 to about 635 nm and the second light source, e.g. the argon laser or LED has an excitation wavelength in the range of about 510 to about 535 nm. For instance, the detection system comprises a first light source, e.g. an argon laser or a LED, which has an excitation wavelength of about 635 nm and a second light source, e.g. an argon laser or a LED, which has an excitation wavelength of about 525 nm.

[0246] The detection system may be also capable of distinguishing the individual size or shape of a microsphere from the individual size or shape of another microsphere, thereby allowing individual identification of the microsphere.

[0247] The detection system may be one of the group consisting of MAGPIX?, Luminex 200?, and FLEXMAP 3D? (Luminex Corp. Austin, Tex.). In a preferred embodiment the detection system is the MAGPIX? (Luminex Corp. Austin, Tex.).

[0248] The detection system may be operated by a specific software, including the xPONENT? software (Luminex Corp. Austin, Tex.).

[0249] The detection system may be capable of detecting both, the signal from the detectable label of the reporter Ab, as well as the signal from the detectable label of the microsphere.

[0250] The detection system may be capable of analyzing one microsphere after the other thereby identifying the microsphere by detecting the signal from the detectable label of the microsphere and detecting the signal from the detectable label of the reporter antibody such as flow cytometry based detection systems (e.g. Luminex 200? and FLEXMAP 3D?). The flow cytometry based detection systems Luminex 200? and FLEXMAP 3D? include two lasers each one for irradiation of the detectable label of the microsphere and the detectable label of the reporter Ab. As flow cytometry based detection systems are not capturing the microspheres with a magnet, the Luminex 200? and FLEXMAP 3D? systems are compatible with both, magnetic microspheres such as the MagPlex? microspheres and non-magnetic microspheres such as the Microplex? microspheres. The Luminex 200? and FLEXMAP 3D? systems detect signals from the microspheres and reporter Abs by avalanche photodiodes (APD) in combination with photomultipliers (PMT).

[0251] Alternatively, the detection system may be capable of analyzing multiple microspheres at once. Therefore, a monolayer of magnetic microspheres is captured by a magnet and the microspheres are excited with two LEDs, one LED for excitement of the detectable label of the microsphere and the other LED for excitement of the detectable label of the reporter Ab. The signals from the microspheres and reporter Abs are recorded by a CCD imager, which allows identification of each microsphere and the corresponding antigen to which the microsphere is coupled to. An example for a LED-based detection system is the MAGPIX? instrument. As analyses with the MAGPIX? instrument involves capture of the microspheres with a magnet, the MAGPIX? instrument is solely compatible with magnetic microspheres such as MagPlex? microspheres.

Methods for Determining Flavivirus-Reactive Complement-Fixing Antibodies

[0252] The present invention is directed to methods for determining flavivirus-reactive complement-fixing Abs. Concerning the microsphere complex, the reporter and pre-reporter Ab, as well as the detection system reference is made to the sections above termed Microsphere complex, Reporter and Pre-reporter Ab, and Detection system. In addition, certain specific embodiments are also outlined in this section and shall, however, not be taken as limiting.

[0253] In one aspect the method for determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in a sample from a subject according to the present invention comprises the steps of: [0254] Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; [0255] Step 2: contacting an amount of complement component 1q (C1q) with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow binding of the C1q to the heavy chain constant region of the complement-fixing antibodies; [0256] Step 3: contacting an amount of a reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the reporter antibody to the C1q, wherein the reporter antibody binds to the C1q with the variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and [0257] Step 4: detecting a signal from the reporter antibody bound to the C1q in step 3, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

[0258] In one embodiment of the first aspect, the method for determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in a sample from a subject comprises the steps of: [0259] Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; [0260] Step 2: contacting an amount of C1q with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow binding of the C1q to the heavy chain constant region of the complement-fixing antibodies; [0261] Step 3.1: contacting an amount of a pre-reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the pre-reporter antibody to the C1q, wherein the pre-reporter antibody binds to the C1q with the variable region of the pre-reporter antibody; [0262] Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C1q in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and [0263] Step 4: detecting a signal from the reporter antibody bound to the pre-reporter antibody in step 3.2, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

[0264] According to one embodiment of the present invention, the method for determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in a sample from a subject comprises the further steps of: [0265] Step 5: determining the presence and/or amount of the reporter antibody from the signal of step 4; and [0266] Step 6: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 5.

[0267] In a second aspect, the present invention provides a method for determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in a sample from a subject comprising the steps of: [0268] Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; [0269] Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow the formation of complement component fragment C3d (C3d); [0270] Step 3: contacting an amount of a reporter antibody with the C3d formed in step 2 to allow binding of the reporter antibody to the C3d, wherein the reporter antibody binds to the C3d with the variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and [0271] Step 4: detecting a signal from the reporter antibody bound to the C3d in step 3, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

[0272] In one embodiment of the second aspect the method comprises the steps of: [0273] Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; [0274] Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow the formation of C3d; [0275] Step 3.1: contacting an amount of a pre-reporter antibody with the C3d formed in step 2 to allow binding of the pre-reporter antibody to the C3d, wherein the pre-reporter antibody binds to the C3d with the variable region of the pre-reporter antibody; [0276] Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C3d in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and [0277] Step 4: detecting a signal from the reporter antibody bound to the pre-reporter antibody in step 3.2, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

[0278] In a more preferred embodiment the method comprises the further steps of: [0279] Step 5: determining the presence and/or amount of the reporter antibody from the signal of step 4; and [0280] Step 6: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 5.

[0281] According to one embodiment contacting in step 1 is carried out for about 30 to 90 minutes. In specific embodiments contacting in step 1 is carried out for about 60 minutes.

[0282] According to one embodiment contacting in step 2 is carried out for about 10 to 50 minutes. In specific embodiments contacting in step 2 is carried out for about 30 minutes.

[0283] According to one embodiment contacting in step 3.1 is carried out for about 10 to 50 minutes. In specific embodiments contacting in step 3.1 is carried out for about 30 minutes.

[0284] According to one embodiment contacting in step 3.2 is carried out for about 10 to 50 minutes. In specific embodiments contacting in step 3.2 is carried out for about 30 minutes.

[0285] In one specific embodiment contacting in step 1 is carried out for about 30 to 90 minutes, contacting in step 2 is carried out for about 10 to 50 minutes, contacting in step 3.1 is carried out for about 10 to 50 minutes, and contacting in step 3.2 is carried out for about 10 to 50 minutes.

[0286] In a more specific embodiment contacting in step 1 is carried out for about 60 minutes, contacting in step 2 is carried out for about 30 minutes, contacting in step 3.1 is carried out for about 30 minutes, and contacting in step 3.2 is carried out for about 30 minutes.

[0287] In one embodiment of the present invention, the detectable label to which the reporter antibody is attached to is a fluorescence label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof. In a preferred embodiment the detectable label is phycoerythrin.

[0288] In one embodiment the flavivirus is selected from the group consisting of dengue virus, zika virus, West Nile virus, Japanese encephalitis virus, Tick-Borne encephalitis virus, Yellow Fever virus, Murray Valley encephalitis virus, and St. Louis encephalitis virus.

[0289] In other embodiments the antigen is selected from the group consisting of virus like particle (VLP), non-structural protein 1, envelope protein, pre-membrane protein, membrane protein, capsid protein, non-structural protein 2A, non-structural protein 2B, non-structural protein 3, non-structural protein 4A, non-structural protein 4B, and non-structural protein 5 and any derivative thereof.

[0290] In specific embodiments the flavivirus antigen is a DENV VLP. The DENV VLP can be of any serotype (DENV1-4). In other specific embodiments the flavivirus antigen is DENV NS1. The DENV NS1 can be of any serotype (DENV1-4).

[0291] In other specific embodiments the flavivirus antigen is a ZIKV VLP. In other specific embodiments the flavivirus antigen is ZIKV NS1.

[0292] In certain embodiments, the C1q is present within a complement-component serum, which may be human complement-component serum. In certain embodiments, the C1q is purified from plasma. Purification can be carried out by any protein purification method known in the art, such as filtration, centrifugation, chromatographic separation, or buffer exchange. In other embodiments, the C1q is of human origin. In specific embodiments the C1q is purified human C1q. In general, the origin of the C1q is dependent on the origin of complement-fixing Abs in the sample. For instance, for detection of human complement-fixing Abs, human C1q is suitable.

[0293] In certain embodiments, the complement competent serum is a commercially obtainable complement competent serum. Alternatively, a human serum may be obtained and used.

[0294] In certain embodiments, the sample is heat-inactivated. Heat-inactivation can be carried out at about 56? C. for about 30 minutes. In general, heat-inactivation can be varied according to the type of sample to be analyzed.

[0295] In other embodiments the sample is a blood sample, in particular a plasma or serum sample. In general, the method can be carried out with either matrices i.e. serum or plasma. Preferably, the sample was obtained from an individual immunized with a Dengue vaccine. Alternatively, the sample may have been obtained from a patient suffering from Dengue disease. The sample may have been obtained from the patient suffering from Dengue disease at any time after infection, such as for instance, 1 or 2 weeks after infection.

[0296] In other embodiments the sample is a urine sample.

[0297] In specific embodiments, the sample is a heat-inactivated serum or plasma sample.

[0298] In one embodiment the subject is selected from the group consisting of mouse, primate, non-human primate, human, rabbit, cat, rat, horse, and sheep. In preferred embodiments the subject is a non-human primate. In more preferred embodiments the subject is human.

[0299] In other embodiments the subject is seronegative for the flavivirus. In other embodiments the subject is seropositive for the flavivirus. In specific embodiments the subject is seronegative for DENY. In other specific embodiments the subject is seropositive for DENY. In other specific embodiments the subject is seronegative for ZIKV. In other specific embodiments the subject is seropositive for ZIKV.

[0300] In some embodiments of the present invention the subject is vaccinated with a flavivirus vaccine.

[0301] In one embodiment of the present invention, the signal from the reporter antibody detected in step 4 is resulting from the detectable label to which the reporter antibody is attached. In a specific embodiment, the signal in step 4 is a fluorescence signal. In even more specific embodiments, the signal in step 4 is a fluorescence signal resulting from phycoerythrin. The signal in step 4 can be detected upon irradiation with a light source as present in any suitable detection system.

[0302] Within the meaning of the invention, in embodiments of the first aspect of the present invention wherein no pre-reporter Ab is applied, the signal from the reporter antibody in step 4 is resulting from a reporter antibody which is bound to C1q, wherein C1q is bound to the complement-fixing Abs bound to the flavivirus antigen coupled to microspheres in a microsphere complex.

[0303] Within the meaning of the invention, in embodiments of the first aspect of the present invention wherein a pre-reporter Ab is applied, the signal from the reporter antibody in step 4 is resulting from a reporter antibody which is bound to the pre-reporter Ab, wherein the pre-reporter Ab is bound to C1q, wherein C1q is bound to the complement-fixing Abs bound to the flavivirus antigen coupled to microspheres in a microsphere complex.

[0304] Within the meaning of the invention, in embodiments of the second aspect of the present invention wherein no pre-reporter Ab is applied, the signal from the reporter antibody in step 4 is resulting from a reporter antibody which is bound to the formed C3d. Within the meaning of the invention, in embodiments of the first aspect of the present invention wherein a pre-reporter Ab is applied, the signal from the reporter antibody in step 4 is resulting from a reporter antibody which is bound to the pre-reporter Ab, wherein the pre-reporter Ab is bound to the formed C3d. In specific embodiments of the present invention the presence and/or amount of reporter Ab in step 5 is determined by comparing the signal of step 4 to a standard curve, wherein the standard curve comprises signals resulting from known amounts of reporter Ab.

[0305] In specific embodiments of the invention the presence and/or amount of flavivirus-reactive complement-fixing Abs in the sample in step 6 is determined based on the amount of the reporter Ab determined in step 5 as the amount of reporter Ab is in direct proportion to the complement-fixing Abs present within a sample.

Methods for the Concomitant Determination of Complement-Fixing Antibodies Reactive to Different Flaviviruses

[0306] The present invention is further directed to methods for the concomitant determination complement-fixing Abs reactive to different flaviviruses. Concerning the microsphere complex, the reporter and pre-reporter Ab, as well as the detection system reference is made to the sections above termed Microsphere complex, Reporter and Pre-reporter Ab, and Detection system. In addition, certain specific embodiments are also outlined in this section and shall, however, not be taken as limiting.

[0307] In one aspect of the present invention the method for the concomitant determination of the presence and/or amount of complement-fixing antibodies reactive to different flaviviruses in a sample from a subject comprises the steps of: [0308] Step 1: contacting an amount of at least two microsphere complexes with the sample, [0309] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen [0310] to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen, [0311] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, [0312] Step 2: contacting an amount of C1q with the complement-fixing antibodies bound to the first and/or the second flavivirus antigen in step 1 to allow binding of the C1q to the heavy chain constant region of the complement-fixing antibodies, [0313] Step 3: contacting an amount of a reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the reporter antibody to the C1q, wherein the reporter antibody binds to the C1q with the variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, [0314] Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and [0315] simultaneously detecting a signal from the reporter antibody bound to the C1q in step 3 of the at least one microsphere upon irradiation with a second light source, [0316] Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and [0317] Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

[0318] In certain embodiments the method for the concomitant determination of the presence and/or amount of complement-fixing antibodies reactive to different flaviviruses in a sample from a subject comprises the steps of: [0319] Step 1: contacting an amount of at least two microsphere complexes with the sample, [0320] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen [0321] to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen, [0322] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, [0323] Step 2: contacting an amount of C1q with the complement-fixing antibodies bound to the first and/or the second flavivirus antigen in step 1 to allow binding of the C1q to the heavy chain constant region of the complement-fixing antibodies, [0324] Step 3.1: contacting an amount of a pre-reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the pre-reporter antibody to the C1q, wherein the pre-reporter antibody binds to the C1q with the variable region of the pre-reporter antibody, [0325] Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C1q in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, [0326] Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and [0327] simultaneously detecting a signal from the reporter antibody bound to the C1q in step 3 of the at least one microsphere upon irradiation with a second light source, [0328] Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and [0329] Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

[0330] In some embodiments the method for the concomitant determination of the presence and/or amount of complement-fixing antibodies reactive to different flaviviruses in a sample from a subject comprises the further steps of: [0331] Step 7: determining the presence and/or amount of reporter antibody from the summarized signal in step 6, and [0332] Step 8: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of reporter antibody determined in step 7.

[0333] In a second aspect of the present invention, the method for the concomitant determination of the presence and/or amount of complement-fixing antibodies reactive to different flaviviruses in a sample from a subject comprises the steps of: [0334] Step 1: contacting an amount of at least two microsphere complexes with the sample, [0335] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen [0336] to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen, [0337] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, [0338] Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow the formation of C3d, [0339] Step 3: contacting an amount of a reporter antibody with the C3d formed in step 2 to allow binding of the reporter antibody to the C3d, wherein the reporter antibody binds to the C3d with the variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, [0340] Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and [0341] simultaneously detecting a signal from the reporter antibody bound to the C3d in step 3 of the at least one microsphere upon irradiation with a second light source, [0342] Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and [0343] Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

[0344] In one embodiment of this aspect the method comprises the steps of: [0345] Step 1: contacting an amount of at least two microsphere complexes with the sample, [0346] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen [0347] to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen, [0348] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, [0349] Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the first and/or the second flavivirus antigen in step 1 to allow the formation of C3d, [0350] Step 3.1: contacting an amount of a pre-reporter antibody with the C3d formed in step 2 to allow the formation of C3d and to allow binding of the pre-reporter antibody to the C3d, wherein the pre-reporter antibody binds to the C3d with the variable region of the pre-reporter antibody, [0351] Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C3d in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, [0352] Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and simultaneously detecting a signal from the reporter antibody bound to the C3d in step 3 of the at least one microsphere upon irradiation with a second light source, [0353] Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and [0354] Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample.

[0355] In one preferred embodiment of this aspect the method further comprises the steps of: [0356] Step 7: determining the presence and/or amount of the reporter antibody from the summarized signal in step 6, and [0357] Step 8: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 7.

[0358] In certain embodiments the first detectable label is at least one fluorescent dye. In other embodiments the second detectable label is at least one fluorescent dye. In other embodiments the first and the second detectable labels are each at least one fluorescent dye. Within the meaning of the invention the first and the second detectable label are different labels. In preferred embodiments the at least one fluorescent dye is selected from the group consisting of squaraine, phthalocyanine, naphthalocyanine, and any derivative thereof. For instance, the first microsphere comprises squarine, the second microsphere comprises phthalocyanine.

[0359] In certain embodiments the emission signal of the detectable label of the at least one microsphere detected under step 4 upon irradiation with a first light source corresponds to the emission signal of the first detectable label.

