Method for the detection of an IgM antibody specific for a flavivirus in a sample

Abstract

Disclosed is a method for the detection of an IgM antibody specific for a flavivirus in a sample, comprising the steps of (a) contacting the sample with a solid support comprising immobilised IgM-binding molecules, (b) allowing binding of IgM antibodies in the sample to the IgM binding molecules on the solid support so that the IgM antibodies are also immobilised on the solid support, and (c) detecting IgM antibodies specific for a flavivirus by allowing binding of a complex comprising (i) an antiparallel dimer of soluble flavivirus Protein E (sE) and (ii) a marker and identifying the binding of the complex to the specific flavivirus IgM antibody by detecting the marker; and a kit suitable for performing the method.

Claims

1. A method for the detection of an IgM antibody specific for a flavivirus in a sample, comprising: (a) contacting the sample with a solid support comprising immobilised IgM-binding molecules, (b) allowing binding of IgM antibodies in the sample to the IgM binding molecules on the solid support so that the IgM antibodies are also immobilised on the solid support, and (c) premixing an antiparallel dimer of soluble flavivirus Protein E (sE) with a marker for formation of a complex comprising the sE dimer and the marker, wherein the antiparallel dimer of sE comprises a tag and wherein the tag links the marker to the antiparallel dimer of sE, and (d) detecting IgM antibodies specific for a flavivirus by allowing binding of the preformed complex and identifying the binding of the complex to the specific flavivirus IgM antibody by detecting the marker.

2. The method according to claim 1, wherein the IgM-binding molecules are anti-IgM antibodies or IgM-binding fragments thereof.

3. The method according to claim 1, wherein the flavivirus is selected from group tick-borne encephalitis virus (TBEV) or dengue virus (DENV).

4. The method according to claim 1, wherein the sample is a serum or plasma sample, or a cerebrospinal fluid (CSF) sample.

5. The method according to claim 1, wherein the tag is selected from Strep-tag or His-tag.

6. The method according to claim 1, wherein the marker is selected from an affinity marker, a fluorescence marker, a radioactive marker, a nucleic acid marker, a chromogenic marker, a luminescence marker, a magnetic marker, or combinations thereof.

7. The method according to claim 1, wherein the complex comprises a labelled antibody and/or a Strep-tag binding protein.

8. The method according to claim 1, wherein the solid support is a microtiter plate or other plastic containers or surfaces, a biological microchip, a bead, a disc, a magnetic particle, a fiber optic sensor, a glass slide, or a membrane, preferably a nitrocellulose membrane, a polytetrafluorethylene membrane, a cellulose acetate membrane or a cellulose nitrate membrane.

9. The method according to claim 1, wherein detecting the marker comprises the addition of further substances thereby generating a signal or activating the marker to elicit a signal and measuring the signal.

10. The method according to claim 1, wherein sE of a flavivirus distantly related to the important human pathogenic flaviviruses is applied as a control.

11. The method according to claim 1, wherein the method is performed in an automated system.

12. A kit comprising: a solid support comprising immobilised IgM-binding molecules; and a complex comprising: (i) an antiparallel dimer of soluble flavivirus Protein E (sE); and (ii) a marker, for performing the method according to claim 1.

13. The kit of claim 12, further defined as comprising means to detect the marker.

14. The kit of claim 12, further defined as comprising an automated detection system.

15. The kit of claim 12, wherein at least the solid support and/or the complex and/or the sE and the marker are packed in a sterile wrap.

16. The method of claim 3, wherein the flavivirus is selected from DENV serotype 1 (DEN1), DENV serotype 2 (DEN2), DENV serotype 3 (DEN3), and DENV serotype 4 (DEN4), and Zika virus (ZV).

17. The method of claim 7, wherein the complex comprises a labelled antibody specific for a tag of the antiparallel dimer of sE and/or a Strep-tag binding protein comprising a marker further defined as an affinity marker, a fluorescence marker, a radioactive marker, a nucleic acid marker, a chromogenic marker, a luminescence marker, a magnetic marker, or combination thereof.

18. The method of claim 10, wherein the control is sE of Rio Bravo, Modoc, Yokose, Entebbe bat, Barkedji, or Lammi viruses.

19. The kit of claim 13, further defined as comprising means for detecting an affinity marker, a fluorescence marker, a radioactive marker, a chromogenic marker, a luminescence marker, or a magnetic marker.

20. The kit of claim 14, wherein the automated detection system is further defined as comprising a sample conveying device, a detection device, a computer processing unit and/or a display for displaying the signal from the detection unit processed by computer software programs executed with the computer processing unit.

Description

(1) The present invention is further disclosed by the following examples and the figures, yet without being restricted thereto.

(2) FIG. 1: Illustration of antigens used in the present invention; A: Schematic representation of a flavivirus particle in immature (left) and mature (right) form. Viral proteins: Eenvelope; prMprecursor of membrane protein; Mmembrane protein; Ccapsid protein; sEC-terminally truncated soluble form of E lacking the so-called stem and the transmembrane anchor. B: Ribbon diagram of the crystal structure of tick-borne encephalitis (TBE) virus sE. Upper panel: top view (showing the antiparallel dimeric structure of this protein); lower panel: side view. Color coding of the three sE domains: Domain I (DI)red; domain II (DII)yellow; domain III (DIII)blue. Color coding of the three sE domains: Domain I (DI)grey; domain II (DII)light grey; domain III (DIII)dark grey. C: Ribbon diagrams of monomeric soluble forms of E in their side views (1.sup.st panel: sE monomer (TBE); 2.sup.nd panel: sEFP (TBE sE monomer in which amino acids 98 to 111 were replaced by a GGGG-linker); 3.sub.rd panel: DI+DII monomer (TBE); 4.sup.th panel: pr+sE monomer (DEN2), FPfusion peptide).

(3) FIG. 2: Schematic representation of the test principle in the present invention; MAC (IgM antibody capture) ELISA format used in the invention; HRPhorseradish peroxidase.

(4) FIG. 3: Titration curves of a serum pool of recent human TBE virus infections in three different MAC ELISAs. Black solid line: Assay according to the invention. Grey solid line: Conventional assay according to references (4), (17). Black dotted line: Assay according to the invention but using sequential addition of the detector components.

(5) FIG. 4: MAC ELISAs using monomeric and dimeric E antigens in the detector complex. A: A 1:100 dilution of a serum pool of recent human TBE virus infections was analyzed in the inventive assay using the TBE sE dimer (black column), TBE sE FP, TBE DI+II and TBE pr-sE (grey-shaded columns) as antigens in the detector complex. B: Four serum samples from recent human WN virus infections were analyzed at a dilution of 1:100 in the inventive assay format using the monomeric WN sE in the detector complex (black columns) in comparison to a MAC ELISA using purified whole WN virus particles as an antigen (grey columns). HRPhorseradish peroxidase; mabmonoclonal antibody

(6) FIG. 5: Results of TBE IgM determinations of 31 human serum samples from recent TBE virus infections in the inventive assay (black dots) compared to the conventional MAC ELISA using purified infectious TBE virus (open squares), expressed in arbitrary units. Nsnot significant. The bars indicate the mean+/ 95% confidence intervals. Serum dilution1:1000.

(7) FIG. 6: Demonstration of the oligomeric structure of recombinant sE proteins from TBE, Zika, WN, and DEN4 viruses. SDS-PAGE of recombinant sE proteins without () and after chemical cross-linking with DMS (+). Staining with Coomassie Blue.

(8) FIG. 7: Results of the analysis of serum samples of recent flavivirus infections using the inventive assay with a panel of flavivirus sE antigens as indicated on the x-axis. A: TBE virus infection; B: Zika virus infection; C: Dengue 2 virus infection; D: Dengue 3 virus infection; E: Zika and Dengue 2 virus double infections; F: Dengue 3 virus infection. Serum dilution1:100.

(9) FIG. 8: Results of the analysis of serum samples of recent dengue infections using the inventive assay with dengue serotype 1 to 4 sE antigens as indicated on the x-axis. A: Dengue 1 virus infection; B: Dengue 2 virus infection; C: Dengue 3 virus infection; D: Dengue 4 virus infection. Serum dilution1:100.