[0360] In certain embodiments the emission signal of the detectable label of the at least one microsphere detected under step 4 upon irradiation with a first light source corresponds to the emission signal of the second detectable label.

[0361] In specific embodiments the first light source is a laser or a LED. The wavelength of the first light source may be in the range of about 400 to about 700 nm. In a preferred embodiment the wavelength of the first light source may be in the range of about 600 to about 650 nm. In a more preferred embodiment the wavelength of the first light source may be in the range of about 615 to about 640 nm. In an even more preferred embodiment the wavelength of the first light source may be in the range of about 620 to about 635 nm. For instance, the wavelength of the first light source is about 635 nm.

[0362] In certain embodiments the third detectable label is a fluorescence label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof. In preferred embodiments the fluorescence label is phycoerythrin.

[0363] In one embodiment of the present invention, the signal from the reporter antibody detected in step 4 is resulting from the third detectable label to which the reporter antibody is attached. In a specific embodiment, the signal in step 4 is a fluorescence signal. In even more specific embodiments, the signal in step 4 is a fluorescence signal resulting from phycoerythrin.

[0364] The signal from the reporter antibody in step 4 can be detected upon irradiation with a second light source. In specific embodiments the second light source is a laser or a LED. The wavelength of the second light source may be in the range of about 300 to about 700 nm. In a preferred embodiment the wavelength of the first light source may be in the range of about 500 to about 600 nm. In a more preferred embodiment the wavelength of the first light source may be in the range of about 505 to about 540 nm. In an even more preferred embodiment the wavelength of the first light source may be in the range of about 510 to about 535 nm. For instance, the wavelength of the first light source is about 525 nm.

[0365] Detection of the signal from the reporter antibody and recording of the emission signal of the detectable label of at least one microsphere is carried out simultaneously, i.e. in one analysis step. Therefore, the at least one microsphere is irradiated with the first light source followed by detecting the emission signal of the detectable label of the at least one microsphere in a first step, directly followed by irradiation of the at least one microsphere with the second light source followed by detecting the signal from the reporter antibody in a second step. It is also possible, that the at least one microsphere is irradiated with the second light source followed by detecting the signal from the reporter antibody in a first step, directly followed by irradiation of the at least one microsphere with the first light source followed by detecting the emission signal of the detectable label of the at least one microsphere in a second step.

[0366] Within the meaning of the invention, in embodiments of the first aspect of the present invention, wherein no pre-reporter Ab is applied, the signal from the reporter antibody in step 4 is resulting from a reporter antibody which is bound to C1q, wherein C1q is bound to the complement-fixing Abs bound to the flavivirus antigen coupled to microspheres in a microsphere complex.

[0367] Within the meaning of the invention, in embodiments of the first aspect of the present invention wherein a pre-reporter Ab is applied, the signal from the reporter antibody in step 4 is resulting from a reporter antibody which is bound to the pre-reporter Ab, wherein the pre-reporter Ab is bound to C1q, wherein C1q is bound to the complement-fixing Abs bound to the flavivirus antigen coupled to microspheres in a microsphere complex.

[0368] Within the meaning of the invention, in embodiments of the second aspect of the present invention wherein no pre-reporter Ab is applied, the signal from the reporter antibody in step 4 is resulting from a reporter antibody which is bound to the formed C3d. Within the meaning of the invention, in embodiments of the first aspect of the present invention wherein a pre-reporter Ab is applied, the signal from the reporter antibody in step 4 is resulting from a reporter antibody which is bound to the pre-reporter Ab, wherein the pre-reporter Ab is bound to the formed C3d.

[0369] In one embodiment step 4 is repeated until at least 30 microspheres coupled to the same flavivirus antigen are identified. In another embodiment step 4 is repeated until at least 40 microspheres coupled to the same flavivirus antigen are identified. In another embodiment step 4 is repeated until at least 50 microspheres coupled to the same flavivirus antigen are identified. In another embodiment step 4 is repeated until at least 60 microspheres coupled to the same flavivirus antigen are identified. In another embodiment step 4 is repeated until at least 80 microspheres coupled to the same flavivirus antigen are identified. In another embodiment step 4 is repeated until at least 100 microspheres coupled to the same flavivirus antigen are identified.

[0370] For instance, if one microsphere is coupled to a DENV1 VLP and another microsphere is coupled to a DENV2 VLP, step 4 is repeated until at least 30, or at least 40, or at least 50, or at least 60, or at least 80, or at least 100 microspheres that are coupled to DENV1 VLP and until at least 30, or at least 40, or at least 50, or at least 60, or at least 80, or at least 100 microspheres that are coupled to DENV2 VLP are identified.

[0371] Within the meaning of step 6, summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen refers to the addition of the detected signals for all single microspheres coupled to the same flavivirus antigen.

[0372] The summarized signal from the reporter antibody of step 5 for the number of microspheres coupled to the same flavivirus antigen identified under step 4 is representative for all microspheres coupled to the same flavivirus antigen in the experiment.

[0373] In certain embodiments, the flaviviruses are selected from the group consisting of dengue virus, zika virus, West Nile virus, Japanese encephalitis virus, Tick-Borne encephalitis virus, Yellow Fever virus, Murray Valley encephalitis virus, and St. Louis encephalitis virus.

[0374] In certain embodiments, the antigens are selected from the group consisting of virus like particle (VLP), non-structural protein 1, envelope protein, pre-membrane protein, membrane protein, capsid protein, non-structural protein 2A, non-structural protein 2B, non-structural protein 3, non-structural protein 4A, non-structural protein 4B, and non-structural protein 5 and any derivative thereof.

[0375] Within the meaning of the invention the first flavivirus antigen and the second flavivirus antigen are different. For instance, the first flavivirus antigen is a DENV VLP and the second flavivirus antigen is a ZIKV VLP. Another example would be that the first flavivirus antigen is a DENV VLP and the second flavivirus antigen is a DENV NS1. Another example would be that the first flavivirus antigen is a DENV serotype 1 VLP and the second flavivirus antigen is a DENV serotype 2 VLP.

[0376] The method of the present invention provides the possibility to contact any number of microsphere complexes with the sample in step 1, wherein each microsphere complex comprises a microsphere coupled to a different flavivirus antigen and wherein each microsphere complex comprises a microsphere with a different detectable label resulting in a different emission signal e.g. a different emission intensity upon irradiation thereby providing the possibility to identify the different microspheres and thereby the different antigens.

[0377] In a specific embodiment, in step 1, an amount of a first microsphere complex comprising a first microsphere coupled to a dengue 1 virus VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a dengue 2 virus VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a dengue 3 virus VLP, and an amount of a fourth microsphere complex comprising a fourth microsphere coupled to a dengue 4 virus VLP is contacted with the sample.

[0378] In other specific embodiments, in step 1, an amount of a first microsphere complex comprising a first microsphere coupled to a dengue 1 virus VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a dengue 2 virus VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a dengue 3 virus VLP, an amount of a fourth microsphere complex comprising a fourth microsphere coupled to a dengue 4 virus VLP, an amount of a fifth microsphere complex comprising a fifth microsphere coupled to a zika virus VLP, an amount of a sixth microsphere complex comprising a sixth microsphere coupled to a dengue 1 virus non-structural protein 1, an amount of a seventh microsphere complex comprising a seventh microsphere coupled to a dengue 2 virus non-structural protein 1, an amount of an eighth microsphere complex comprising an eighth microsphere coupled to a dengue 3 virus non-structural protein 1, an amount of a ninth microsphere complex comprising a ninth microsphere coupled to a dengue 4 virus non-structural protein 1, and an amount of a tenth microsphere complex comprising a tenth microsphere coupled to a zika virus non-structural protein 1 is contacted with the sample.

[0379] According to one embodiment contacting in step 1 is carried out for about 30 to 90 minutes. In specific embodiments contacting in step 1 is carried out for about 60 minutes.

[0380] According to one embodiment contacting in step 2 is carried out for about 10 to 50 minutes. In specific embodiments contacting in step 2 is carried out for about 30 minutes.

[0381] According to one embodiment contacting in step 3.1 is carried out for about 10 to 50 minutes. In specific embodiments contacting in step 2 is carried out for about 30 minutes.

[0382] According to one embodiment contacting in step 3.2 is carried out for about 10 to 50 minutes. In specific embodiments contacting in step 2 is carried out for about 30 minutes.

[0383] In one specific embodiment contacting in step 1 is carried out for about 30 to 90 minutes, contacting in step 2 is carried out for about 10 to 50 minutes, contacting in step 3.1 is carried out for about 10 to 50 minutes, and contacting in step 3.2 is carried out for about 10 to 50 minutes.

[0384] In a more specific embodiment contacting in step 1 is carried out for about 60 minutes, contacting in step 2 is carried out for about 30 minutes, contacting in step 3.1 is carried out for about 30 minutes, and contacting in step 3.2 is carried out for about 30 minutes.

[0385] In certain embodiments, the C1q is present within a complement-component serum, which may be human complement-component serum. In certain embodiments, the C1q is purified from plasma. Purification can be carried out by any protein purification method known in the art, such as filtration, centrifugation, chromatographic separation, or buffer exchange. In other embodiments, the C1q is of human origin. In specific embodiments the C1q is purified human C1q. In general, the origin of the C1q is dependent on the origin of complement-fixing Abs in the sample. For instance, for detection of human complement-fixing Abs, human C1q is suitable.

[0386] In certain embodiments, the complement competent serum is a commercially obtainable complement competent serum. Alternatively, a human serum may be obtained and used.

[0387] In certain embodiments, the sample is heat-inactivated. Heat-inactivation can be carried out at about 56? C. for about 30 minutes. In general, heat-inactivation can be varied according to the type of sample to be analyzed.

[0388] In some embodiments the sample is a blood sample, in particular a plasma or serum sample. In general, the method can be carried out with either matrices i.e. serum or plasma.

[0389] In other embodiments the sample is a urine sample.

[0390] In specific embodiments, the sample is a heat-inactivated serum or plasma sample.

[0391] In one embodiment the subject is selected from the group consisting of mouse, primate, non-human primate, human, rabbit, cat, rat, horse, and sheep. In preferred embodiments the subject is a non-human primate. In more preferred embodiments the subject is human.

[0392] In other embodiments the subject is seronegative for the flavivirus. In other embodiments the subject is seropositive for the flavivirus. In specific embodiments the subject is seronegative for DENY. In other specific embodiments the subject is seropositive for DENY.

[0393] In some embodiments of the present invention the subject is vaccinated with a flavivirus vaccine.

[0394] In specific embodiments of the present invention the presence and/or amount of reporter Ab in step 7 is determined by comparing the summarized signal of step 6 to a standard curve, wherein the standard curve comprises signals resulting from known amounts of reporter Ab.

[0395] In specific embodiments of the invention the presence and/or amount of flavivirus-reactive complement-fixing Abs in the sample in step 8 is determined based on the amount of the reporter Ab determined in step 7 as the amount of reporter Ab is in direct proportion to the complement-fixing Abs present within a sample. Thereby, the amount of complement-fixing Abs reactive to the different flaviviruses can be determined.

In Vitro Methods for Diagnosing a Flavivirus Infection

[0396] The present invention is further directed to in vitro methods for diagnosing a flavivirus infection. Concerning the determination of the amount of flavivirus-reactive complement-fixing Abs, reference is made to the sections above termed Methods for determining flavivirus-reactive complement-fixing antibodies and Methods for the concomitant determination of complement-fixing antibodies reactive to different flaviviruses. In addition, certain specific embodiments are also outlined in this section and shall, however, not be taken as limiting.

[0397] In one aspect of the present invention the in vitro method for diagnosing a flavivirus infection in a subject comprises determining the amount of flavivirus-reactive complement-fixing antibodies in the sample according to the methods of the first aspect of the present invention as described above, wherein the presence of flavivirus-reactive complement-fixing antibodies in the sample is indicative for a flavivirus infection.

[0398] In a second aspect of the present invention the in vitro method for diagnosing a flavivirus infection in a subject comprises determining the amount of flavivirus-reactive complement-fixing antibodies in the sample according to the methods of the second aspect of the present invention as described above, wherein the presence of flavivirus-reactive complement-fixing antibodies in the sample is indicative for a flavivirus infection.

[0399] In some embodiments, the flavivirus is selected from the group consisting of dengue virus, zika virus, West Nile virus, Japanese encephalitis virus, Tick-Borne encephalitis virus, Yellow Fever virus, Murray Valley encephalitis virus, and St. Louis encephalitis virus. In preferred embodiments the flavivirus is DENV or ZIKV.

[0400] In some embodiments, the subject is infected by a dengue virus serotype selected from the group of dengue virus serotype 1, dengue virus serotype 2, dengue virus serotype 3, and dengue virus serotype 4.

[0401] In some embodiments, the subject is infected by at least two different flaviviruses. For instance, the subject can be infected by DENV and ZIKV. The infection may be acute or convalescent. For instance, the subject has been infected by DENV first and was then infected by ZIKV several months later. The in vitro method for diagnosing of the present application is capable of diagnosing the at least two different flavivirus infections e.g. the DENV and the ZIKV infection. Consequently, the in vitro method for diagnosing of the present application is capable of determining whether a subject was infected with one or more flaviviruses and by which flaviviruses the subject was infected.

[0402] In some embodiments, the subject is infected by at least two different dengue virus serotypes selected from the group of dengue virus serotype 1, dengue virus serotype 2, dengue virus serotype 3, and dengue virus serotype 4. For instance, the subject can be infected by DENV serotype 1 and DENV serotype 2. The infection may be acute or convalescent. For instance, the subject has been infected by DENV serotype 1 first and was then infected by DENV serotype 2 several months later. The in vitro method for diagnosing of the present application is capable of diagnosing the at least two different dengue virus serotype infections e.g. the DENV serotype 1 and the DENV serotype 2 infection. Consequently, the in vitro method for diagnosing of the present application is capable of determining whether a subject was infected with one or more DENV serotypes and by which DENV serotypes the subject was infected.

[0403] In certain embodiments, the sample is heat-inactivated. Heat-inactivation can be carried out at about 56? C. for about 30 minutes. In general, heat-inactivation can be varied according to the type of sample to be analyzed.

[0404] In other embodiments the sample is a blood sample, in particular a plasma or serum sample. In general, the method can be carried out with either matrices i.e. serum or plasma.

[0405] In other embodiments the sample is a urine sample.

[0406] In specific embodiments, the sample is a heat-inactivated serum or plasma sample.

[0407] In one embodiment the subject is selected from the group consisting of mouse, primate, non-human primate, human, rabbit, cat, rat, horse, and sheep. In preferred embodiments the subject is a non-human primate. In more preferred embodiments the subject is human.

[0408] In other embodiments the subject is seronegative for the flavivirus. In other embodiments the subject is seropositive for the flavivirus. In specific embodiments the subject is seronegative for DENV. In other specific embodiments the subject is seropositive for DENV.

[0409] In some embodiments of the present invention the subject is vaccinated with a flavivirus vaccine.

[0410] In one embodiment the flavivirus infection is acute. In another embodiment the flavivirus infection is convalescent.

Kits for Detecting Flavivirus-Reactive Complement-Fixing Antibodies

[0411] The present invention is further directed to kits for detecting flavivirus-reactive complement-fixing antibodies. Concerning the microsphere complex, as well as the reporter and pre-reporter Ab, reference is made to the sections above termed microsphere complex and Reporter and Pre-reporter Ab. In addition, certain specific embodiments are also outlined in this section and shall, however, not be taken as limiting.

[0412] In a first aspect of the invention, the kit for detecting flavivirus-reactive complement-fixing antibodies in a sample comprises: [0413] an amount of at least one microsphere complex comprising a microsphere coupled to a flavivirus antigen as described above, [0414] an amount of C1q, and [0415] an amount of a reporter antibody, wherein the reporter antibody is attached to a detectable label.

[0416] In one embodiment, the kit for detecting flavivirus-reactive complement-fixing antibodies in a sample comprises: [0417] an amount of at least two microsphere complexes, [0418] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen as described above and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen as described above, [0419] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and [0420] wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label [0421] an amount of C1q, and [0422] an amount of a reporter antibody, wherein the reporter antibody binds to the C1q with the variable region of the reporter antibody, and wherein the reporter antibody is attached to a detectable label.

[0423] In a second aspect the present invention provides a kit for detecting flavivirus-reactive complement-fixing antibodies in a sample comprising: [0424] an amount of at least one microsphere complex comprising a microsphere coupled to a flavivirus antigen, [0425] an amount of a complement competent serum, and [0426] an amount of a reporter antibody, wherein the reporter antibody binds to the C3d with the variable region of the reporter antibody, and wherein the reporter antibody is attached to a detectable label.