(10) FIG. 9: Titration curves of a serum pool of recent human TBE virus infections in four different MAC ELISAs. Black solid line: Assay according to the invention. Black dotted line: Assay according to the invention but using sequential addition of the detector components. Grey solid line: Conventional TBE MAC ELISA, using the same recombinant, strep-tagged sE as in the inventive complex, but a Biotin-labeled E-protein specific monoclonal antibody (mab) and streptavidin-HRP for detection. Grey dashed line: Conventional TBE MAC ELISA, using an untagged sE dimer (isolated form purified TBE virus, Heinz et al. 1991, J Virol 65 (10):5579-5583), and a Biotin-labeled E-protein specific monoclonal antibody (mab) and streptavidin-HRP for detection.

EXAMPLES

Abbreviations

(11) DI, II, III domain I, II, III DEN dengue DMS dimethyl suberimidate ELISA enzyme linked immunosorbent assay FP fusion peptide HRP horseradish peroxidase JE Japanese encephalitis MAC ELISA IgM antibody capture ELISA PBS phosphate buffered saline RB Rio Bravo SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis sE soluble E TBE tick-borne encephalitis WN West Nile
Materials and Methods
Human Serum Samples.

(12) Serum samples of patients were sent to the Department of Virology, Medical University of Vienna, and stored at 20 C.

(13) Virus Production and Inactivation.

(14) Virus production was carried out essentially as described in references (3, 19). In brief, primary chicken embryo cells were infected with TBE virus, strain Neudrfl (GenBank # U27495) or WN virus, strain NY99 (GenBank # AF196835). 24-48 hours p.i. the cell supernatant was harvested and clarified by centrifugation. WN virus was inactivated with formalin (1:2000) for 24 h at 37 C. Virus-containing suspensions were concentrated by ultracentrifugation and purified by rate zonal followed by equilibrium sucrose density gradient centrifugation.

(15) Production of Recombinant E Proteins.

(16) The recombinant antigens were produced in the Drosophila Expression System (Invitrogen) with a double strep-tag as described in references (5, 18-20). The expression vector pT389 encodes the export signal sequence Bip, an enterokinase cleavage site and the double strep-tag. Drosophila Schneider 2 cells were stably transfected using blasticidin for selection. Protein expression was induced by the addition of CuSO.sub.4 and supernatants were harvested 7-10 days after induction. Antigens were purified via affinity chromatography with Streptactin columns (IBA) according to the manufacturer's instructions.

(17) The following recombinant proteins were expressed and used as antigens in the inventive MAC-ELISA:

(18) TBE virus: sEamino acids E 1 to 400; TBE virus DI+IIamino acids E 1 to 302; TBE virus sE FPamino acids 1 to 400 (amino acids 98-111 were replaced by a GGGG-linker); TBE virus pr+sE [amino acids sE 1 to 400; prM 1 to 129 with a mutation in the furin cleavage site as described in reference (2); prM and sE were connected by the tobacco etch virus protease cleavage site as described in reference (7)]

(19) WN virus: sEamino acids E 1 to 400; DEN viruses: sEamino acids E 1 to 399 for DENV1, 2 and 4; 1 to 397 for DENV3; Zika virus: sEamino acids E 1 to 408; Rio Bravo: sEamino acids E 1 to 395 TBE (Neudrfl); GenBank # U27495 WN (NY99); GenBank # AF196835 Dengue 1 (FGA/89); GenBank # AF226687 Dengue 2 (16681); GenBank # U87411 Dengue 3 (CH53489); GenBank # DQ863638 Dengue 4 (11070); GenBank # NC_002640 Zika (H/PF/2013); GenBank # KJ776791 Rio Bravo (RiMAR); GenBank # AF144692
Labeling of Virus and Mab.

(20) Purified infectious TBE virus and the WN specific monoclonal antibody were labeled with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit (Innova Biosciences) according to the manufacturer's instructions.

(21) Chemical Cross-Linking and SDS-PAGE

(22) Chemical cross-linking of recombinant proteins was performed essentially as described previously (1). Briefly, 10 mM dimethyl suberimidate (DMS; Pierce) was added to the recombinant proteins in triethanolamine buffer pH 8.0 and incubated for 30 minutes at room temperature. Cross-linking was stopped by the addition of ethanolamine to a final concentration of 10 mM. Proteins were precipitated with trichloroacetic acid, subjected to SDS-PAGE using 5% polyacrylamide gels under non-reducing conditions as described in reference (9) and stained with Coomassie blue R-250.

(23) MAC ELISA

(24) For all MAC ELISA formats Nunc MaxiSorp plates were coated overnight at 4 C. with 50 l/well of rabbit immunoglobulin against human chains (DAKO) diluted 1:1,000 in carbonate buffer (pH 9.6; 0.159% Na.sub.2CO.sub.3 and 0.293% NaHCO.sub.3). After removal of the coating solution serum samples in PBS (phosphate buffered saline) buffer pH 7.4 (containing 2% Tween 20 and 2% sheep serum) were added and incubated for 45 minutes at 37 C.

(25) Serum samples were then removed and after three washing steps with PBS pH 7.4 different detection systems were applied as follows: a) Conventional TBE MAC ELISA (4, 17): Peroxidase-labeled infectious TBE virus was incubated for 30 minutes at 37 C. b) Conventional WN MAC ELISA: Formalin-inactivated WN virus was incubated for 30 minutes at 37 C. followed by a 30-minutes incubation with a WN virus specific peroxidase-labeled monoclonal antibody. c) Preformed inventive complex: sE proteins and Streptactin-HRP (IBA) were mixed in pre-determined optimal concentrations, incubated for 30 minutes at 500 rpm at room temperature and either stored at 80 C. or directly used. The complex was incubated for 30 minutes at 37 C. d) Sequential addition of the components of the inventive complex in the same concentrations as used in the complex: sE was incubated for 30 minutes at 37 C. followed by a 30-minutes incubation with Streptactin-HRP. e) Conventional TBE MAC ELISA, using the same recombinant, strep-tagged sE as in the inventive complex, but a Biotin-labeled E-protein specific monoclonal antibody (mab) and streptavidin-HRP for detection: sE was incubated for 30 minutes at 37 C. followed by a 30-minute incubation with the mab and a 30-minutes incubation with the streptavidin-HRP. f) Conventional TBE MAC ELISA, using an untagged sE dimer (isolated form purified TBE virus, Heinz 1991), and a Biotin-labeled E-protein specific monoclonal antibody (mab) and streptavidin-HRP for detection: sE was incubated for 30 minutes at 37 C. followed by a 30-minutes incubation with the mab and a 30-minutes incubation with the streptavidin-HRP.

(26) Then the plates were washed three times and 50 l substrate (o-phenylenediamine; Sigma) were added. The enzyme was allowed to react for 30 minutes at room temperature in the dark and stopped by the addition of 100 l 2N H.sub.2SO.sub.4.

(27) Results

(28) Demonstration that 1.) the MAC ELISA according to the invention is equivalent to a MAC ELISA in which HRP-labeled infectious TBE virus is used for detection and that 2.) this performance is only achieved when the detector components are added as a preformed complex and not when the individual components are added sequentially.

(29) In order to assess the quality of the assay according to the invention, we compared its performance with a) that of an established MAC ELISA that uses purified infectious HRP-labeled tick-borne encephalitis (TBE) virus as detector (4, 17) and b) an assay format in which the individual components of the inventive detector complex were added sequentially. As can be seen in FIG. 3, the sensitivity of IgM detection with the inventive assay in a pool of serum samples from recent TBE virus infections was equivalent to the conventional assay. In contrast, a much lower signal was obtained when the individual detector components (sE dimer and HRP-streptactin) were added sequentially (FIG. 3).

(30) FIG. 3 shows titration curves of a serum pool of recent human TBE virus infections in three different MAC ELISAs (Black solid line: Assay according to the invention; Grey solid line: Conventional assay according to references (4, 17); Black dotted line: Assay according to the invention but using sequential addition of the detector components.

(31) Demonstration that the excellent performance of the MAC ELISA according to the invention is dependent on the anti-parallel dimeric structure of sE used in the detector complex.

(32) For analyzing the structural requirements of the antigen used in the detector complex, we compared the TBE virus sE dimer with different monomeric forms of this protein. This included 1.) sE in which a sequence element required for dimerization (amino acids 98 to 111, fusion peptide FP) in domain II was removed (sE FP), 2.) a construct comprised only of domains I+II (DI+II) and 3.) a monomeric sE in complex with pr (pr+sE).

(33) The results in FIG. 4A show that the performance of the assays with the monomeric forms (grey-shaded columns, FIG. 4A) is dramatically reduced compared to the inventive assay using dimeric sE (black column, FIG. 4A).