[0427] In an embodiment of the second aspect the kit comprises an amount of at least two microspheres complexes, [0428] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen, [0429] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and [0430] wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label.

[0431] In some embodiments, the flavivirus is selected from the group consisting of dengue virus, Zika virus, West Nile virus, Japanese encephalitis virus, Tick-Borne encephalitis virus, Yellow Fever virus, Murray Valley encephalitis virus, and St. Louis encephalitis virus.

[0432] In some embodiments, the antigen is selected from the group consisting of virus like particle (VLP), non-structural protein 1, envelope protein, pre-membrane protein, membrane protein, capsid protein, non-structural protein 2A, non-structural protein 2B, non-structural protein 3, non-structural protein 4A, non-structural protein 4B, and non-structural protein 5 and any derivative thereof.

[0433] The kit of the present invention may comprise any number of microsphere complexes, wherein each microsphere complex comprises a microsphere coupled to a different flavivirus antigen and wherein each microsphere complex comprises a microsphere with a different detectable label resulting in a different emission signal e.g. a different emission intensity upon irradiation thereby providing the possibility to identify the different microspheres and thereby the different antigens.

[0434] In specific embodiments the kit comprises an amount of a first microsphere complex comprising a first microsphere coupled to a dengue-1 virus VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a dengue-2 virus VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a dengue-3 virus VLP, and an amount of a fourth microsphere complex comprising a fourth microsphere coupled to a dengue-4 virus VLP.

[0435] In other specific embodiments the kit comprises an amount of a first microsphere complex comprising a first microsphere coupled to a dengue-1 virus VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a dengue-2 virus VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a dengue-3 virus VLP, an amount of a fourth microsphere complex comprising a fourth microsphere coupled to a dengue-4 virus VLP, an amount of a fifth microsphere complex comprising a fifth microsphere coupled to a zika virus VLP, an amount of a sixth microsphere complex comprising a sixth microsphere coupled to a dengue-1 virus non-structural protein 1, an amount of a seventh microsphere complex comprising a seventh microsphere coupled to a dengue-2 virus non-structural protein 1, an amount of an eighth microsphere complex comprising an eighth microsphere coupled to a dengue-3 virus non-structural protein 1, an amount of a ninth microsphere complex comprising a ninth microsphere coupled to a dengue-4 virus non-structural protein 1, and an amount of a tenth microsphere complex comprising a tenth microsphere coupled to a zika virus non-structural protein 1.

[0436] In certain embodiments, the C1q is present within a complement-component serum, which may be human complement-component serum. In certain embodiments, the C1q is purified from plasma. Purification can be carried out by any protein purification method known in the art, such as filtration, centrifugation, chromatographic separation, or buffer exchange. In other embodiments, the C1q is of human origin. In specific embodiments the C1q is purified human C1q. In general, the origin of the C1q is dependent on the origin of complement-fixing Abs in the sample. For instance, for detection of human complement-fixing Abs, human C1q is suitable.

[0437] In certain embodiments, the complement competent serum is a commercially obtainable complement competent serum. Alternatively, a human serum may be obtained and used. In certain embodiments of the first aspect of the invention, the reporter antibody binds to the C1q with the variable region of the reporter antibody, when the kit does not further comprise a pre-reporter antibody.

[0438] In other embodiments the kit further comprises an amount of a pre-reporter antibody, wherein the pre-reporter antibody binds to the C1q with the variable region of the pre-reporter antibody and the reporter antibody binds to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody.

[0439] In a second aspect, when the assay is based on the detection of the formation of C3d due to the formation of flavivirus antigen/complement-fixing antibody complexes in the presence of complement competent serum, the reporter Ab is capable of binding to the formed C3d. Preferably, the reporter Ab is specific for C3d, more preferably for human C3d.

[0440] Within the embodiments of the present invention, the reporter Ab is attached to a detectable label. In preferred embodiments, the reporter Ab is attached to the detectable label by the heavy chain constant region of the reporter Ab.

[0441] In a third aspect, when applied together with a pre-reporter Ab, the reporter Ab is capable of binding to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody. The reporter Ab is attached to a detectable label, preferably with the heavy chain constant region of the reporter antibody.

[0442] In some embodiments the detectable label to which the reporter antibody is attached to is a fluorescence label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof. In preferred embodiments the fluorescence label is phycoerythrin.

[0443] In certain embodiments the first detectable label is at least one fluorescent dye. In other embodiments the second detectable label is at least one fluorescent dye.

[0444] In other embodiments the first and the second detectable labels are each at least one fluorescent dye. Within the meaning of the invention the first and the second detectable label are different labels. In preferred embodiments the fluorescent dye is selected from the group consisting of squaraine, phthalocyanine, naphthalocyanine, and any derivative thereof. For instance, the first microsphere comprises squarine, the second microsphere comprises phthalocyanine.

[0445] The kits may further contain a suitable container for the mixture of the components of the kit.

EXAMPLES

[0446] The following Examples are included to demonstrate certain aspects and embodiments of the invention as described in the claims. It should be appreciated by those of skill in the art, however, that the following description is illustrative only and should not be taken in any way as a restriction of the invention.

Example 1: Coupling of Flavivirus Antigens to Microspheres

[0447] Microspheres used for coupling were MagPlex? microspheres (Luminex Corporation, Austin, Texas). MagPlex? microspheres are superparamagnetic polystyrene microspheres with surface carboxylic acid groups. The microspheres were delivered in a volume of 4 to 4.1 mL with an average concentration of 1.2 to 1.3?10.sup.7 microspheres per mL (microspheres/mL). MagPlex? microspheres are available in several unique regions, i.e. the microspheres comprise one or more fluorescent dyes having a defined emission signal (the detectable label) in order to distinguish the microspheres from microspheres of other unique regions. As the coupling mechanism involving the surface carboxyl groups is independent of the specific feature of the microspheres, MagPlex? microspheres of different unique regions may be exchanged according to variations in experimental set-ups.

[0448] DENV antigens for coupling to microspheres were DENV1 VLP (0.46 mg/mL liquid stock in 10 mM sodium phosphate, 20 mM sodium citrate, 154 mM sodium chloride pH 7.4; The Native Antigen Company, Product Code: DENV1-VLP-500, Batch No. 19040109), DENV2 VLP (0.52 mg/mL liquid stock in 10 mM sodium phosphate, 20 mM sodium citrate, 154 mM sodium chloride pH 7.4; The Native Antigen Company, Product Code: DENV2-VLP-500, Batch No. 19040816), DENV3 VLP (0.72 mg/mL liquid stock in 10 mM sodium phosphate, 20 mM sodium citrate, 154 mM sodium chloride pH 7.4; The Native Antigen Company, Product Code: DENV3-VLP-500, Batch No. 18111415), and DENV4 VLP (0.53 mg/mL liquid stock in Dulbecco's phosphate-buffered saline (DPBS) pH 7.4, 30% sucrose; The Native Antigen Company, Product Code: DENV4-VLP-500, Batch No. 19061911).

[0449] DENV1-4 VLPs are consisting of DENV prM, M, and E protein produced in human embryonic kidney (HEK 293) cells. For production of DENV1-4 VLPs, the C-terminal 20% of DENV E protein were replaced by the corresponding Japanese encephalitis virus (JEV) SA-14 sequence (EMBL-EBI accession No: M55506.1; E protein amino acids 399-497 (DENV1 VLP), 397-495 (DENV2 VLP), 399-492 (DENV3 VLP), 400-495 (DENV4 VLP)). The replaced sequence corresponds to the transmembrane and intraparticle portion of the protein. DENV1 VLP was produced using the sequence from strain Puerto Rico/US/BID-V853/1998 (SEQ ID NO: 1 and 5; GenBank accession No. EU482592.1, Uniprot No. B1PNU3). DENV2 VLP was produced using the sequence from strain Thailand/16681/84 (SEQ ID NO: 2 and 6; EMBL-EBI accession No: U87411.1, Uniprot No. P29990.1). DENV3 VLP was produced using the sequence from strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (SEQ ID NO: 3 and 7; GenBank accession No. AY099336.1, Uniprot No. Q6YMS4.1). DENV4 VLP was produced using the sequence from strain Dominica/814669/1981 (SEQ ID NO: 4 and 8; EMBL-EBI accession No: AF326825.1, Uniprot No. P09866.2).

[0450] ZIKV VLPs (The Native Antigen Company, Product Code: ZIKV-VLP) comprise prM, M, and E protein of ZIKV strain Z1106033 isolated in Suriname (Asian genotype; Enfissi et al., Lancet 2016, 387(10015):227-228; GenBank Accession No. KU312312.1 and ALX35659.1, SEQ ID NO: 9 and 10), include amino acids 123-819 and are produced in HEK 293 cells.

[0451] DENV1-4 NS1 (The Native Antigen Company, Product Code: DENVX4-NS1) and ZIKV NS1 (The Native Antigen Company, Product Code: ZIKVSU-NS1) are produced in human embryonic kidney (HEK 293) cells. DENV1 NS1 was produced using the sequence from strain Nauru/Western Pacific/1974 (SEQ ID NO: 11 and 12; GenBank accession No. AY145121). The sequence of DENV1 NS1 as coupled to the microspheres is shown in SEQ ID NO: 13. DENV2 NS1 was produced using the sequence from strain Thailand/16681/84 (SEQ ID NO: 2 and 6; EMBL-EBI accession No: U87411.1, Uniprot No. P29990.1). The sequence of DENV2 NS1 as coupled to the microspheres is shown in SEQ ID NO: 14. DENV3 NS1 was produced using the sequence from strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 (SEQ ID NO: 3 and 7; GenBank accession No. AY099336.1, Uniprot No. Q6YMS4.1). The sequence of DENV3 NS1 as coupled to the microspheres is shown in SEQ ID NO: 15. DENV4 NS1 was produced using the sequence from strain Dominica/814669/1981 (SEQ ID NO: 4 and 8; EMBL-EBI accession No: AF326825.1, Uniprot No. P09866.2). The sequence of DENV4 NS1 as coupled to the microspheres is shown in SEQ ID NO: 16. ZIKV NS1 was produced using the sequence (amino acids 795-1146) from strain Suriname Z110603 (SEQ ID NO: 9 and 10; GenBank Accession No. KU312312.1 and ALX35659.1) and buffered in PBS, pH 7.4.

[0452] VLPs are a desirable reagent for coupling to the microspheres because of their ease of manufacture, antigenic fidelity, and lack of safety concerns. Further, for evaluation of complement fixing antibodies against all DENV serotypes produced upon vaccination with live attenuated viruses comprising E and prM structural proteins, VLPs are favorable as they are a good surrogate for the whole virion. Moreover, antibodies against structural antigens i.e. E and prM protein can be detected by the application of one single antigen (the VLP).

[0453] Different microspheres comprising one or more fluorescent dyes having a specific emission signal (different unique regions) were applied for coupling of the different antigens to provide the possibility to distinguish the microspheres according to their coupled antigens when analyzed within one sample (capability to multi-plex). For example, DENV1 VLP was coupled to a set of MagPlex? microspheres of region 76, DENV2 VLP was coupled to a set of MagPlex? microspheres of region 14, DENV3 VLP was coupled to a set of MagPlex? microspheres of region 25, and DENV4 VLP was coupled to a set of MagPlex? microspheres of region 33. DENV1 NS1 was coupled to a set of MagPlex? microspheres of region 45, DENV2 NS1 was coupled to a set of MagPlex? microspheres of region 65, DENV3 NS1 was coupled to a set of MagPlex? microspheres of region 66, and DENV4 NS1 was coupled to a set of MagPlex? microspheres of region 67. ZIKV VLP was coupled to a set of MagPlex? microspheres of region 47, ZIKV NS1 was coupled to a set of MagPlex? microspheres of region 36.

Coupling of Flavivirus Antigens to Microspheres

[0454] The uncoupled stocks of MagPlex? microsphere suspensions (1.2 to 1.3?10.sup.7 microspheres/mL, Luminex Corporation, Austin, Texas) were resuspended by vortexing (30 sec) and 12.5?10.sup.6 microspheres of each stock were transferred to 5 mL microcentrifuge tubes and placed into a 5 mL tubes magnetic separator (Life Technologies). Separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was carefully removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. Afterwards, the tubes were removed from the magnetic separator and the microspheres were resuspended in 500 ?L distilled H.sub.2O (dH.sub.2O) by vortexing and sonication for approximately 20 sec. The tubes were again placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The microspheres were resuspended in 400 ?l of activation buffer (0.1 M sodium phosphate (monobasic) pH 6.2) and mixed by vortexing and sonication for 20 sec. Then, 50 ?L of 50 mg/mL N-hydroxysulfosuccinimide (Sulfo-NHS; 50 mg of Sulfo-NHS in 1000 ?L of dH.sub.2O; Thermo Fisher Scientific) were added to each microsphere tube and gentle mixing was carried out by vortexing (5 sec). Further, 50 ?L of 50 mg/mL 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; 10 mg EDC in 200 ?L of dH.sub.2O; Thermo Fisher Scientific) were added to each microsphere tube and gentle mixing was carried out by vortexing (5 sec). Samples were incubated for 20 min at room temperature (18-24? C.) with gentle mixing by vortexing after 10 min. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The tubes were removed from the magnetic separator and the microspheres were resuspended in 500 ?L of 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 6.00 for DENV1-4 VLP, DENV1-4 NS1 proteins, and ZIKV NS1 protein (Boston Bioproducts, Cat. No. BBMS-60, Lot. No. F03K118) or at pH 7.00 for ZIKV VLP by vortexing and sonication for 20 sec. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The tubes were removed from the magnetic separator and the microspheres were resuspended in 500 ?L of corresponding 50 mM MES buffer by vortexing and sonication for 20 sec. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator.

[0455] Afterwards, 1000 ?L of a solution of each one of the DENV1-4 VLPs, DENV1-4 NS1 proteins, ZIKV VLP and ZIKV NS1 (diluted in 50 mM MES buffer at pH 6.0 for all NS1 proteins (DENV and ZIKV), as well as DENV1-4 VLPs and at pH 7.0 for ZIKV VLP) were transferred to a different 5 mL tube containing the activated microspheres to result in a ratio of 5 ?g antigen per 10.sup.6 microspheres in a total volume of 1000 ?L. The mixture was vortex for 20 sec. For coupling, samples were incubated for 2 hours under rotation at room temperature. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The tubes were removed from the magnetic separator and the microspheres were resuspended in 1 mL of 1% (v/v) bovine serum albumin (BSA) in 1-fold PBS pH 7.4 (prepared by diluting a 10% BSA stock) by vortexing for approximately sec. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The tubes were removed from the magnetic separator and the microspheres were resuspended in 1 mL of 1% BSA in 1-fold PBS pH 7.4 by vortexing for approximately 20 sec. The tubes were placed into the magnetic separator and separation of the microspheres from the suspension occurred for 30-60 sec. Supernatant was removed without disrupting the microsphere pellet while the tubes were still positioned in the magnetic separator. The tubes were removed from the magnetic separator and the microspheres were resuspended in 2000 ?L of 1% BSA in 1-fold PBS pH 7.4 by vortexing for approximately 20 sec. The microspheres were kept in the 2.0 mL tubes. In order to count the microspheres recovered after the coupling reaction, the microsphere suspension was diluted 2-fold in 1% BSA in 1-fold PBS pH 7.4 (e.g. 15 ?L microsphere suspension diluted with 15 ?L of 1% BSA in 1-fold PBS pH 7.4). The number of microspheres recovered after the coupling reaction was determined using an automated cell counter (Countes II, Thermo Fisher Scientific, Cat. No. AMQAX1000) by correlating the determined dead cells concentration provided by the cell counter to the microspheres. The coupled microspheres were stored at 2-8? C. in the dark (blocking step) separately for each antigen. Previous to use, the coupled microspheres were allowed to pre-warm for at least 30 min at room temperature.

Optimization of Coupling Conditions

[0456] As the coupling efficiency, as well as the integrity of the antigen after the coupling procedure is dependent on the buffer used, different buffer conditions were evaluated for coupling of the VLPs and NS1 proteins to the microspheres. Coupling carried out as described above, except that the buffer was varied. Optimization of the coupling procedure is important in order to ensure that the three-dimensional structure of the antigen is not disturbed. The buffer conditions may vary dependent on the type of antigen used.