(34) In addition, we analyzed the inventive assay format in another flavivirus system [West Nile (WN) virus] which like Japanese encephalitis (JE) virusin contrast to TBE and other flaviviruses (5, 8, 10, 11, 15)yields a monomeric form of sE upon production as a recombinant protein (6, 8, 13). Consistent with the results obtained with the monomeric TBE sE forms (FIG. 4A), very low signals were observed with the monomeric WN virus sE compared to a MAC ELISA using purified whole virus particles (FIG. 4B).

(35) These results demonstrate that the quality of the inventive assay is dependent on the native antiparallel structure of the flavivirus E antigen in the detector complex.

(36) FIG. 4 shows MAC ELISAs using monomeric and dimeric E antigens in the detector complex (A: A 1:100 dilution of a serum pool of recent human TBE virus infections was analyzed in the inventive assay using the TBE sE dimer (black column), TBE sE FP, TBE DI+II and TBE pr-sE (grey-shaded columns) as antigens in the detector complex; B: Four serum samples from recent human WN virus infections were analyzed at a dilution of 1:100 in the inventive assay format using the monomeric WN sE in the detector complex (black columns) in comparison to a MAC ELISA using purified whole WN virus particles as an antigen (grey columns); HRPhorseradish peroxidase; mabmonoclonal antibody).

(37) Demonstration that the MAC ELISA according to the invention is equivalent to the conventional format using a panel of 31 human serum samples from recent TBE virus infections.

(38) To compare the sensitivity of the inventive assay format with that of a conventional MAC ELISA using purified infectious TBE virus, we analyzed a panel of 31 serum samples from recent human TBE virus infections in both assays, using a positive standard serum for determining arbitrary TBE IgM units as described (17). The evaluation of the results revealed no statistically significant difference and complete equivalence of both assays (FIG. 5).

(39) FIG. 5 shows the results of TBE IgM determinations of 31 human serum samples from recent TBE virus infections in the inventive assay (black dots) compared to the conventional MAC ELISA using purified infectious TBE virus (open squares), expressed in arbitrary units (Nsnot significant. The bars indicate the mean+/ 95% confidence intervals. Serum dilution1:1000).

(40) Demonstration that the MAC ELISA according to the invention also works for other important human-pathogenic flaviviruses that yield dimeric recombinant sE proteins and allows the sero-diagnosis of recent human TBE, dengue and Zika virus infections with high specificity.

(41) The invention is dependent on the use of the native antiparallel structure of sE. The formation of such dimers upon recombinant protein expression differs between flaviviruses, specifically the recombinant sE proteins of JE and WN are monomeric (6, 8, 13); compare FIG. 1). However, sE proteins of other important human-pathogenic flaviviruses can potentially form dimers (FIG. 6) and could thus be used directly in the inventive assay format (FIG. 7).

(42) For this purpose, we expressed and purified recombinant sE proteins of dengue (DEN) and Zika viruses and analyzed their oligomeric structure by cross-linking with DMS in comparison to the sE proteins of TBE (predominantly dimeric) and WN (predominantly monomeric) viruses. As can be seen in FIG. 6, both DEN4 and Zika sE proteins yielded cross-linking patterns consistent with a dimeric structure, similar to TBE virus. The results obtained with DEN serotypes 1, 2 and 3 sE proteins were similar to that of DEN4.

(43) FIG. 6 demonstrates the oligomeric structure of recombinant sE proteins from TBE, Zika, WN, and DEN4 viruses (SDS-PAGE of recombinant sE proteins without () and after chemical cross-linking with DMS (+). Staining with Coomassie Blue).

(44) To demonstrate the suitability of DEN and Zika virus sE antigens for the inventive assay, we analyzed human serum samples from recent infections with these viruses in parallel with sEs of TBE, DEN serotypes 1 to 4, Zika and Rio Bravo viruses. The latter sE is used as a cross-reactive control antigen that is distantly related to all relevant human-pathogenic flaviviruses (16) and allows the determination of the contribution of broadly flavivirus cross-reactive antibodies to the assay signal. Examples of results obtained with such serum samples in inventive MAC ELISAs using Dengue serotype 1,2,3,4; Zika; Rio Bravo and TBE sE as antigens are shown in FIG. 7. The data reveal the excellent performance of the assay, allowing a type-specific serological diagnosis of important flavivirus infections of humans. Panel E shows that even recent double infections with dengue and Zika viruses can be resolved.