[0457] Different buffer conditions (50 mM MES at pH 5.00, 6.00, and 7.00) for coupling were examined and the signal to noise ratio was evaluated in a DENV-quadruplex complement-fixing assay set-up essentially as described under Example 2. Therefore, a negative control, i.e. a serum sample lacking DENV antibodies, and a control sample, i.e. a plasma sample derived from a human subject living in DENV endemic areas in Colombia with high amounts of anti-DENV complement fixing Abs were tested (FIG. 1A, exemplarily shown for DENV2 VLP). The assay shows, that a pH of 6.00 results in the highest fluorescence signal from the high control sample. In addition, also a pH of 7.00 shows a high signal to noise ratio. In contrast, a lower pH of 5.00 did not result in high signals indicating that the pH is not suitable for VLP coupling. For comparison, when coupling single proteins such as NS1, the optimum pH values seem to be different (FIG. 1B, exemplarily shown for DENV2 NS1). While for the VLP a coupling pH of 7.00 results in high fluorescence signals, for the NS1 protein the same pH does solely result in low fluorescence signals, indicating significant differences in coupling efficacies depending on the size, as well as three-dimensional structure of the antigen. Satisfyingly, no background signal was observed from the negative control sample neither for the VLP, nor for the NS1 protein independent of the buffer pH.

[0458] As described above, for routine coupling of DENV1-4 VLPs and DENV1-4 NS1 50 mM MES at pH 6.00 was applied. Of note, the optimum coupling pH for ZIKV VLP was 50 mM MES at pH 7.00 indicating that also virus-specific effects have an impact on the coupling efficiency.

Example 2: Evaluation of a DENV-Quadruplex Complement-Fixing Assay Set-Up

[0459] The DENV1-4 VLP-coupled microspheres of Example 1 were applied to develop a DENV-quadruplex complement-fixing assay set-up (FIG. 2) as described in the following.

[0460] For evaluation of the complement-fixing assay, a reference sample, as well as control samples were analyzed. The reference sample consists of pooled plasma samples from human subjects living in DENV endemic areas in Colombia with high levels of anti-DENV complement-fixing Abs (ABO Pharmaceuticals, Lot. VBU-01140-148). Control samples comprised plasma derived from human subjects living in DENV endemic areas in Colombia, including high, medium, and low amounts of anti-DENV complement fixing Abs (ABO Pharmaceuticals, Lot. No. PARS_82 (high control), Lot. No. PARS_96 (medium control), Lot. No. VBU-01140-189 (low control)). In addition, a negative control sample was included consisting of serum lacking any anti-DENV Abs (Bioreclamation, Lot.-No. BRH1140253). In general, the assay can be performed with either matrices i.e. serum or plasma samples. All samples were stored at ?80? C. prior to use. The samples were thawed and heat inactivated in a 56?1? C. water bath (Thermo Fisher, Isotemp 210, Cat.-No. 15-462-10Q) for 30?5 minutes prior to testing. Heat-inactivation is important to denature the temperature sensitive complement proteins within the sample and thereby to avoid assay interference.

[0461] In a next step, the DENV1-4 VLP-coupled microspheres of Example 1 were vortexed gently to break up clumping of the microspheres and ensure a uniform suspension. The microspheres were combined by dilution in assay buffer to result in a final concentration of 25 microspheres/?L for each DENV serotype and vortexed gently. Assay buffer consisted of phosphate buffered saline (PBS) with 1% (v/v) bovine serum albumin (BSA), diluted from a 10% stock (Fisher Scientific, Cat-No. 37525) and was stored at 2-8? C. for up to one month. The assay buffer was allowed to pre-warm for at least 30 minutes at room temperature prior to dilution of the microspheres. 50 ?L of the microsphere suspension containing all four DENV serotype antigens were pipetted per well into a 96-well polystyrene microplate (solid black flat bottom plate, in the following referred to as assay plate; Corning, Cat.-No. 3915) resulting in 1250 microspheres per DENV-serotype per well. The plate was sealed with a foil plate seal (ThermoFisher, Cat.-No. AB0558) and stored at room temperature until the samples were diluted.

[0462] Heat-inactivated samples were serially diluted (8 dilutions final) using assay buffer pre-warmed to room temperature at least 30 minutes before testing. 50 ?L per sample dilution were transferred into the assay plate per well to the microspheres in duplicates. Sample and microsphere suspension were mixed thoroughly by pipetting up and down 3-5 times. Next, the plate was sealed with a foil plate seal and incubated for 60?5 min at room temperature on a plate shaker (Heidolph, Titramax 1000, Cat.-No. 544-12200-00) at 600 rpm.

[0463] Afterwards, the plate was washed with wash buffer (PBS with 0.05% (v/v) Tween-20) using the Luminex Flat 96 Mag setting on a plate washer (BioTek ELx405, Cat.-No. 7100745S). After decanting of residual wash buffer, 50 ?L/well of purified human C1q at a concentration of 4.0 ?g/mL in assay buffer were added. Human C1q (Quidel, Cat.-No. A400, Lot.-No. 142550) is purified (>95%) from plasma, reconstituted with 40% (v/v) glycerol in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at 1 mg/mL and stored at ?80? C. prior to use. After C1q addition, the plate was sealed with a foil plate seal and incubated for 30?5 min at room temperature on a plate shaker at 600 rpm. By using purified C1q the exact amount of C1q applied in the assay can be controlled. In contrast, by using complement-component human serum the C1q concentration in the serum could vary between different lots of the serum. In addition, also the binding of C1q could be interfered by other serum components. Thus, the use of purified C1q improves reproducibility of the assay.

[0464] After incubation with C1q, the plate was washed with wash buffer as described above. After decanting of residual wash buffer, a pre-reporter Ab, i.e. sheep IgG anti-human C1q was added 50 ?L/well at a 6.4 ?g/mL dilution in assay buffer. Polyclonal purified sheep IgG anti-human C1q Ab (Bio-Rad, Cat.-No. AHP033, Lot.-No. 148553) recognizes human C1q. The Ab is solubilized in glycine buffer saline from manufacturer to 5.1 mg/mL and stored at 4? C. prior to use. After Ab addition, the plate was sealed with a foil plate seal and incubated for 30?5 min at room temperature on a plate shaker at 600 rpm.

[0465] After incubation with anti-C1q Ab, the plate was washed with wash buffer as described above. After decanting of residual wash buffer, a reporter Ab, i.e. PE-conjugated donkey IgG anti-sheep IgG was added 50 ?L/well at a 10 ?g/mL dilution in assay buffer. R-Phycoerythrin F(ab).sub.2 fragment donkey anti-sheep IgG Ab (Jackson ImmunoResearch, Cat.-No. 713-116-147) is sold freeze-dried and was reconstituted with 1 mL deionized water according to the manufacturer to result in a 500 ?g/mL solution. After Ab addition, the plate was sealed with a foil plate seal and incubated for 30?5 min at room temperature on a plate shaker at 600 rpm.

[0466] Finally, the plate was washed with wash buffer as described above. After decanting of residual wash buffer, 100 ?L/well of assay buffer were added to the plate. The plate was covered with foil plate seal and shaken at 600 rpm for 5 min in order to resuspend the microspheres prior to read. At this point, the plate may also be stored at 2-8? C. overnight for analysis on the following day. If refrigerated overnight, the plate was shaken at room temperature for at least 30 min at 600 rpm.

[0467] The plate was analyzed in a MAGPIX Luminex plate reader with the xPONENT 4.2 software (Luminex Corp., Cat.-No. MAGPIX-XPONENT). The Luminex acquisition mode was set to 50 ?L sample volume and 50 microspheres of each DENV serotype per well. The number of microspheres determined by the acquisition mode specifies that the Luminex reader needs to acquire at least 50 microspheres of each DENV serotype per well in order to determine the representative (statistically) mean signal of all microspheres of each DENV serotype per well.

[0468] For data analysis, each DENV serotype antigen is independently evaluated resulting in four standalone immunoassays. Data were analyzed and plotted using GraphPad Prism 8 version 8.1.0 (GraphPad Software, Inc). Mean Fluorescent Intensity (MFI; MFI values are the raw data reported by the MAGPIX? reader) values were plotted in dependence of log.sub.10-transformed serum dilutions (e.g. 5-fold dilution resulting in log.sub.10(5)=0.7). Sigmoidal fitting was performed according to a dose-response curve (Sigmoidal, 4PL, X=Log(concentration)). The equation used for the non-linear regression was log(agonist) vs. responseVariable slope. The MFI signal threshold of the reference sample equivalent to the EC25 concentration was calculated by subtracting the estimated bottom from top signals and multiplying the result by 0.25 (signal equivalent to 25% of effective concentration). The MFI signal threshold was then interpolated on the 4PL curves of both the reference sample and the control samples (as well as corresponding test samples) to determine the sample dilution equivalent to the EC25 signal of the reference (interpolated dilution). The interpolated dilutions are then divided by the interpolated dilution calculated in the reference to result in relative dilutions. Further, relative dilutions were multiplied by a constant (EC50 of the reference against each DENV serotype) for the corresponding DENV serotype (i.e. 468 for DENV-1 VLP, 345 for DENV-2 VLP, 369 for DENV-3 VLP and 257 for DENV-4 VLP) to result in the complement-fixing antibody titers in the samples in EU/mL. The EU/mL concentration is a relative arbitrary concentration based on the levels of complement-fixing antibodies found in the reference sample.

Signals Resulting from Different DENV VLPs

[0469] MFI values of the different sample dilutions and corresponding dose-response curve fits of reference and control samples are presented for all four DENV VLPs in FIGS. 3 and 4. Satisfyingly, for all four DENV VLPs MFI signals increased for all the samples except for the negative control with decreasing dilution factor. In addition, MFI signals increased from low to high titer control samples. Moreover, negative control serum did result in MFI signals close to zero independent of the dilution.

Limit of Detection (LOD)

[0470] The LOD was determined by serial dilution of the reference sample in assay buffer and determination of the lowest complement-fixing antibody concentration for which the relative error (% RE=(obtained titer?expected titer)*100/obtained titer) is above 25% in one independent run using duplicates per dilution (Table 1). In summary, the LOD ranged from 0.46 EU/mL for DENV1 VLP to 0.72 EU/mL for DENV3 VLP.

TABLE-US-00001 TABLE 1 Complement-Fixing Antibody Titers in EU/mL dependent on the sample dilution and DENV VLP. Presented are the expected titers, as well as the obtained titers in both replicates. The relative error was calculated (% RE). The LOD was determined as the lowest Ab concentration for which the relative error is above 25% (highlighted values). DENV1 VLP DENV2 VLP (Complement-Fixing Abs in EU/mL) (Complement-Fixing Abs in EU/mL) Sample Obtained Obtained Obtained Obtained Dilution Expected Repl. 1 Repl. 2 % RE Expected Repl. 1 Repl. 2 % RE Not dil. 468.10 345.043 1:2 234.05 275.61 343.52 32% 172.52 180.98 223.70 17% 1:4 117.02 113.77 127.49 3% 86.26 82.14 104.77 8% 1:8 58.51 74.96 60.98 16% 43.13 41.52 43.14 ?2% 1:16 29.26 29.27 26.60 ?5% 21.57 20.53 17.86 ?11% 1:32 14.63 15.92 16.57 11% 10.78 9.70 11.09 ?4% 1:64 7.31 7.65 7.05 0% 5.39 5.62 5.92 7% 1:128 3.66 4.06 3.06 ?3% 2.70 2.90 2.15 ?6% 1:256 1.83 1.72 1.50 ?12% 1.35 1.52 1.27 4% 1:512 0.91 1.23 0.88 15% 0.67 0 0.94 ?31% 1:1024 0.46 0.66 44% 0.34 0 ?100% 1:2048 0.228 0 0.365 ?20% 0.168 0 ?100% 1:4096 0.114 0 ?100% 0.084 0 ?100% DENV3 VLP DENV4 VLP (Complement-Fixing Abs in EU/mL) (Complement-Fixing Abs in EU/mL) Sample Obtained Obtained Obtained Obtained Dilution Expected Repl. 1 Repl. 2 % RE Expected Repl. 1 Repl. 2 % RE Not dil. 368.64 257.19 1:2 184.32 194.14 227.57 14% 128.59 128.64 142.64 5% 1:4 92.16 91.76 103.76 6% 64.30 63.02 61.29 ?3% 1:8 46.08 46.82 45.83 1% 32.15 29.65 30.91 ?6% 1:16 23.04 22.20 21.00 ?6% 16.07 15.05 13.35 ?12% 1:32 11.52 10.86 11.94 ?1% 8.04 7.37 7.80 ?6% 1:64 5.76 5.09 5.31 ?10% 4.02 4.03 4.03 0% 1:128 2.88 2.92 2.47 ?6% 2.01 1.94 1.66 ?10% 1:256 1.44 1.33 1.31 ?8% 1.00 1.08 0.98 2% 1:512 0.72 0 0.76 ?47% 0.50 0 0.62 ?38% 1:1024 0.36 0 ?100% 0.25 0 ?100% 1:2048 0.18 0 ?100% 0.125 0 ?100% 1:4096 0.09 0 ?100% 0.063 0 ?100%

Lower Limit of Quantification (LLOQ)

[0471] The LLOQ was determined by serial dilution of the reference sample in human IgG-depleted serum and determination of the lowest complement-fixing antibody concentration in which the relative error (% RE) is above 25% in five independent runs (Tables 2-5). The LLOQ for all DENV VLPs was determined to be 3.00 EU/mL.

TABLE-US-00002 TABLE 2 Expected and obtained complement-fixing Ab titers (EU/mL) for DENV1 VLP dependent on the sample dilution. Presented are the expected titers, as well as the obtained titers in the five independent runs. The average obtained titer of the five independent runs was calculated, as well as the standard deviation (SD), the percent coefficient of variation (% CV), and the relative error (% RE). As the signal threshold was not reached, titers in rows highlighted in grey were calculated based on extrapolation of the titration curves and, therefore, were considered not detectable. Expected Obtained Titer (EU/mL) Sample Titer Run Run Run Run Run % % Dilution (EU/mL) #1 #2 #3 #4 #5 Average SD RE CV Not dil. 468.10 1:2 234.05 257.16 229.55 184.00 187.70 214.78 214.64 30.39 ?8% 14% 1:4 117.02 126.08 121.01 112.95 94.45 94.72 109.84 14.70 ?6% 13% 1:8 58.51 64.37 59.34 50.52 49.12 50.84 54.84 6.68 ?6% 12% 1:16 29.26 28.31 28.39 23.02 25.29 26.03 26.21 2.25 ?10% 9% 1:32 14.63 12.95 14.87 12.77 12.46 12.68 13.15 0.98 ?10% 7% 1:64 7.31 7.63 6.95 8.66 5.99 7.78 7.40 1.00 1% 13% 1:128 3.66 3.69 3.36 4.02 2.90 3.68 3.53 0.43 ?3% 12% 1:256 1.83 1.69 1.68 1.79 1.09 1.61 1.57 0.28 ?14% 18% 1:512 0.91 0.87 0.87 NC ?5% NC 1:1024 0.46 NC NC NC NC NC = not calculated.

TABLE-US-00003 TABLE 3 Expected and obtained complement-fixing Ab titers (EU/mL) for DENV2 VLP dependent on the sample dilution. Presented are the expected titers, as well as the obtained titers in the five independent runs. The average obtained titer of the five independent runs was calculated, as well as the standard deviation (SD), the percent coefficient of variation (% CV), and the relative error (% RE). As the signal threshold was not reached, titers in rows highlighted in grey were calculated based on extrapolation of the titration curves and, therefore, were considered not detectable. Expected Obtained Titer (EU/mL) Sample Titer Run Run Run Run Run % % Dilution (EU/mL) #1 #2 #3 #4 #5 Average SD RE CV Not dil. 345.043 1:2 172.52 216.39 177.48 133.24 140.42 161.12 165.73 33.25 ?4% 20% 1:4 86.26 99.61 82.69 81.97 67.33 64.81 79.28 14.00 ?8% 18% 1:8 43.13 49.65 39.87 35.38 34.54 34.70 38.83 6.43 ?10% 17% 1:16 21.57 22.04 19.70 17.02 18.20 19.40 19.27 1.88 ?11% 10% 1:32 10.78 9.22 10.95 9.82 9.23 9.64 9.77 0.71 ?9% 7% 1:64 5.39 5.35 5.21 6.48 4.62 6.53 5.64 0.84 5% 15% 1:128 2.70 2.78 2.55 2.97 2.22 3.13 2.73 0.36 1% 13% 1:256 1.35 1.54 1.33 1.52 0.76 1.57 1.34 0.34 0% 25% 1:512 0.67 0.87 0.51 0.95 0.77 0.23 15% 30% 1:1024 0.34 NC NC NC NC NC = not calculated.