(45) Sequential infections with different DEN serotypes are especially prone to the development of broadly flavivirus cross-reactive antibodies that can pose problems in the sero-diagnosis of such infections (12, 14). Including an independent flavivirus antigen that does not play a role as human pathogen (sE from Rio Bravo virus) allows the detection of the background cross-reactive fraction and can thus help to identify the recently infecting DEN virus by comparing the extents of signals in assays with all four serotypes (FIG. 7F).

(46) FIG. 7 shows the results of the analysis of serum samples of recent flavivirus infections using the inventive assay with a panel of flavivirus sE antigens as indicated on the x-axis (A: TBE virus infection; B: Zika virus infection; C: Dengue 2 virus infection; D: Dengue 3 virus infection; E: Zika and Dengue 2 virus infections; F: Dengue 3 virus infection; serum dilution1:100).

(47) Further examples for the serotype-specific diagnosis of dengue virus infections using the inventive assay are shown in FIG. 8.

(48) FIG. 8 shows the results of the analysis of serum samples of recent dengue infections using the inventive assay with dengue serotype 1 to 4 sE antigens as indicated on the x-axis (A: Dengue 1 virus infection; B: Dengue 2 virus infection; C: Dengue 3 virus infection; D: Dengue 4 virus infection; serum dilution1:100).

(49) Demonstration that the MAC ELISA according to the invention is superior compared to conventional MAC ELISA formats that also use dimeric forms of sE

(50) In order to demonstrate the improved technical effect of the inventive assay, we compared its performance with a) that of the assay according to the invention but using sequential addition of the detector components, b) a conventional TBE MAC ELISA using the same recombinant, strep-tagged sE as in the inventive complex, but a Biotin-labeled E-protein specific monoclonal antibody (mab) and streptavidin-HRP for detection and c) a conventional TBE MAC ELISA, using an untagged sE dimer (isolated form purified TBE virus, Heinz 1991), and a Biotin-labeled E-protein specific monoclonal antibody (mab) and streptavidin-HRP for detection. As can be seen in FIG. 9, the sensitivity of IgM detection with the inventive assay in a pool of serum samples from recent human TBE virus infections is surprisingly more sensitive than detection with similar methods described in the art.

(51) The results in FIG. 9 show that adding the detector components as a preformed complex according to the invention (FIG. 9 black solid line) drastically increases the performance in comparison to sequential addition of the detector components (FIG. 9 black dotted line). In addition, the results demonstrate that with conventional MAC ELISAs using the same recombinant sE antigen (FIG. 9 grey solid line) the performance of IgM detection was much lower compared to the assay according to the invention. Same results were obtained with conventional MAC ELISAs using untagged sE dimers isolated from TBE virus (FIG. 9 grey dashed line).

(52) The data therefore reveal that the present invention provides a highly sensitive IgM detection assay whose performance could be dramatically improved over existing conventional MAC ELISAs using sE dimers.

(53) In view of this disclosure, the present invention provides the following preferred embodiments:

(54) 1. A method for the detection of an IgM antibody specific for a flavivirus in a sample, comprising the steps of (a) contacting the sample with a solid support comprising immobilised IgM-binding molecules, (b) allowing binding IgM antibodies in the sample to the IgM binding molecules on the solid support so that the IgM antibodies are also immobilised on the solid support, and (c) detecting IgM antibodies specific for a flavivirus by allowing binding of a complex comprising

(55) (i) an antiparallel dimer of soluble flavivirus Protein E (sE) and (ii) a marker
and identifying the binding of the complex to the specific flavivirus IgM antibody by detecting the marker.

(56) 2. Method according to embodiment 1, wherein the IgM-binding molecules are anti-IgM antibodies or IgM-binding fragments thereof.

(57) 3. Method according to embodiment 1 or 2 wherein the flavivirus is selected from group tick-borne encephalitis virus (TBEV), dengue virus (DENV), especially DENV serotype 1 (DEN1), DENV serotype 2 (DEN2), DENV serotype 3 (DEN3), and DENV serotype 4 (DEN4), and Zika virus (ZV).

(58) 4. Method according to any one of embodiments 1 to 3, wherein the sample is a serum or plasma sample, or a cerebrospinal fluid (CSF) sample.

(59) 5. Method according to any one of embodiments 1 to 4, wherein the antiparallel dimer of sE comprises a tag and wherein the tag links the marker to the antiparallel dimer of sE, the tag being preferably selected from Strep-tag or His-tag.

(60) 6. Method according to any one of embodiments 1 to 5, wherein the marker is selected from an affinity marker, a fluorescence marker, a radioactive marker, a nucleic acid marker, a chromogenic marker, a luminescence marker, a magnetic marker, or combinations thereof.

(61) 7. Method according to any one of embodiments 1 to 6, wherein the complex comprises a labelled antibody, especially an antibody being specific for a tag of the antiparallel dimer of sE; a Strep-tag binding protein, especially a Strep-tag binding protein comprising a marker according to embodiment 6.

(62) 8. Method according to any one of embodiments 1 to 7, wherein the solid support is a microtiter plate or other plastic containers or surfaces, a biological microchip, a bead, a disc, a magnetic particle, a fiber optic sensor, a glass slide, or a membrane, preferably a nitrocellulose membrane, a polytetrafluorethylene membrane, a cellulose acetate membrane or a cellulose nitrate membrane.

(63) 9. Method according to any one of embodiments 1 to 8, wherein detecting the marker comprises the addition of further substances thereby generating a signal or activating the marker to elicit a signal and measuring the signal.

(64) 10. Method according to any one of embodiments 1 to 9, wherein sE of a flavivirus distantly related to the important human pathogenic flaviviruses, preferably sE of Rio Bravo, Modoc, Yokose, Entebbe bat, Barkedji, or Lammi viruses, especially sE of Rio Bravo, is applied as a control.

(65) 11. Method according to any one of embodiments 1 to 10, wherein the method is performed in an automated system.

(66) 12. Method according to embodiment 10, wherein the automated system is characterised by being performed in a commercially available automated system, preferably in the Architect Immunoassay Analyzer (Abbott); Vidas Immunoanalyzer (Biomerieux); Cobas and Elecsys automated Immunoassay-Analyzers (Roche Diagnostics); Liaision Analyzer (Diasorin); or Euroimmun Analyzers (Euroimmun).

(67) 13. Kit for performing the method according to any one of embodiments 1 to 12, comprising: a solid support comprising immobilised IgM-binding molecules and a complex comprising (i) an antiparallel dimer of soluble flavivirus Protein E (sE) and (ii) a marker.

(68) 14. Kit according to embodiment 13, further comprising means to detect the marker, preferably means for detecting an affinity marker, a fluorescence marker, a radioactive marker, a chromogenic marker, a luminescence marker, or a magnetic marker.

(69) 15. Kit according to embodiment 13 or 14, further comprising a sample or a container containing a sample.

(70) 16. Kit according to any one of embodiments 13 to 15, further comprising a standard comprising IgM antibodies specific for a flavivirus.

(71) 17. Kit according to any one of embodiments 13 to 16, wherein the kit further comprises sE of a flavivirus distantly related to the important human pathogenic flaviviruses, preferably sE of Rio Bravo, Modoc, Yokose, Entebbe bat, Barkedji, or Lammi viruses, especially sE of Rio Bravo.

(72) 18. Kit according to any one of embodiments 13 to 17, wherein the kit further comprises an automated detection system, preferably comprising a sample conveying device, a detection device, a computer processing unit and/or a display for displaying the signal from the detection unit processed by computer software programs executed with the computer processing unit.

(73) 19. Kit according to any one of embodiments 13 to 18, wherein the kit is combined with a commercially available automated system, preferably with the Architect Immunoassay Analyzer (Abbott); Vidas Immunoanalyzer (Biomerieux); Cobas and Elecsys automated Immunoassay-Analyzers (Roche Diagnostics); Liaision Analyzer (Diasorin); or Euroimmun Analyzers (Euroimmun).

(74) 20. Kit according to any one of embodiments 13 to 19 wherein at least the solid support and/or the complex and/or the sE and the marker are packed in a sterile wrap, preferably in a sterile transparent plastic wrap.

REFERENCES

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