TABLE-US-00004 TABLE 4 Expected and obtained complement-fixing Ab titers (EU/mL) for DENV3 VLP dependent on the sample dilution. Presented are the expected titers, as well as the obtained titers in the five independent runs. The average obtained titer of the five independent runs was calculated, as well as the standard deviation (SD), the percent coefficient of variation (% CV), and the relative error (% RE). As the signal threshold was not reached, titers in rows highlighted in grey were calculated based on extrapolation of the titration curves and, therefore, were considered not detectable. Expected Obtained Titer (EU/mL) Sample Titer Run Run Run Run Run % % Dilution (EU/mL) #1 #2 #3 #4 #5 Average SD RE CV Not dil. 368.64 1:2 184.32 211.80 177.43 157.61 168.53 174.03 177.88 20.40 ?4% 11% 1:4 92.16 104.33 94.66 93.56 80.86 76.36 89.96 11.28 ?2% 13% 1:8 46.08 53.64 45.65 40.78 41.95 41.84 44.77 5.29 ?3% 12% 1:16 23.04 23.68 22.19 20.25 22.14 20.53 21.76 1.40 ?6% 6% 1:32 11.52 10.75 11.90 10.94 10.86 10.33 10.96 0.58 ?5% 5% 1:64 5.76 6.05 5.41 6.30 4.70 5.96 5.68 0.64 ?1% 11% 1:128 2.88 3.03 2.64 2.92 2.30 2.90 2.76 0.29 ?4% 11% 1:256 1.44 1.21 1.12 1.20 0.63 1.02 1.04 0.24 ?28% 23% 1:512 0.72 0.49 0.49 NC ?31% NC 1:1024 0.36 NC NC NC NC NC = not calculated.

TABLE-US-00005 TABLE 5 Expected and obtained complement-fixing Ab titers (EU/mL) for DENV4 VLP dependent on the sample dilution. Presented are the expected titers, as well as the obtained titers in the five independent runs. The average obtained titer of the five independent runs was calculated, as well as the standard deviation (SD), the percent coefficient of variation (% CV), and the relative error (% RE). As the signal threshold was not reached, titers in rows highlighted in grey were calculated based on extrapolation of the titration curves and, therefore, were considered not detectable. Expected Obtained Titer (EU/mL) Sample Titer Run Run Run Run Run % % Dilution (EU/mL) #1 #2 #3 #4 #5 Average SD RE CV Not dil. 257.19 1:2 128.19 151.94 122.52 101.84 108.87 121.68 121.37 19.19 ?6% 16% 1:4 64.30 72.98 63.56 62.17 52.93 52.54 60.84 8.49 ?5% 14% 1:8 32.15 37.13 30.94 26.45 27.23 27.58 29.86 4.41 ?7% 15% 1:16 16.07 16.07 14.53 13.25 13.92 14.38 14.43 10.4 ?10% 7% 1:32 8.04 7.04 8.04 7.25 6.90 7.12 7.27 0.45 ?9% 6% 1:64 4.02 4.14 3.82 4.56 3.38 4.42 4.06 0.48 1% 12% 1:128 2.01 2.14 1.86 2.15 1.68 2.17 2.00 0.22 0% 11% 1:256 1.00 1.01 0.89 0.93 0.56 0.85 0.85 0.17 ?16% 20% 1:512 0.50 0.53 0.43 0.39 0.45 0.08 ?11% 17% 1:1024 0.25 NC NC NC NC NC = not calculated.

Assay Linearity

[0472] Assay linearity was evaluated by plotting the median value of the obtained concentration of the five independent runs performed for determination of the LLOQ for each reference sample dilution and DENV VLP against the expected concentration (FIGS. 5 and 6). Linear regression of the obtained and expected concentrations was performed and the slope together with the 95% confidence interval (CI) was calculated. The obtained and expected concentrations correlated well and in a linear manner with slopes close to one. The 95% Confidence Intervals (CI) were 0.8663 to 0.9731 for DENV1 VLP (slope of 0.9197), 0.8798 to 1.034 for DENV2 VLP (slope of 0.9571), 0.9193 to 1.013 for DENV3 VLP (slope of 0.9662), and 0.8856 to 1.006 for DENV4 VLP (slope of 0.9459).

Assay Precision

[0473] To evaluate the assay precision high, medium and low control samples were tested five times per run. In total, two different operators performed each two runs. Complete sigmoidal curves are exemplarily shown for one run and the DENV2 VLP antigen (FIGS. 7 and 8). Satisfyingly, fluorescence signals for the five replicates were highly similar independent of the signal intensity and sample type. In general, complement-fixing Ab titers were highly similar independent of the run, the control sample, the DENV VLP, and the operator (FIGS. 9 and 10).

[0474] For determination of intra-assay precision, the percent coefficient of variation (% CV=standard deviation/mean*100) of the complement-fixing Ab concentration was calculated for each sample within each run. For determination of inter-assay precision, the percent coefficient of variation (% CV) of the complement-fixing Ab concentration was calculated in between the runs.

[0475] Intra-assay precision was consistently below 20% for all control samples and DENV VLPs (FIGS. 11 and 12). Inter-assay precision was below 20% for all control samples and DENV VLPs, except for the DENV1 VLP signals from the low control sample, which was solely slightly above 20% (FIG. 13).

[0476] In conclusion, the DENV-quadruplex complement-fixing assay was successfully set-up and validated.

Example 3: Comparison of the DENV-Quadruplex Assay Set-Up with the Singleplex Assay Set-Up

[0477] In order to further evaluate the performance of the developed DENV-quadruplex complement-fixing assay set-up of Example 2, the quadruplex assay was compared to the corresponding singleplex complement-fixing assay set-ups solely comprising one DENV serotype VLP per assay. The singleplex assay was performed mutatis mutandis as described for the quadruplex assay under Example 2. The final microsphere concentration in the assay was the same for each serotype independent of singleplex or multiplex set-up. Complement fixing antibody titers were determined in the high, medium, and low titer control samples described in Example 2. In addition, a medium-high titer control sample (ABO Pharmaceuticals, Lot No. PARS_73) was included (Table 6).

TABLE-US-00006 TABLE 6 Complement-fixing antibody titers (EU/mL) detected in the high (H), medium-high (MH), medium (M), and low (L) titer control samples using the quadruplex (QP) and the singleplex (SP) assay set-up for all DENV VLPs. The titers were compared using percent coefficient of variation (% CV). Complement-Fixing Antibody Titers (EU/mL) DENV1 VLP DENV2 VLP DENV3 VLP DENV4 VLP Sample QP SP % CV QP SP % CV QP SP % CV QP SP % CV H 248 298 13% 191 207 6% 214 178 13% 154 160 3% MH 144 157 6% 82 88 5% 81 101 16% 60 61 1% M 27 29 4% 16 20 16% 19 18 6% 17 19 8% L 5 7 24% 4 6 34% 4 4 1% 3 4 15%

[0478] In summary, the titers were consistent and showed good correlation, further validating the performance of the quadruplex complement-fixing assay.

Example 4: Detection and Quantification of DENV-Reactive Complement-Fixing Abs in Human Serum Samples Vaccinated with a Tetravalent DENV Vaccine

[0479] Virus-reactive complement-fixing Abs can be produced upon natural virus infection or vaccination. Complement fixation improves antigen-specific B cell clone selection and affinity maturation by sequestration (via complement receptors) of antigens in germinal centers of secondary lymphoid organs and thus contributes to antibody production against the viral pathogen.

[0480] As the determination of complement-fixing Abs is an important tool to characterize the quality of the immune response after infection or vaccination, the quadruplex assay set-up developed under Example 2 was further applied to analyze human serum test samples for the presence and amount of DENV1-4-reactive complement-fixing Abs in the following. The samples were derived from subjects vaccinated with a tetravalent DENV vaccine candidate, comprising live-attenuated viruses of all four serotypes (TAK-003 vaccine from Takeda Vaccines, Inc.). The components of the tetravalent DENV vaccine are based on DENV1 strain 16007 Thailand (NCBI No. AAF59976.1), DENV2 strain 16681 Thailand (Uniprot No. P29990.1), DENV3 strain 16562 Philippines (Uniprot No. A0A173DS74), and DENV4 strain 1036 Indonesia (Uniprot No. A0A1Z1XCD1). Of note, the DENV2 strain used for tetravalent DENV vaccine production was the same as the one used for VLP production (cf. Example 1). The other strains used for tetravalent DENV vaccine production show high homology to the E protein sequences of the strains used for VLP preparation (97%, 97%, and 99% sequence identity of corresponding E protein sequences of DENV1, DENV3, and DENV4 strains).

[0481] Per analysis run, the reference sample as well as negative, high, medium, and low control samples of Example 2 were included. In addition, five human serum test samples were analyzed (samples 1 to 5). The assay and data analysis was carried out as described under Example 2, including heat inactivation of all samples. For data analysis, the human serum test samples were analyzed as described for the control samples.

[0482] FIG. 14 shows the MFI values of the different sample dilutions of reference and human serum test samples exemplarily for DENY-1 VLP-coupled microspheres. Data analysis revealed final sample titers of 468.0 EU/mL for the reference sample, 214.8 EU/mL for sample 1, 94.7 EU/mL for sample 2, 50.8 EU/mL for sample 3, 26.0 EU/mL for sample 4, and 12.7 EU/mL for sample 5 of DENY-1-reactive complement-fixing Abs (cf. Table 7).

TABLE-US-00007 TABLE 7 Data analysis and evaluation of DENV-1-reactive complement-fixing Ab titers. The MFI signal threshold from the reference sample was calculated by subtracting the estimated bottom (MFI = 1,039) from top signals (MFI = 31,950) and multiplying the result by 0.25 (signal equivalent to 25% of effective concentration), resulting in a threshold of 7,727.75. Signal at Signal at Complement- maximum minimum Interpolated Relative Fixing Abs EC.sub.50 dilution dilution Sample dilution dilution [EU/mL] value R.sup.2 [MFI] [MFI] Reference 1,037.38 1.000 468.00 520.10 0.9953 1,366.50 31,529.50 Sample 1 476.10 0.459 214.78 256.90 0.9959 4,957.00 31,187.00 Sample 2 209.95 0.202 94.72 109.20 0.9909 2,179.00 31,638.00 Sample 3 112.70 0.109 50.84 51.27 0.9882 1,501.75 31,651.50 Sample 4 57.69 0.056 26.03 30.08 0.9993 626.50 31,138.50 Sample 5 28.11 0.027 12.68 16.18 0.9990 440.75 27,663.50

[0483] In conclusion, the DENV-quadruplex complement-fixing assay set-up was well suitable for evaluation of samples from subjects vaccinated with the tetravalent DENV vaccine and resulted in a robust determination of complement fixing Abs.

Example 5: Comparison Between Seropositive and Seronegative Subjects

[0484] With a validated DENV-quadruplex complement-fixing assay set-up in hand, serum samples from both, seronegative and seropositive subjects from two different studies before and after vaccination with the tetravalent DENV vaccine from Example 4 were evaluated (FIGS. 15-18). 28 seronegative and 11 seropositive subjects were tested in study #1 (FIGS. 15 and 16) and 11 seronegative and 10 seropositive subjects were tested in study #2 (FIGS. 17 and 18). The titers of complement-fixing Abs against all DENV serotypes increased upon vaccination, indicating that the vaccine induced the production of Abs directed against all serotypes that can fix complement system.

[0485] Complement-fixing Ab titers prior to vaccination (samples at day 0 in FIGS. 15 and 16 and samples at day 1 in FIGS. 17 and 18, respectively) are in line with the serostatus of the subjects, i.e. close to zero for the seronegative cohort and around 10 for the seropositive cohort indicating that the assay can be applied to distinguish whether a subject has been infected with DENV or not.

[0486] Altogether, this data further validate a reliable assay performance and suitability of the assay for analysis of samples derived from subjects prior and post vaccination, independent of the serostatus of the subjects prior to vaccination.

Example 6: Comparison of Microneutralization Test (MNT) and Complement-Fixing Assay

[0487] The DENV-quadruplex complement-fixing antibody assay of Example 2 was compared to the microneutralization test (MNT), which is a gold standard technique. Although the MNT is widely applied, the assay is time-consuming as for instance one step requires infection of and incubation with cells in culture.

[0488] To compare both assays, 53 samples derived from DENV seropositive and seronegative non-vaccinated subjects (aged 1.5 to 45 years living in dengue endemic areas) were evaluated. Out of the 53 samples, 14 samples were derived from subjects aged 1-5 years, 20 samples were derived from subjects aged 6-11 years, 12 samples were derived from subjects aged 12-20 years, and 7 samples were derived from subjects older than 20 years. The DENV-quadruplex complement-fixing antibody assay was carried out as described under Example 2, the MNT was carried out by heat-inactivating control and testing samples at 56? C. for 30 minutes. For each of the four serotype-specific MNT assays, a 30 ?L heat-inactivated sample was serially diluted two-fold from 1:5 to 1:10240 and mixed with equal amounts of diluted dengue virus control for each serotype to give final dilution ratios of 1:10 to 1:20480. The virus control diluted in an equal amount of 10% (v/v) Dulbecco's Modified Eagle Medium (DMEM) provided the baseline foci count used to measure 50% neutralization. Neutralization occurred overnight at 2-8? C. This serum/virus mixture was then inoculated in triplicate on Vero cells that had been seeded onto 96-well plates at 2.5?10.sup.5 cells/mL approximately 24 hours previously. Absorbance was allowed to proceed for 90 minutes at 37? C. and ended with the addition of an overlay of 1% (w/v) methyl cellulose in DMEM. The infected cells were then incubated at 34? C.?2? C., 5?3% CO.sub.2 for different time periods according to dengue serotype (DENV1 and DENV3: 66?2 hours; DENV2: 70?2 hours; DENV4: 46?2 hours). Thereafter, the cells were fixed with methanol for 60 minutes at ??20? C. or acetone for 20 minutes at room temperature. Infectious centers (immunofoci) were developed using a primary anti-dengue antibody (incubated at 2-8? C. for 18?4 hours), peroxidase-conjugated secondary antibody (incubated at 37? C.?2? C. for 90-120 minutes), and a precipitant substrate (2-amino-9-ethyl carbazole, Sigma). The peroxidase substrate formed a colored precipitate upon oxidation, allowing the immunofoci to be visualized. The antibody titer of each sample was determined by photographing the plates and counting the immunofoci in individual wells using a ViruSpot Plaque Counter (Autoimmun Diagnostika GmbH). Short viral amplification periods and microscopic visualization allowed for robust counting up to 136 foci. The percentage neutralization reduction was determined by comparing the number of immunofoci in averaged virus control-containing wells to the immunofoci number in serum sample wells. The MNT50 was calculated through linear interpolation using the equation MNT50=10{circumflex over ()}[(50?c)/m], where c is the slope and m is the intercept. The linear interpolation utilized foci counts for the wells above and below the 50% virus control foci average. As described under Example 2, the LLOQ for the complement-fixing assay was 3.00 EU/mL, which was applied as a cut-off value for seropositivity. For the MNT, an MNT.sub.50 titer of equal to or greater than 10 was applied as a cut-off for seropositivity.

[0489] In general, a good concordance in serostatus classification between the results from the MNT and the complement-fixing assay was observed (Table 8).

TABLE-US-00008 TABLE 8 Evaluation of samples from subjects that were seropositive or seronegative for a specific DENV serotype by the MNT and complement-fixing assay set-up. The cut-off titer for the MNT was determined as a MNT.sub.50 of equal to or greater than 10, the cut-off titer for the complement-fixing assay as a titer of equal to or greater than 3.00 EU/mL. In summary, samples of 53 subjects were analyzed. The numbers show the percentage of seropositive and seronegative subjects as predicted by either the MNT or the complement-fixing assay. Complement-fixing MNT antibodies Titers ?10 Titers <10 Titers ?3.00 Titers <3.00 Viruses Total (%) (%) EU/mL (%) EU/mL (%) DENV1 53 17 (32) 36 (68) 15 (28) 38 (72) DENV2 14 (26) 39 (74) 14 (26) 39 (74) DENV3 16 (30) 37 (70) 13 (25) 40 (75) DENV4 12 (23) 41 (77) 13 (25) 40 (75)

[0490] FIG. 26 and Table 9 depict the overall distribution of antibody titers in the samples, as well as geometric mean antibody titers, respectively, against all DENV serotypes by both assays. Geometric mean MNT50 titers ranged from 12 (DENV4) to 20 (DENV2) and the complement-fixing antibody geometric mean titers ranged from 4 EU/mL (DENV2-4) to 5 EU/mL (DENV1, Table 9).

TABLE-US-00009 TABLE 9 Neutralizing (MNT50) and complement-fixing antibody titers against all four dengue virus serotypes from the 53 samples analyzed. Minimum, maximum, as well as geometric mean (GeoMean) titers, together with 95% confidence interval (CI) were determined for both, the neutralizing and complement-fixing antibodies. Complement-fixing antibody MNT50 Titers titers (EU/mL) Min- Max- GeoMean Min- Max- GeoMean Virus imum imum (95% CI) imum imum (95% CI) DENV1 5 20480 19 (10, 35) 2 1915 5 (3, 7) DENV2 5 10919 20 (10, 40) 2 1565 4 (3, 7) DENV3 5 5120 15 (9, 26) 2 1564 4 (3, 7) DENV4 5 1442 12 (7, 20) 2 730 4 (3, 6) MNTdengue microneutralization assay; MNT50 LLOQ is 10; GeoMeangeometric mean; CIconfidence interval

[0491] Correlation analysis between complement-fixing antibody titers and microneutralization titers was carried out using Log.sub.10-transformed values using JMP version 13.1.0 software (SAS Institute, Inc.). When the relationship between the MNT50 and the complement-fixing antibodies was investigated, moderate (R.sup.2=0.675 for DENV1) to high (R.sup.2=0.902 for DENV3) correlations were observed (FIG. 27).

[0492] When compared to the MNT, specificity and sensitivity of the complement-fixing assay was above 80% for each serotype (Table 10), indicating that the performance of the complement-fixing assay is similar to the gold standard dengue MNT.

TABLE-US-00010 TABLE 10 Sensitivity and specificity of the complement-fixing assay relative to the DENV MNT. The numbers indicate the true positives (samples with MNT50 ?10) and true negatives (samples with MNT50 <10) of the complement-fixing assay. Sensitivity of the complement-fixing assay was calculated based on the percentage of samples with an MNT50 ?10 (seropositive) that were equal to or above the threshold titer for positivity of 3.00 EU/mL. Specificity of the complement-fixing assay was calculated based on the percentage of samples with an MNT50 <10 (seronegative) that were below the threshold titer for positivity of 3.00 EU/mL. For instance, all 39 DENV2-positive samples according to the MNT were also predicted to be positive by the complement-fixing assay resulting in 100% sensitivity and specificity. Number of Samples Comple- Comple- ment >3.00 Sensitivity ment <3.00 Specificity Viruses MNT50 ?10 EU/mL (%) MNT50 <10 EU/mL (%) DENV1 17 14 82% 36 35 97% DENV2 14 14 100% 39 39 100% DENV3 16 13 81% 37 37 100% DENV4 12 11 92% 41 39 95%

[0493] To illustrate the accuracy of the DENV-quadruplex complement-fixing assay set-up for diagnosis of a DENV infection, we evaluated the data in terms of a Receiver operating characteristic (ROC) curve. Therefore, the sensitivity, meaning the percentage of true positive samples i.e. samples from seropositive subjects with an MNT50 titer of ?10 being also classified as positive in the complement assay is plotted on the y-axis and 1-specificity, meaning the percentage of true negative samples i.e. samples from seronegative subjects with an MNT50 titer of <10 being also classified as negative in the complement assay is plotted on the x-axis for several complement-fixing antibody titer thresholds (from <3 EU/mL to approximately 1000 EU/mL; FIG. 19). Then, the area under the curve (AUC) is calculated, which represents the accuracy of a diagnostic candidate test relative to the gold standard, i.e. the MNT. The AUC (95% confidence interval) was 0.9346 (0.8440 to 1.000) for DENV1, 1.000 (1.000 to 1.000) for DENV2, 0.9688 (0.8990 to 1.000) for DENV3, and 0.9167 (0.7603 to 1.000) for DENV4. An AUC over 0.9 represents an excellent diagnostic test, indicating that the DENV-quadruplex complement-fixing assay set-up is well suitable for diagnosing DENV infection.

Example 7: Complement-Fixing Antibodies Evaluated in a 10-Plex Assay Set-Up

[0494] In a next step a 10-plex assay set-up including DENV1-4 VLPs, DENV1-4 NS1 proteins, as well as Zika virus (ZIKV) VLP, and ZIKV NS1 protein was developed. The goal was to determine complement-fixing Abs directed against structural and non-structural proteins of DENV and ZIKV in one single experiment. Antigens were coupled to the microspheres as described under Example 1. The assay was carried out mutatis mutandis as described under Example 2, with the exception that no titer in EU/mL was calculated (the samples were evaluated looking at MFI signal only).

[0495] Complement-fixing Abs were determined in samples collected in the Dominican Republic in 2016 to 2017 from subjects suffering from febrile illness consistent with DENV and ZIKV infections (panel #1), as well as in samples collected in Colombia either before or after 2016 from subjects 8-40 days post febrile illness consistent with DENV and ZIKV infection (panel #2 and #3). In addition, samples, which did not contain flavivirus Abs were evaluated as negative controls (FIGS. 20-21).

[0496] Negative controls resulted in fluorescence values similar to the blank, validating the overall 10-plex assay set-up. High signals were observed for samples #1 to 3 for DENV1-4 VLPs and DENV1-4 NS1 proteins. Lower signals were detected for the ZIKV VLP and NS1 protein. In general, the complement-fixing assay was successfully transferred to a set-up comprising 10 different antigens.

[0497] Moreover, the DENV1-4 VLP signals from the 10-plex assay were similar to the signals recorded from the quadruplex assay set-up using DENV1-4 VLPs. Consequently, the number of antigens in the complement-fixing assay set-up of the present invention can be increased to any desired number as the results of the assay for specific antigens are not altered dependent on the number of antigens included.

Example 8: Application of the DENV-Quadruplex Complement-Fixing Assay Set-Up to Distinguish Between DENV Serotype Infections

[0498] In a next step, it was evaluated if the DENV-quadruplex complement-fixing assay set-up developed under Example 2 is suitable for distinguishing between infections with different DENV serotypes. Therefore, 16 non-human primates (adult male cynomolgus macaques 5-7 years, weighing 6-9 kg of Mauritius origin and na?ve to DENV) were infected subcutaneously with each one of the four DENV serotypes. For infection, 2.47e7 PFU/mL DENV1 strain Western Pacific, 3.11e6 PFU/mL DENV2 strain New Guinea C, 2.94e6 PFU/mL DENV3 strain Sleman/78, and 5.30e6 PFU/mL DENV4 strain 1228 were used per animal. All monkeys were negative for Hepatitis B virus, Simian Retrovirus, Simian Immunodeficiency virus, Simian T Lymphotropic virus, Mycobacterium Tuberculosis, Simian Varicella virus, Malaria, Salmonella, Shigella, Yersinia and internal parasites. Of note, human C1q cross-reacts with monkey complement-fixing Abs and can therefore be used for evaluation of monkey samples.

[0499] Complement-fixing Ab titers for each animal and DENV serotype were determined (FIG. 22B) and averaged for animals infected with the same serotype (FIG. 23). The titers of complement-fixing Abs for the infecting serotype were higher for each animal. Complement-fixing Abs were also cross-reactive amongst the different serotypes. The amount of cross-reactive complement-fixing Abs observed was consistent with the inter-serotype homology of the envelope protein (FIG. 22A).

[0500] Both serotype-specific and cross-reactive Abs are involved in complement fixation. The less similar the envelope amino acid sequence among the DENV serotypes, the less cross-reactive Abs are produced (e.g. the amounts of DENV4 cross-reactive Abs produced in DENV1-3 infected animals were the lowest). However, the data show that the DENV quadruplex complement-fixing assay is able to distinguish between infections caused by different DENV serotypes.

Example 9: Application of the 10-Plex Assay Set-Up to Distinguish Between Flavivirus Infections

[0501] As Abs are often cross-reactive amongst different flaviviruses, the capacity of the complement-fixing assay of the present invention to distinguish between infections by different flaviviruses was evaluated. Therefore, the 10-plex assay set-up developed under Example 7 was applied to detect complement-fixing Abs directed against ZIKV NS1.

[0502] Non-human primates were infected with each one DENV serotype as described under Example 8 and complement fixing Abs directed against ZIKV NS1 were monitored for one year after infection. Then, non-human primates were infected with ZIKV and complement-fixing Abs directed against NS1 were further evaluated (FIG. 24A). Interestingly, none of the DENV infections, independent of the serotype, resulted in increasing ZIKV NS1-reactive complement-fixing Abs. However, after ZIKV infection, signal intensity significantly raised for all animals, indicating that the complement fixing assay is very specific and can also be used to reliably distinguish between ZIKV and DENV infection.

[0503] Further, the complement-fixing Abs in human samples with either Abs directed against West Nile Virus (WNV) or against ZIKV were evaluated in the 10-plex assay set-up to detect complement-fixing Abs directed against ZIKV NS1. In addition, a negative control sample lacking flavivirus-directed Abs was included. Satisfyingly, the assay was also able to distinguish between complement-fixing Abs binding to ZIKV and WNV (FIG. 24B) in line with the non-human primate data.

[0504] Altogether, this data indicate that samples can be evaluated for complement-fixing Abs directed against one specific flavivirus without interference from Abs generated upon infection with another flavivirus using NS1 as the antigen coupled to the microspheres in the complement-fixing assay set-up. This can be very useful in practice as multiple infections with different flavivirus are commonly observed as flaviviruses are co-circulating in several areas. The complement-fixing assay described in the present application is therefore also suitable for diagnosing by which flavivirus a subject was infected.

Example 10: Application of the DENV-Quadruplex Complement-Fixing Assay Set-Up to Distinguish Between Flavivirus Infections

[0505] In a next step, it was evaluated if the DENV-quadruplex complement-fixing assay set-up developed under Example 2 is selective complement-fixing Abs generated upon DENV infection.

[0506] Therefore, four rhesus macaques per group (2-3 year old male flavivirus na?ve Indian Rhesus macaques, weighing 3-6 kg) were vaccinated subcutaneously with one of a YFV (Stamaril?, Sanofi Pasteur), ZIKV (purified inactivated zika vaccine, PIZV, Takeda; see, for instance, WO 2019/090228), JEV (Ixiaro?, Valenva), WNV (Innovator?, Fort Dodge), or TBEV (Encepur?, GlaxoSmithKline) vaccine either in one dose (YFV vaccine at 1000 international units) or in two doses administered four weeks apart (ZIKV vaccine at 10 ?g per dose; JEV vaccine at 6 AU (?460 ng) per dose; 0.5 mL of WNV vaccine per dose; TBEV vaccine at 1.5 ?g per dose). All monkeys were negative for Hepatitis B virus, Simian Retrovirus, Simian Immunodeficiency virus, Simian T Lymphotropic virus, Mycobacterium tuberculosis, Simian Varicella virus, Malaria, Salmonella, Shigella, Yersinia and internal parasites. Serum was collected for all animals on day 1 (prior to vaccination on the day of vaccination), day 57, and day 169 and analyzed by the DENV-quadruplex complement-fixing assay set-up. Except for ZIKV vaccinated animals, no or only low titers against all four DENV VLPs were observed, indicating a good specificity of the assay even in the presence of complement-fixing antibodies and other non-complement-fixing antibodies directed against various flaviviruses (FIG. 25). However, also for the ZIKV vaccinated animals signal decreased over time for most animals. This data further indicate, that vaccination against other flaviviruses except ZIKV (and probably also natural infection by other flaviviruses except ZIKV) does not induce complement-fixing antibodies that are capable of fixing and activating complement on DENV. However, also the cross-reactive complement-fixing Abs induced by vaccination against ZIKV wane overtime.

[0507] In summary, this data underline the suitability of the DENV-quadruplex complement-fixing assay set-up to reliable distinguish between flavivirus infections independent of the infection being acute or convalescent.

Example 11: Comparison of Total IgG ELISA and Complement-Fixing Assay

[0508] The complement-fixing assay set-up was further compared to a dengue total binding IgG enzyme linked immunosorbent assay (ELISA). Therefore, the 53 samples from children and adults analyzed under Example 6 were further analyzed with the DENV-quadruplex complement-fixing assay set-up as described above under Example 2 or with a total IgG ELISA set-up as described below.

[0509] To quantitate DENV1, DENV2, DENV3 and DENV4 total binding IgG using an antigen capture ELISA, 96-well MaxiSorp microplates (Nunc) were coated with monoclonal antibody clone 4G2 (Absolute Antibody) prepared in 0.1M carbonate/bicarbonate buffer (Sigma) by overnight incubation at 4? C. Afterwards, microplates were blocked with SuperBlock T20 (Thermo Scientific) for 1 hour at 37? C. and washed with 1?PBS 0.1% (v/v) Tween 20 (PBS-T). Microplates were incubated with either serotype present in a tetravalent dengue vaccine (TAK-003 or DENvax, Takeda; for review, see, for instance, Osorio et al., 2011, Vaccine 29: 7251-7260) for 1.5 hours at 37? C. and washed with PBS-T. Serially diluted serum samples were then added to the microplate and incubated for 1 hr at 37? C. Following washes with PBS-T, horseradish peroxidase (HRP)-conjugated anti-IgG secondary antibody (Abcam) was added to the microplate and incubated for 1 hr at 37? C. After a final washing cycle with PBS-T, microplates were developed with ABTS (2,2-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) peroxidase substrate (Seracare) for 15 minutes at room temperature. The reaction was stopped with 1?ABTS stop solution (Seracare) and microplates were read at 405 nm using a SpectraMax Plus 384 microplate reader (Molecular Devices). DENV-specific antibody levels were calculated relative to a reference standard using GraphPad Prism version 7 (Graph Pad Software). Serotype-specific total IgG levels are shown in FIG. 28.

[0510] Correlation analysis between complement-fixing antibody titers and total IgG titers was carried out using Log.sub.10-transformed values using JMP version 13.1.0 software (SAS Institute, Inc.). Moderate correlation was observed for the total IgG ELISA and the complement-fixing assay set-up (FIG. 29), indicating that total binding IgG produced in response to natural virus exposure are functional to activate complement.

Example 12: The Complement-Fixing Assay Set-Up Based on C1q Fixation Translates to C3d Deposition

[0511] In a next step, it was evaluated whether the complement-fixing assay set-up based on the fixation of C1q as described above translates to complement C3d deposition (see also FIG. 30A), an early marker of complement system activation known to be involved in increased B cell responses.

[0512] The C3d deposition assay set-up (see also FIG. 30B) was carried out as described above for the C1q fixation set-up outlined under Example 2, unless otherwise noted. In particular, incubation temperatures and shaking, as well as microsphere counting was carried out as described above. In addition, also all samples evaluated in the C3d deposition assay were heat-inactivated as described above for the C1q fixation set-up. For the C3d deposition assay, 50 ?L of VLP-coupled microspheres were combined with 50 ?L of diluted samples and incubated for 1 hour as described under Example 2. Afterwards, the microspheres were washed with wash buffer (PBS supplemented with 0.05% (v/v) Tween-20) using the plate washer (BioTek, model ELx405). The antigen-antibody immunocomplex formed was detected by adding 50 ?L/well of complement-competent human serum (Complement Technology) diluted 160-fold in 1?PBS and incubating for 15 min at 37? C. Following washing with wash buffer, the C3d deposition on the VLP-coupled microspheres by antigen-specific sample antibodies was detected by adding 50 ?L/well of a pre-reporter antibody, i.e. mouse anti-human C3d IgG1 monoclonal antibody (Quidel) at 1.25 ?g/mL and incubating for 30 min. After washing with wash buffer, microspheres were incubated with 50 ?L/well of an anti-mouse IgG reporter antibody conjugated to phycoerythrin (Jackson Immunoresearch) at 10 ?g/mL for 30 min. Afterwards, the microspheres were washed twice with PBS-T, reconstituted with 100 ?L/well of assay buffer and read on the Magpix plate reader (Luminex Corporation) as described above under Example 2. C3d deposition antibody titers (or also shortly referred to as C3d deposition) were determined mutatis mutandis as described above for the complement-fixing antibody titers derived from the C1q-based assay. Correlation analysis between complement-fixing antibody titers based on C1q-fixation and C3d deposition antibody titers was carried out in GraphPad Prism version 8.1.0 (GraphPad Software, Inc.) using log.sub.10-transformed values.

[0513] For comparison of the two set-ups (i.e. the C1q-based and the C3d-deposition DENV-quadruplex complement-fixing assay set-ups), a panel of 9 samples from healthy subjects who were seropositive to DENY, with a wide range of complement-fixing antibody levels, was used to measure C1q-based complement-fixing antibody titers (as described above under Example 2), as well as C3d deposition (FIGS. 31-34). The 9 serum samples collected from adults living in DENY-endemic areas in Colombia from 2015-2016 were purchased from ABO Pharmaceuticals. In addition, high, medium, and low control samples, comprising high, medium, and low titers of DENY-reactive complement-fixing Abs were examined (see, for instance, also Example 2). The overall pattern of C3d deposition levels was similar to complement-fixing antibody titers. Both biomarkers were highly correlated, with a correlation coefficient (R.sup.2) and slope close to 1.000 irrespective of the DENV serotype (FIG. 35), validating that the C1q-based complement-fixing assay set-up can be used as a surrogate marker for complement system activation by antigen-specific antibodies.

SEQUENCE LISTING

[0514] Genomic sequences of flaviviruses are presented in the form of DNA in the sequence listing. As flaviviruses are RNA viruses it is clear to the person skilled in the art that the thymidine residues need to be replaced by uridine. Table 11 gives an overview of sequences of the present application.

Table 11 Overview of sequences of the present application

TABLE-US-00011 SEQ ID Sequence NO: Organism Strain Type 1 Dengue virus Puerto Rico/US/BID-V853/1998 Amino Acid serotype 1 2 Dengue virus Thailand/16681/84 Amino Acid serotype 2 3 Dengue virus Sri Lanka D3/H/IMTSSA- Amino Acid serotype 3 SRI/2000/1266 4 Dengue virus Dominica/814669/1981 Amino Acid serotype 4 5 Dengue virus Puerto Rico/US/BID-V853/1998 Genomic serotype 1 6 Dengue virus Thailand/16681/84 Genomic serotype 2 7 Dengue virus Sri Lanka D3/H/IMTSSA- Genomic serotype 3 SRI/2000/1266 8 Dengue virus Dominica/814669/1981 Genomic serotype 4 9 Zika virus Suriname Z1106033 Amino Acid 10 Zika virus Suriname Z1106033 Genomic 11 Dengue virus Nauru/Western Pacific/1974 Amino Acid serotype 1 12 Dengue virus Nauru/Western Pacific/1974 Genomic serotype 1 13 Dengue virus NS1 Amino Acid serotype 1 14 Dengue virus NS1 Amino Acid serotype 2 15 Dengue virus NS1 Amino Acid serotype 3 16 Dengue virus NS1 Amino Acid serotype 4

Items of the Invention

Microsphere Complex

[0515] 1. A microsphere complex comprising a microsphere coupled to a flavivirus antigen. [0516] 2. The microsphere complex according to item 1, wherein the flavivirus is selected from the group consisting of dengue virus, zika virus, West Nile virus, Japanese encephalitis virus, Tick-Borne encephalitis virus, Yellow Fever virus, Murray Valley encephalitis virus, and St. Louis encephalitis virus. [0517] 3. The microsphere complex according to any one of item 1 or 2, wherein the antigen is selected from the group consisting of virus like particle (VLP), non-structural protein 1, envelope protein, pre-membrane protein, membrane protein, capsid protein, non-structural protein 2A, non-structural protein 2B, non-structural protein 3, non-structural protein 4A, non-structural protein 4B, and non-structural protein 5 and any derivative thereof. [0518] 4. The microsphere complex according to item 3, wherein the antigen is a virus like particle. [0519] 5. The microsphere complex according to item 3, wherein the antigen is a non-structural protein 1. [0520] 6. The microsphere complex according to any one of items 1 to 5, wherein the flavivirus is a zika virus. [0521] 7. The microsphere complex according to any one of items 1 to 5, wherein the flavivirus is a West Nile virus. [0522] 8. The microsphere complex according to any one of items 1 to 5, wherein the flavivirus is a dengue virus. [0523] 9. The microsphere complex according to item 8, wherein the dengue virus is a dengue-1 virus. [0524] 10. The microsphere complex according to item 8, wherein the dengue virus is a dengue-2 virus. [0525] 11. The microsphere complex according to item 8, wherein the dengue virus is a dengue-3 virus. [0526] 12. The microsphere complex according to item 8, wherein the dengue virus is a dengue-4 virus.

VLP

[0527] 13. The microsphere complex of item 1, wherein the microsphere is coupled to a dengue-1 virus VLP. [0528] 14. The microsphere complex of item 13, wherein the dengue-1 virus VLP is derived from dengue-1 virus strain Puerto Rico/US/BID-V853/1998 characterized by SEQ ID NO: 1. [0529] 15. The microsphere complex of item 13, wherein the dengue-1 virus VLP comprises the envelope protein, the membrane protein, and the pre-membrane protein which are at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to corresponding parts of SEQ ID NO: 1. [0530] 16. The microsphere complex of item 1, wherein the microsphere is coupled to a dengue-2 virus VLP. [0531] 17. The microsphere complex of item 16, wherein the dengue-2 virus VLP is derived from dengue-2 virus strain Thailand/16681/84 characterized by SEQ ID NO: 2. [0532] 18. The microsphere complex of item 16, wherein the dengue-2 virus VLP comprises the envelope protein, the membrane protein, and the pre-membrane protein which are at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to corresponding parts of SEQ ID NO: 2. [0533] 19. The microsphere complex of item 1, wherein the microsphere is coupled to a dengue-3 virus VLP. [0534] 20. The microsphere complex of item 19, wherein the dengue-3 virus VLP is derived from dengue-3 virus strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 characterized by SEQ ID NO: 3. [0535] 21. The microsphere complex of item 19, wherein the dengue-3 virus VLP comprises the envelope protein, the membrane protein, and the pre-membrane protein which are at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to corresponding parts of SEQ ID NO: 3. [0536] 22. The microsphere complex of item 1, wherein the microsphere is coupled to a dengue-4 virus VLP. [0537] 23. The microsphere complex of item 22, wherein the dengue-4 virus VLP is derived from dengue-4 virus strain Dominica/814669/1981 characterized by SEQ ID NO: 4. [0538] 24. The microsphere complex of item 22, wherein the dengue-4 virus VLP comprises the envelope protein, the membrane protein, and the pre-membrane protein which are at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to corresponding parts of SEQ ID NO: 4. [0539] 25. The microsphere complex of item 1, wherein the microsphere is coupled to a zika virus VLP. [0540] 26. The microsphere complex of item 25, wherein the zika virus VLP is derived from zika virus strain Suriname Z1106033 characterized by SEQ ID NO: 9. [0541] 27. The microsphere complex of item 25, wherein the zika virus VLP comprises the envelope protein, the membrane protein, and the pre-membrane protein which are at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to corresponding parts of SEQ ID NO: 9. [0542] 28. The microsphere complex of any one of items 13 to 27, wherein the VLP is produced in human cells, in particular human embryonic kidney (HEK293) cells.

NS1

[0543] 29. The microsphere complex of item 1, wherein the microsphere is coupled to a dengue-1 virus non-structural protein 1. [0544] 30. The microsphere complex of item 29, wherein the dengue-1 virus non-structural protein 1 is derived from dengue-1 virus strain Nauru/Western Pacific/1974 characterized by SEQ ID NO: 11. [0545] 31. The microsphere complex of item 29, wherein the dengue-1 virus non-structural protein 1 is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to the corresponding part of SEQ ID NO: 11, in particular the dengue-1 virus non-structural protein 1 is shown in SEQ ID NO: 13. [0546] 32. The microsphere complex of item 1, wherein the microsphere is coupled to a dengue-2 virus non-structural protein 1. [0547] 33. The microsphere complex of item 32, wherein the dengue-2 virus non-structural protein 1 is derived from dengue-2 virus strain Thailand/16681/84 characterized by SEQ ID NO: 2. [0548] 34. The microsphere complex of item 32, wherein the dengue-2 virus non-structural protein 1 is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to the corresponding part of SEQ ID NO: 2, in particular the dengue-2 virus non-structural protein 1 is shown in SEQ ID NO: 14. [0549] 35. The microsphere complex of item 1, wherein the microsphere is coupled to a dengue-3 virus non-structural protein 1. [0550] 36. The microsphere complex of item 35, wherein the dengue-3 virus non-structural protein 1 is derived from dengue-3 virus strain Sri Lanka D3/H/IMTSSA-SRI/2000/1266 characterized by SEQ ID NO: 3. [0551] 37. The microsphere complex of item 35, wherein the dengue-3 virus non-structural protein 1 is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to the corresponding part of SEQ ID NO: 3, in particular the dengue-3 virus non-structural protein 1 is shown in SEQ ID NO: 15. [0552] 38. The microsphere complex of item 1, wherein the microsphere is coupled to a dengue-4 virus non-structural protein 1. [0553] 39. The microsphere complex of item 38, wherein the dengue-4 virus non-structural protein 1 is derived from dengue-4 virus strain Dominica/814669/1981 characterized by SEQ ID NO: 4. [0554] 40. The microsphere complex of item 38, wherein the dengue-4 virus non-structural protein 1 is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to the corresponding part of SEQ ID NO: 4, in particular the dengue-4 virus non-structural protein 1 is shown in SEQ ID NO: 16. [0555] 41. The microsphere complex of item 1, wherein the microsphere is coupled to a zika virus non-structural protein 1. [0556] 42. The microsphere complex of item 41, wherein the zika virus non-structural protein 1 is derived from zika virus strain Suriname 21106033 characterized by SEQ ID NO: 9. [0557] 43. The microsphere complex of item 41, wherein the zika virus non-structural protein 1 is at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 100% identical to the corresponding part of SEQ ID NO: 9.

Microsphere

[0558] 44. The microsphere complex of any one of items 1 to 43, wherein the microsphere is a polystyrene microsphere. [0559] 45. The microsphere complex of any one of items 1 to 44, wherein the microsphere is magnetic. [0560] 46. The microsphere complex of any one of items 1 to 45, wherein the microsphere has a diameter in the range from about 0.01 to about 100 ?m, preferably in the range from about 1 to about 20 ?m, more preferably from about 5 to about 7 ?m, most preferably the microsphere has a diameter of about 6.5 ?m. [0561] 47. The microsphere complex of any one of items 1 to 46, wherein the microsphere contains carboxylate groups at the microsphere surface. [0562] 48. The microsphere complex of item 47, wherein coupling of the microsphere to the flavivirus antigen occurs by formation of an amide bond between a carboxylate group of the microsphere and an amine group of the flavivirus antigen. [0563] 49. The microsphere complex of any one of items 1 to 48, wherein the microsphere comprises a detectable label. [0564] 50. The microsphere complex of item 49, wherein the detectable label is at least one fluorescent dye. [0565] 51. The microsphere complex of item 50, wherein the microsphere can be identified by the emission signal of the at least one fluorescent dye. [0566] 52. The microsphere complex of any one of item 50 or 51, wherein the at least one fluorescent dye is selected from the group consisting of squaraine, phthalocyanine, naphthalocyanine, and any derivative thereof. [0567] 53. The microsphere complex of any one of items 1 to 48, wherein the microsphere can be identified by its diameter. [0568] 54. The microsphere complex of any one of items 1 to 48, wherein the microsphere can be identified by its shape.

Method for Determining the Presence and/or Amount of Flavivirus-Reactive Complement-Fixing Antibodies

[0569] 55. A method for determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in a sample from a subject comprising the steps of: [0570] Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen according to any one of items 1 to 54 with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; [0571] Step 2: contacting an amount of complement component 1q (C1q) with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow binding of the C1q to the heavy chain constant region of the complement-fixing antibodies; [0572] Step 3: contacting an amount of a reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the reporter antibody to the C1q, wherein the reporter antibody binds to the C1q with the variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and [0573] Step 4: detecting a signal from the reporter antibody bound to the C1q in step 3, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample. [0574] 56. The method according to item 55, comprising the further steps of: [0575] Step 5: determining the presence and/or amount of the reporter antibody from the signal of step 4; and [0576] Step 6: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 5. [0577] 57. The method according to item 55, comprising the steps of: [0578] Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen according to any one of items 1 to 54 with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; [0579] Step 2: contacting an amount of C1q with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow binding of the C1q to the heavy chain constant region of the complement-fixing antibodies; [0580] Step 3.1: contacting an amount of a pre-reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the pre-reporter antibody to the C1q, wherein the pre-reporter antibody binds to the C1q with the variable region of the pre-reporter antibody; [0581] Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C1q in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and [0582] Step 4: detecting a signal from the reporter antibody bound to the pre-reporter antibody in step 3.2, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample. [0583] 58. The method according to item 57, wherein the method further comprises the steps of: [0584] Step 5: determining the presence and/or amount of the reporter antibody from the signal of step 4; and [0585] Step 6: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 5. [0586] 59. The method according to any one of item 57 or 58, wherein [0587] contacting in step 1 is carried out for about 30 to 90 minutes, [0588] contacting in step 2 is carried out for about 10 to 50 minutes, [0589] contacting in step 3.1 is carried out for about 10 to 50 minutes, and [0590] contacting in step 3.2 is carried out for about 10 to 50 minutes. [0591] 60. The method according to item 59, wherein [0592] contacting in step 1 is carried out for about 60 minutes, [0593] contacting in step 2 is carried out for about 30 minutes, [0594] contacting in step 3.1 is carried out for about 30 minutes, and [0595] contacting in step 3.2 is carried out for about 30 minutes. [0596] 61. The method according to any one of items 55 to 60, wherein the signal from the reporter antibody in step 4 is resulting from the detectable label to which the reporter antibody is attached. [0597] 62. The method according to any one of items 55 to 61, wherein the detectable label to which the reporter antibody is attached to is a fluorescence label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof. [0598] 63. The method according to item 62, wherein the detectable label is phycoerythrin.

Method for the Concomitant Determination of the Presence and/or Amount of Complement-Fixing Antibodies Reactive to Different Flaviviruses

[0599] 64. A method for the concomitant determination of the presence and/or amount of complement-fixing antibodies reactive to different flaviviruses in a sample from a subject comprising the steps of: [0600] Step 1: contacting an amount of at least two microsphere complexes with the sample, [0601] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen according to any one of items 1 to 54 and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen according to any one of items 1 to 54 [0602] to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen, [0603] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, [0604] Step 2: contacting an amount of C1q with the complement-fixing antibodies bound to the first and/or the second flavivirus antigen in step 1 to allow binding of the C1q to the heavy chain constant region of the complement-fixing antibodies, [0605] Step 3: contacting an amount of a reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the reporter antibody to the C1q, wherein the reporter antibody binds to the C1q with the variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, [0606] Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and [0607] simultaneously detecting a signal from the reporter antibody bound to the C1q in step 3 of the at least one microsphere upon irradiation with a second light source, [0608] Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and [0609] Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample. [0610] 65. The method according to item 64, wherein the method further comprises the steps of: [0611] Step 7: determining the presence and/or amount of the reporter antibody from the summarized signal in step 6, and [0612] Step 8: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 7. [0613] 66. The method according to item 64, comprising the steps of: [0614] Step 1: contacting an amount of at least two microsphere complexes with the sample, [0615] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen according to any one of items 1 to 54 and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen according to any one of items 1 to 54 [0616] to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen, [0617] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, [0618] Step 2: contacting an amount of C1q with the complement-fixing antibodies bound to the first and/or the second flavivirus antigen in step 1 to allow binding of the C1q to the heavy chain constant region of the complement-fixing antibodies, [0619] Step 3.1: contacting an amount of a pre-reporter antibody with the C1q bound to the complement-fixing antibodies in step 2 to allow binding of the pre-reporter antibody to the C1q, wherein the pre-reporter antibody binds to the C1q with the variable region of the pre-reporter antibody, [0620] Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C1q in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, [0621] Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and [0622] simultaneously detecting a signal from the reporter antibody bound to the C1q in step 3 of the at least one microsphere upon irradiation with a second light source, [0623] Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and [0624] Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample. [0625] 67. The method according to item 66, wherein the method further comprises the steps of: [0626] Step 7: determining the presence and/or amount of reporter antibody from the summarized signal in step 6, and [0627] Step 8: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of reporter antibody determined in step 7. [0628] 68. The method according to any one of item 66 or 67, wherein [0629] contacting in step 1 is carried out for about 30 to 90 minutes, [0630] contacting in step 2 is carried out for about 10 to 50 minutes, [0631] contacting in step 3.1 is carried out for about 10 to 50 minutes, and [0632] contacting in step 3.2 is carried out for about 10 to 50 minutes. [0633] 69. The method according to item 68, wherein [0634] contacting in step 1 is carried out for about 60 minutes, [0635] contacting in step 2 is carried out for about 30 minutes, [0636] contacting in step 3.1 is carried out for about 30 minutes, and [0637] contacting in step 3.2 is carried out for about 30 minutes. [0638] 70. The method according to any one of items 64 to 69, wherein the signal from the reporter antibody in step 4 is resulting from the third detectable label. [0639] 71. The method according to any one of items 64 to 70, wherein the third detectable label is a fluorescence label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof. [0640] 72. The method according to item 71, wherein the fluorescence label is phycoerythrin. [0641] 73. The method according to any one of items 64 to 72, wherein the first detectable label is at least one fluorescent dye. [0642] 74. The method according to any one of items 64 to 73, wherein the second detectable label is at least one fluorescent dye. [0643] 75. The method according to item 73 or 74, wherein the at least one fluorescent dye is selected from the group consisting of squaraine, phthalocyanine, naphthalocyanine, and any derivative thereof. [0644] 76. The method according to any one of items 64 to 75, wherein in step 1, an amount of a first microsphere complex comprising a first microsphere coupled to a dengue 1 virus VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a dengue 2 virus VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a dengue 3 virus VLP, and an amount of a fourth microsphere complex comprising a fourth microsphere coupled to a dengue 4 virus VLP is contacted with the sample. [0645] 77. The method according to any one of items 64 to 75, wherein in step 1, an amount of a first microsphere complex comprising a first microsphere coupled to a dengue 1 virus VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a dengue 2 virus VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a dengue 3 virus VLP, an amount of a fourth microsphere complex comprising a fourth microsphere coupled to a dengue 4 virus VLP, an amount of a fifth microsphere complex comprising a fifth microsphere coupled to a zika virus VLP, an amount of a sixth microsphere complex comprising a sixth microsphere coupled to a dengue 1 virus non-structural protein 1, an amount of a seventh microsphere complex comprising a seventh microsphere coupled to a dengue 2 virus non-structural protein 1, an amount of an eighth microsphere complex comprising an eighth microsphere coupled to a dengue 3 virus non-structural protein 1, an amount of a ninth microsphere complex comprising a ninth microsphere coupled to a dengue 4 virus non-structural protein 1, and an amount of a tenth microsphere complex comprising a tenth microsphere coupled to a zika virus non-structural protein 1 is contacted with the sample. [0646] 78. The method according to any one of items 55 to 77, wherein the C1q is human C1q. [0647] 79. The method according to any one of claims 55 to 78, wherein the C1q is purified. [0648] 80. The method according to any one of items 55 to 79, wherein the subject is seropositive for the flavivirus. [0649] 81. The method according to any one of items 55 to 79, wherein the subject is seronegative for the flavivirus. [0650] 82. The method according to any one of items 55 to 80, wherein the subject is vaccinated with a flavivirus vaccine.

[0651] In Vitro Method for Diagnosing a Flavivirus Infection [0652] 83. An in vitro method for diagnosing a flavivirus infection in a subject comprising the steps of: [0653] Step 1: providing a sample obtained from the subject, [0654] Step 2: determining the amount of flavivirus-reactive complement-fixing antibodies in the sample according to any one of items 55 to 82, wherein the presence of flavivirus-reactive complement-fixing antibodies in the sample is indicative for a flavivirus infection. [0655] 84. The method according to item 83, wherein the flavivirus infection is acute. [0656] 85. The method according to item 83, wherein the flavivirus infection is convalescent. [0657] 86. The method according to any one of items 83 to 85, wherein the flavivirus is selected from the group consisting of dengue virus, zika virus, West Nile virus, Japanese encephalitis virus, Tick-Borne encephalitis virus, Yellow Fever virus, Murray Valley encephalitis virus, and St. Louis encephalitis virus. [0658] 87. The method according to any one of items 83 or 86, wherein the subject is infected by a dengue virus serotype selected from the group of dengue virus serotype 1, dengue virus serotype 2, dengue virus serotype 3, and dengue virus serotype 4. [0659] 88. The method according to any one of items 83 to 86, wherein the subject is infected by at least two different flaviviruses, such as zika virus and dengue virus. [0660] 89. The method according to any one of items 83 to 86, wherein the subject is infected by at least two different dengue virus serotypes selected from the group of dengue virus serotype 1, dengue virus serotype 2, dengue virus serotype 3, and dengue virus serotype 4. [0661] 90. The method according to any one of items 55 to 89, wherein the sample is heat-inactivated. [0662] 91. The method according to any one of items 55 to 90, wherein the sample is a serum sample or a plasma sample. [0663] 92. The method according to any one of items 55 to 91, wherein the subject is selected from the list consisting of mouse, primate, non-human primate, human, rabbit, cat, rat, horse, and sheep. [0664] 93. The method according to item 92, wherein the subject is human.

[0665] Kit for Detecting Flavivirus-Reactive Complement-Fixing Antibodies [0666] 94. A kit for detecting flavivirus-reactive complement-fixing antibodies in a sample comprising: [0667] an amount of at least one microsphere complex comprising a microsphere coupled to a flavivirus antigen according to any one of items 1 to 54, [0668] an amount of C1q, and [0669] an amount of a reporter antibody, wherein the reporter antibody is attached to a detectable label. [0670] 95. The kit according to item 94, comprising an amount of at least two microspheres complexes, [0671] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen according to any one of items 1 to 54 and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen according to any one of items 1 to 54, [0672] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and [0673] wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label. [0674] 96. The kit according to any one of item 94 or 95, comprising an amount of a first microsphere complex comprising a first microsphere coupled to a dengue 1 virus VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a dengue 2 virus VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a dengue 3 virus VLP, and an amount of a fourth microsphere complex comprising a fourth microsphere coupled to a dengue 4 virus VLP. [0675] 97. The kit according to any one of item 94 or 95, comprising an amount of a first microsphere complex comprising a first microsphere coupled to a dengue 1 virus VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a dengue 2 virus VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a dengue 3 virus VLP, an amount of a fourth microsphere complex comprising a fourth microsphere coupled to a dengue 4 virus VLP, an amount of a fifth microsphere complex comprising a fifth microsphere coupled to a zika virus VLP, an amount of a sixth microsphere complex comprising a sixth microsphere coupled to a dengue 1 virus non-structural protein 1, an amount of a seventh microsphere complex comprising a seventh microsphere coupled to a dengue 2 virus non-structural protein 1, an amount of an eighth microsphere complex comprising an eighth microsphere coupled to a dengue 3 virus non-structural protein 1, an amount of a ninth microsphere complex comprising a ninth microsphere coupled to a dengue 4 virus non-structural protein 1, and an amount of a tenth microsphere complex comprising a tenth microsphere coupled to a zika virus non-structural protein 1. [0676] 98. The kit according to any one of items 94 to 97, wherein the C1q is human C1q. [0677] 99. The kit according to any one of items 94 to 98, wherein the C1q is purified. [0678] 100. The kit according to any one of items 94 to 99, wherein the reporter antibody binds to the C1q with the variable region of the reporter antibody. [0679] 101. The kit according to any one of items 94 to 99, wherein the kit further comprises an amount of a pre-reporter antibody, and wherein the pre-reporter antibody binds to the C1q with the variable region of the pre-reporter antibody and the reporter antibody binds to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody. [0680] 102. The kit according to any one of items 94 to 101, wherein the detectable label to which the reporter antibody is attached to is a fluorescence label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof. [0681] 103. The kit according to item 102, wherein the fluorescence label is phycoerythrin. [0682] 104. The kit according to any one of items 95 to 103, wherein the first detectable label the first microsphere comprises is at least one fluorescent dye. [0683] 105. The method according to any one of items 95 to 104, wherein the second detectable label the second microsphere comprises is at least one fluorescent dye. [0684] 106. The method according to items 104 or 105, wherein the at least one fluorescent dye is selected from the group consisting of squaraine, phthalocyanine, naphthalocyanine, and any derivative thereof.

[0685] Additional Embodiments are Outlined Below: [0686] 1. A method for determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in a sample from a subject comprising the steps of: [0687] Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; [0688] Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow the formation of complement component fragment C3d (C3d); [0689] Step 3: contacting an amount of a reporter antibody with the C3d formed in step 2 to allow binding of the reporter antibody to the C3d, wherein the reporter antibody binds to the C3d with the variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and [0690] Step 4: detecting a signal from the reporter antibody bound to the C3d in step 3, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample. [0691] 2. The method according to item 1, comprising the steps of: [0692] Step 1: contacting an amount of a microsphere complex comprising a microsphere coupled to a flavivirus antigen with the sample to allow binding of the flavivirus-reactive complement-fixing antibodies in the sample to the flavivirus antigen; [0693] Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow the formation of C3d; [0694] Step 3.1: contacting an amount of a pre-reporter antibody with the C3d formed in step 2 to allow binding of the pre-reporter antibody to the C3d, wherein the pre-reporter antibody binds to the C3d with the variable region of the pre-reporter antibody; [0695] Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C3d in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody and wherein the reporter antibody is attached to a detectable label; and [0696] Step 4: detecting a signal from the reporter antibody bound to the pre-reporter antibody in step 3.2, wherein the signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample. [0697] 3. The method of item 1 or 2, comprising the further steps of: [0698] Step 5: determining the presence and/or amount of the reporter antibody from the signal of step 4; and [0699] Step 6: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 5. [0700] 4. The method of any one of items 1 to 3, wherein the antigen is selected from the group consisting of virus like particle (VLP), non-structural protein 1, envelope protein, pre-membrane protein, membrane protein, capsid protein, non-structural protein 2A, non-structural protein 2B, non-structural protein 3, non-structural protein 4A, non-structural protein 4B, and non-structural protein 5 and any derivative thereof, preferably the antigen is VLP. [0701] 5. The method according to any one of items 1 to 4, wherein the detectable label is a fluorescence label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof. [0702] 6. A method for the concomitant determination of the presence and/or amount of complement-fixing antibodies reactive to different flaviviruses in a sample from a subject comprising the steps of: [0703] Step 1: contacting an amount of at least two microsphere complexes with the sample, [0704] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen [0705] to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen; [0706] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, [0707] Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the flavivirus antigen in step 1 to allow the formation of C3d, [0708] Step 3: contacting an amount of a reporter antibody with the C3d formed in step 2 to allow binding of the reporter antibody to the C3d, wherein the reporter antibody binds to the C3d with the variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, [0709] Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and [0710] simultaneously detecting a signal from the reporter antibody bound to the C3d in step 3 of the at least one microsphere upon irradiation with a second light source, [0711] Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and [0712] Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample. [0713] 7. The method according to item 6, comprising the steps of: [0714] Step 1: contacting an amount of at least two microsphere complexes with the sample, [0715] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen [0716] to allow binding of the complement-fixing antibodies reactive to the first and/or the second flavivirus to the first flavivirus antigen and/or the second flavivirus antigen, [0717] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, [0718] Step 2: contacting an amount of a complement competent serum with the complement-fixing antibodies bound to the first and/or the second flavivirus antigen in step 1 to allow the formation of C3d, [0719] Step 3.1: contacting an amount of a pre-reporter antibody with the C3d formed in step 2 to allow the formation of C3d and to allow binding of the pre-reporter antibody to the C3d, wherein the pre-reporter antibody binds to the C3d with the variable region of the pre-reporter antibody, [0720] Step 3.2: contacting an amount of a reporter antibody with the pre-reporter antibody bound to the C3d in step 3.1 to allow binding of the reporter antibody to the pre-reporter antibody, wherein the reporter antibody binds to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody and wherein the reporter antibody is attached to a third detectable label, [0721] Step 4: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label thereby identifying the at least one microsphere and the flavivirus antigen the at least one microsphere is coupled to, and [0722] simultaneously detecting a signal from the reporter antibody bound to the C3d in step 3 of the at least one microsphere upon irradiation with a second light source, [0723] Step 5: repeating step 4 until at least 30 microspheres coupled to the same flavivirus antigen are identified, and [0724] Step 6: summarizing the detected signal from the reporter antibody in step 4 for all identified microspheres coupled to the same flavivirus antigen, wherein the summarized signal is indicative for the presence and/or amount of the reporter antibody and wherein the presence and/or amount of the reporter antibody is indicative for the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample. [0725] 8. The method according to item 6 or 7, wherein the method further comprises the steps of: [0726] Step 7: determining the presence and/or amount of the reporter antibody from the summarized signal in step 6, and [0727] Step 8: determining the presence and/or amount of flavivirus-reactive complement-fixing antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 7. [0728] 9. The method according to any one of items 6 to 8, wherein the antigen is selected from the group consisting of virus like particle (VLP), non-structural protein 1, envelope protein, pre-membrane protein, membrane protein, capsid protein, non-structural protein 2A, non-structural protein 2B, non-structural protein 3, non-structural protein 4A, non-structural protein 4B, and non-structural protein 5 and any derivative thereof, preferably the antigen is VLP. [0729] 10. The method according to any one of items 6 to 9, wherein the third detectable label is a fluorescence label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof. [0730] 11. The method of any one of items 6 to 10, wherein the flavivirus is selected from the group consisting of dengue virus, zika virus, West Nile virus, Japanese encephalitis virus, Tick-Borne encephalitis virus, Yellow Fever virus, Murray Valley encephalitis virus, and St. Louis encephalitis virus, preferably dengue virus [0731] 12. The method of any one of items 6 to 10, wherein the antigen is selected from the group consisting of virus like particle (VLP), non-structural protein 1, envelope protein, pre-membrane protein, membrane protein, capsid protein, non-structural protein 2A, non-structural protein 2B, non-structural protein 3, non-structural protein 4A, non-structural protein 4B, and non-structural protein 5 and any derivative thereof, preferably the antigen is VLP [0732] 13. The method according to any one of items 6 to 12, wherein in step 1, an amount of a first microsphere complex comprising a first microsphere coupled to a dengue 1 virus VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a dengue 2 virus VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a dengue 3 virus VLP, and an amount of a fourth microsphere complex comprising a fourth microsphere coupled to a dengue 4 virus VLP is contacted with the sample. [0733] 14. An in vitro method for diagnosing a flavivirus infection in a subject comprising the steps of: [0734] Step 1: providing a sample obtained from the subject, [0735] Step 2: determining the amount of flavivirus-reactive complement-fixing antibodies in the sample according to any one of items 1 to 13, wherein the presence of flavivirus-reactive complement-fixing antibodies in the sample is indicative for a flavivirus infection. [0736] 15. The method according to item 14, wherein the flavivirus is selected from the group consisting of dengue virus, zika virus, West Nile virus, Japanese encephalitis virus, Tick-Borne encephalitis virus, Yellow Fever virus, Murray Valley encephalitis virus, and St. Louis encephalitis virus, preferably the flavirus is dengue virus. [0737] 16. The method according to item 14 or 15, wherein the subject is infected by at least two different flaviviruses. [0738] 17. The method according to item 14 or 15, wherein the subject is infected by at least two different dengue virus serotypes selected from the group of dengue virus serotype 1, dengue virus serotype 2, dengue virus serotype 3, and dengue virus serotype 4. [0739] 18. The method according to any one of items 1 to 13, wherein the sample is heat-inactivated. [0740] 19. The method according to any one of items 1 to 13, wherein the subject is selected from the group consisting of mouse, primate, non-human primate, human, rabbit, cat, rat, horse, and sheep, preferably the subject is human. [0741] 20. A kit for detecting flavivirus-reactive complement-fixing antibodies in a sample comprising: [0742] an amount of at least one microsphere complex comprising a microsphere coupled to a flavivirus antigen, [0743] an amount of a complement competent serum, and [0744] an amount of a reporter antibody, wherein the reporter antibody is attached to a detectable label. [0745] 21. The kit according to item 20, comprising an amount of at least two microspheres complexes, [0746] wherein the first microsphere complex comprises a first microsphere coupled to a first flavivirus antigen and the second microsphere complex comprises a second microsphere coupled to a second flavivirus antigen, [0747] wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label, and [0748] wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label. [0749] 22. The kit of any one of item 20 or 21, wherein the antigen is selected from the group consisting of virus like particle (VLP), non-structural protein 1, envelope protein, pre-membrane protein, membrane protein, capsid protein, non-structural protein 2A, non-structural protein 2B, non-structural protein 3, non-structural protein 4A, non-structural protein 4B, and non-structural protein 5 and any derivative thereof. [0750] 23. The kit according to any one of items 20 to 22, wherein the flavivirus is selected from the group consisting of dengue virus, zika virus, West Nile virus, Japanese encephalitis virus, Tick-Borne encephalitis virus, Yellow Fever virus, Murray Valley encephalitis virus, and Saint Louis encephalitis virus, preferably the flavivirus is dengue virus. [0751] 24. The kit according to any one of item 20 or 23, comprising an amount of a first microsphere complex comprising a first microsphere coupled to a dengue 1 virus VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a dengue 2 virus VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a dengue 3 virus VLP, and an amount of a fourth microsphere complex comprising a fourth microsphere coupled to a dengue 4 virus VLP. [0752] 25. The kit according to any one of items 20 to 24, wherein the reporter antibody binds to the C3d with the variable region of the reporter antibody. [0753] 26. The kit according to any one of items 20 to 25, wherein the kit further comprises an amount of a pre-reporter antibody, and wherein the pre-reporter antibody binds to the C3d with the variable region of the pre-reporter antibody and the reporter antibody binds to the heavy chain constant region of the pre-reporter antibody with the variable region of the reporter antibody. [0754] 27. The kit according to any one of items 20 to 26, wherein the detectable label to which the reporter antibody is attached to is a fluorescence label selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof.