FLAVIVIRUS DIAGNOSTIC ASSAY

Abstract

The present invention provides a method for detecting viral infection of a subject by a first Flavivirus species, said method comprising: (a) contacting a sample from said subject with a solid phase support, wherein said solid phase support includes a capture means for immobilising antibody present in the sample that binds a first antigen from a first Flavivirus species; (b) challenging said immobilised antibody with: (i) a second antigen from a second (different) Flavivirus species, wherein the binding of said second antigen thereto suppresses (e.g. blocks) any inherent antigenic binding cross-reactivity towards the second Flavivirus species; and (ii) a labelled first antigen from the first Flavivirus species, thereby forming a labelled antigen-antibody complex; and (c) wherein the presence of labelled complex indicates viral infection of the subject by the first Flavivirus species, and wherein the absence of labelled complex indicates no viral infection of the subject by the first Flavivirus species; and kits for performing said method.

Claims

1. A method for detecting viral infection of a subject by a first Flavivirus species, said method comprising: a. contacting a sample from said subject with a solid phase support, wherein said solid phase support includes a capture means for immobilising antibody present in the sample that binds a first antigen from a first Flavivirus species; b. challenging said immobilised antibody with a second antigen from a second (different) Flavivirus species, wherein the binding of said second antigen thereto suppresses (e.g. blocks) any inherent antigenic binding cross-reactivity towards the second Flavivirus species; and c. challenging antibody that remains unblocked with labelled first antigen from the first Flavivirus species, thereby forming a labelled antigen-antibody complex; and d. wherein the presence of labelled complex indicates viral infection of the subject by the first Flavivirus species, and wherein the absence of labelled complex indicates no viral infection of the subject by the first Flavivirus species; e. wherein the first and second antigens are inter-species homologs of the same polypeptide/protein.

2. The method of claim 1, wherein said challenging steps are carried out simultaneously or sequentially.

3. The method of claim 1, wherein said challenging steps are carried out simultaneously.

4. The method of claim 1, wherein said second antigen is unlabelled.

5. The method of claim 1, wherein the capture means for immobilising antibody that binds a first antigen from a first Flavivirus species comprises a first antigen from a first Flavivirus species bound to the solid phase support.

6. The method of claim 1, wherein the capture means for immobilising antibody that binds a first antigen from a first Flavivirus species comprises an anti-human antibody that binds to an Fe region of the antibody that binds a first antigen from a first Flavivirus species.

7. The method of claim 1, wherein the first antigen from the first Flavivirus species is a Zika virus antigen.

8. The method of claim 7, wherein the second antigen is a non-Zika Flavivirus species antigen, and wherein the presence of labelled complex indicates viral infection of the subject by a Zika virus species, and wherein the absence of labelled complex indicates no infection of the subject by the Zika virus species.

9. The method of claim 1, wherein the second antigen from the second Flavivirus species is a Dengue virus antigen.

10. The method of claim 1, wherein the antibody of the labelled antibody-antigen complex is an IgG antibody.

11. The method of claim 1, wherein the antibody of the labelled antibody-antigen complex is an IgM antibody.

12. The method of claim 1, further comprising recording the output of said method on a data readable format.

13. A kit for detecting viral infection of a subject by a first Flavivirus species, said kit comprising: a. a solid-phase support comprising capture means for immobilising antibody present in the sample that binds a first antigen from a first Flavivirus species; b. a labelled first antigen from a first Flavivirus species; and c. an unlabelled second antigen from a second Flavivirus species; wherein the first antigen and the second antigen are NS1 protein, and wherein the first and second Flavivirus species are different species; wherein the first and second antigens are inter-species homologs of the same polypeptide/protein.

14. The kit of claim 13, wherein the capture means for immobilising antibody present in the sample that binds a first antigen from a first Flavivirus species comprises an immobilised first antigen from a first Flavivirus species.

15. The kit of claim 13, wherein the capture means for immobilising antibody present in the sample that binds a first antigen from a first Flavivirus species comprises an anti-human antibody that binds the Fc region of antibody present in the sample that binds a first antigen from a first Flavivirus species.

16. The kit of claim 13, wherein the first Flavivirus species is a Zika virus.

17. The kit of claim 13, wherein the unlabelled second Flavivirus species is a dengue virus.

18. The kit of claim 13, further comprising instructions for use.

19. The method of claim 8, wherein the absence of labelled complex indicates infection by a different non-Zika Flavivirus species.

20. The method of claim 9, wherein the Dengue virus antigen is a dengue 3 virus antigen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0150] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0151] FIG. 1 shows an X by Y plot illustrating the reactivity of 40 serum samples from patients with confirmed acute ZKV infection. Results are derived from a DABA assay of the invention, and are expressed as sample to cut off ratios when tested using un-blocked conjugate diluent and blocked conjugate diluent containing rDV3NS1Ag. Dotted line is a line of interpolated equivalence assuming no difference. The plot displays the reactivity of 40 samples from patients with confirmed ZKV infection using unblocked and blocked conjugate diluents. Samples from patients with proven ZIKA showing a reduced reactivity resulting from the blocked conjugate are circled. S/CO=sample absorbance (S) to cut-off value (CO) ratio.

[0152] FIG. 2 shows an X by Y plot illustrating the reactivity of 36 serum samples from patients with confirmed acute ZKV infection (these samples were also used in the assay demonstrated in FIG. 1). Results are derived from an IgG capture assay of the invention, and are expressed as sample to cut off ratios when tested using un-blocked conjugate diluent and blocked conjugate diluent containing rDV3NS1Ag. Dotted line is a line of interpolated equivalence assuming no difference. The plot displays the reactivity of 36 samples from patients with confirmed ZKV infection using unblocked and blocked conjugate diluents. Samples from patients with proven ZIKA showing a reduced reactivity resulting from the blocked conjugate are circled.

[0153] FIG. 3 shows an X by Y plot illustrating the reactivity of 20 serum samples from patients with confirmed dengue infection. Results are derived from an IgG capture assay of the invention, and are expressed as Raw Optical Densities (ODs) when tested using un-blocked conjugate diluent and blocked conjugate diluent containing rDVmixedNS1Ag. The plot displays the reactivity of samples from patients not infected by ZIKA whose serum reacted with ZIKA antigen NS1Ag using unblocked and blocked conjugate diluents. Control samples from patients recovering from proven ZIKA are circled.

[0154] FIG. 4 shows an X by Y plot illustrating the reactivity of 20 serum samples from patients with confirmed dengue infection (the same samples as used in the assay demonstrated in FIG. 4). Results are derived from a DABA assay of the invention, and are expressed as Raw Optical Densities (ODs) when tested using un-blocked conjugate diluent and blocked conjugate diluent containing rDVmixedNS1Ag. The plot displays the reactivity of samples from patients not infected by ZIKA whose serum reacted with ZIKA antigen NS1Ag using unblocked and blocked conjugate diluents. Control samples from patients recovering from proven ZIKA are circled.

EXAMPLES

Double Antigen Bridging Assay (Daba) Kit & Methods

[0155] Each kit typically contains sufficient materials for 96 tests. The shelf life of each kit is as indicated on the label fixed to the box containing the kit. It is intended that all components are stored at 2-8 C. unless otherwise stated. Typical materials provided (e.g. in a kit) for performing a method of the invention:

TABLE-US-00002 Coated Wells: One plate of 96 wells coated with recombinant ZIKA NS1 antigen. Allow the wells to reach room temperature (18 to 30 C.) before removal from the bag. Place unused wells in the sealable storage bag provided and return to 2-8 C. Negative Control: One bottle containing 500 l of Negative Control. Ready to use. Positive Control One bottle containing 250 l of Positive Control (purified recombinant monoclonal antibody ZKA35-rIgG1 at a concentration of 0.7 g/ml). Ready to use. Sample Diluent: One bottle containing 10 ml of Sample Diluent (coloured Green).Ready to Use Conjugate One bottle containing 10 ml of Conjugate Diluent. Ready to Use. Diluent: Conjugate One bottle containing 100 l of stock 100 times concentrated Concentrate: strength recombinant ZIKA NS1 antigen coupled with horseradish peroxidase in stabiliser solution. Vortex gently then Centrifuge tube to ensure the full volume of Conjugate Concentrate is in bottom of tube prior to opening. Add one volume of Conjugate Concentrate to 99 volumes (ie 1:100 dilution) of required Conjugate Diluent to get the required working strength Conjugate Solution. Additive A One bottle containing 400 l of stabiliser solution with no excess Antigen present. Additive B One bottle containing 300 l Zika NS1 Antigen diluted 1:1 in stabiliser solution at a final concentration of 250 ug/ml Additive C One bottle containing 300 l Dengue3 NS1 Antigen diluted 1:1 in stabiliser solution at a final concentration of 250 ug/ml TMB Substrate- One bottle containing 10 ml of 3,3,5,5- tetramethylbenzidine and Ready to Use: stabilisers in a colourless solution (TMB Substrate-ready to use) Keep substrate away from sunlight. The Substrate Solution should be colourless; if it is purple before being used, it should be discarded and fresh Substrate Solution used. Once opened, the bottle of TMB Substrate is stable refrigerated (2-8 C.) for 30 days, but must be discarded if crystals have formed. Stop Solution (2M One bottle containing 5 ml of 2M sulphuric acid (H.sub.2SO.sub.4) ready to H.sub.2SO.sub.4: use. Wash Fluid: One bottle containing 100 ml of 10 times concentrated strength Wash Buffer Solution. Add one volume of Wash Fluid Concentrate to 9 volumes of distilled or deionised water to give the required volume or dilute the entire contents of one bottle of Wash Fluid to a final volume of 1000 ml. Store the working strength Wash Fluid at 18-30 C. in a closed vessel under which conditions it will retain activity for one month.

[0156] Additional Material and Instruments (not part of kit): [0157] Good quality deionised or distilled water [0158] Clean vessels for wash solution preparation [0159] Microtitre plate cover [0160] Micropipettes and disposable tips capable of delivering 200 L, 100 L, 20 L and 1-5 l volumes. [0161] Waste discard container with disinfectant [0162] EIA plate reader capable of reading optical density at 450 nm (and 620-650 nm). [0163] Incubator, 37 C.

Specimen Collection & Preparation

[0164] Serum and plasma (EDTA, citrated or heparinised) samples are suitable specimens for the test and should be obtained from whole blood using standard laboratory procedure.

[0165] Either fresh serum or plasma samples can be used for this assay. If not used immediately, they can be stored at 2-8 C. for one week. Care should be taken to ensure that the serum samples are clear and not contaminated by microorganisms. Plasma samples collected into EDTA, sodium citrate or heparin may be tested, but highly lipaemic, icteric or haemolysed samples should not be used as they can give false results in the assay. Do not heat inactive samples.

Assay Procedure

Step 1: Reagents Preparation

[0166] Allow all reagents to reach room temperature (18-30 C.) prior to use.

[0167] Check the Wash Buffer concentrate for the presence of salt crystals. If crystals have formed in the solution, re-suspend by warming at 37 C. until the crystals have dissolved. Dilute the stock bottle concentrated wash fluid buffer 10 times with distilled or deionised water. Use a clean vessel to dilute the buffer. It is recommended that working strength buffer be prepared as required on the day of use. Remaining Wash Buffer Concentrate stock should be re-stored at 2-8 C. if not used.

[0168] Prepare the working strength Conjugate Solution (see Materials Provided section). In this example twenty four reactive samples, (72 wells in total), can be interrogated, together with the additional plate strip containing the six control wells as follows.

[0169] Prepare 3 separate tubes of working strength Conjugate solution with the addition of Additive A, B or C as follows: [0170] Conjugate A Tubedispense 3.15 ml of working strength Conjugate solution followed by the addition of 350 l of Additive A (no antigens) to give a total volume of 3.5 ml (volume required for both plate controls and samples). Mix well before Use. [0171] Conjugate B Tubedispense 2.25 ml of working strength Conjugate solution followed by the addition of 250 l of Additive B to give a total volume of 2.5 ml containing a molar excess of cold Zika NS1 Antigen (volume required for samples only). Mix well before Use [0172] Conjugate C Tubedispense 2.25 ml of working strength Conjugate solution followed by the addition of 250 l of Additive C to give a total volume of 2.5 ml containing a molar excess of cold Dengue3 NS1 Antigen (volume required for samples only). Mix well before Use

[0173] All other reagents are provided ready-to-use.

Step 2: Numbering Wells

[0174] Remove and assemble the required number of recombinant ZIKA NS1 antigen coated microwell strips to perform the test. A minimum of 6 wells is optimal for the controls (2 positive controls and 4 negative controls) which are optimally included in each test run. Set the strips needed into strip-holder and only use the required number of strips for the test. (Use the separately provided ZIKA DABA ELISA Front sheet Template to record the sample identity numbers to be tested and procedural information).

Step 3: Adding Controls and Samples

[0175] Following a DABA ELISA Frontsheet Template as a guide, pipette 70 l of Sample diluent and 30 l of the Positive Control into their respective well positions (1A-1B). Pipette 70 l of Sample diluent and 30 l of Negative Control into their respective well positions (1D-1F).

[0176] Set up each sample in triplicate to allow the addition of Conjugate A to one well, Conjugate B to a second well and Conjugate C to a third well.

[0177] Pipette 70 l of Sample diluent and 30 l of each sample into three separate well positions as designated on the DABA ELISA Frontsheet Template (only testing the number of samples in a single test run that can be dispensed into assigned wells within ten minutesthe assay reagent volumes supplied will allow the testing of a total of 24 samples in triplicate).

[0178] Note: use a separate disposal pipette tip for each sample, negative control and positive control to avoid cross-contamination.

Step 4: Incubation

[0179] Cover the plate with a plate sealer and mix gently by tapping the side of the plate strip holder. Incubate at 372 C. in a moist chamber or dry incubator for 602 minutes.

Step 5: Washing

[0180] After the end of the incubation, remove and discard the plate sealer. Wash each well 5 times with diluted wash buffer (see reagent preparation). The wash cycle is carried out as follows: aspirate the contents of the well and dispense at least 300 l per well of wash buffer to form a meniscus. Allow the microwells to soak for 30-60 seconds and then aspirate. Repeat the wash cycle a further four times. Alternatively an automatic plate washer may be used. After the final washing cycle turn down the strips plate onto blotting paper or clean towel and tap the plate to remove any remaining wash buffer.

Step 6: Conjugate

[0181] Dispense 100 l of Conjugate A into the 2 Positive control and 4 Negative control well positions (1A-1F)

[0182] For each sample tested in triplicate: [0183] Dispense 100 l of Conjugate A into each sample well one, [0184] Dispense 100 l of Conjugate B into each sample well two, [0185] Dispense 100 l of Conjugate C into each sample well three,

[0186] Cover the plate with a plate sealer and incubate at 372 C. in a moist chamber or dry incubator for 1202 minutes (If a dry incubator is used do not open the door frequently).

Step 7: Washing

[0187] After the end of the incubation, remove and discard the plate sealer. Wash each well 5 times as in Step 5. After the final washing cycle turn down the strips plate onto blotting paper or clean towel and tap the plate to remove any remaining wash buffer

Step 8: Substrate

[0188] Dispense 100 l of ready to use TMB substrate into each well. This is best performed using a multi-channel pipette.

[0189] Cover the plate with a plate sealer and mix gently by tapping the side of the plate strip holder. Incubate the plate at 372C in a moist chamber or dry incubator for 30 minutes, avoiding light.

[0190] The enzymatic reaction between the substrate and conjugate will produce a blue colour in the positive control and any anti-ZIKA positive sample wells.

Step 9: Stopping the Reaction

[0191] After the end of the incubation, remove and discard the plate sealer. Add 50 l of stop solution into each well. This is best performed using a multi-channel pipette and the stop solution should be added using the same timing and sequence that was used to add the substrate solution. Mix gently by tapping the side of the plate strip holder. Following addition of stop solution, an intensive yellow colour will develop in the positive control and any anti-ZIKA positive sample wells.

Step 10: Measure the Absorbance

[0192] Calibrate the plate reader with the blank well and read the absorbance at 450 nm within 10 minutes after stopping the reaction (if a dual filter instrument is used, set the reference wavelength at 630 or between 620 and 650 nm on the spectrophotometric plate reader). Calculate the cut-off value and evaluate the results.

Interpretation of Results & Quality Control

[0193] Each microplate is optimally considered separately when calculating and interpreting results of the assay, regardless of the number of plates concurrently processed. The results are calculated by relating each sample optical density (OD) value to the cut-off value (CO) of the plate.

Calculation of Cut-Off Value:

[0194]
Cut-off value (CO)=NC.sub.mean+0.1

NC.sub.mean=the mean absorbance value for 4 negative controls

[0195] If one of the negative control values does not meet the Quality Control range specifications, it should be discarded and the mean value calculated again using the remaining values.

Quality Control Range

[0196] Each sample absorbance test OD result (S) is valid if the Quality Control criteria are verified as below:

[0197] The absorbance value OD of each negative control must be less than 0.100.

[0198] The absorbance value OD of each positive control must be greater than 0.800.

Interpretation of Results

[0199] Negative results (S/CO1): Samples giving an absorbance less or equal to the cut-off value are considered negative, that is, no NS1 antibodies to ZIKA Virus have been detected using this ELISA kit.

[0200] Positive results (S/CO>1): Samples giving absorbance greater than the cut-off value are positive for this assay, that is, NS1 antibodies to ZIKA Virus have been detected with this ELISA kit. [0201] Samples containing antibodies specific to Zika NS1 Antigen will be blocked by the addition of Conjugate B, thus will show an absorbance less than the sample control well (wells with the addition of Conjugate A.) [0202] Samples containing antibodies specific to Dengue3 NS1 Antigen will be blocked by the addition of Conjugate C, thus will show an absorbance less than the sample control well (wells with the addition of Conjugate A).

Immunoglobulin G (IgG) Capture Enzyme Immunoassay Kit & Methods

[0203] Each kit typically contains sufficient materials for 96 tests. The shelf life of each kit is as indicated on the label fixed to the box containing the kit. All components must be stored at 2 to 8 C. unless otherwise stated.

TABLE-US-00003 Coated Wells: One plate of 96 wells coated with anti-human IgG antibody. Allow the wells to reach room temperature (18 to 30 C.) before removal from the bag. Place unused wells in the sealable storage bag provided and return to 2 to 8 C. Transport One bottle containing 40 ml of phosphate buffered saline, protein Medium: stabiliser and detergent. Negative Control: One bottle containing 1.8 ml of Negative Control. Ready to Use. Positive Control: One bottle containing 1 ml of Positive Control (purified recombinant monoclonal antibody ZKA35-rIgG1 at a concentration of 3.56 g/ml) Ready to Use. Conjugate One bottle containing 10 ml of phosphate buffered saline, protein Diluent: stabiliser and detergent. Conjugate One bottle containing 100 l of stock 100 times concentrated Concentrate: strength recombinant ZIKA NS1 antigen coupled with horseradish peroxidase in stabiliser solution. Vortex gently then Centrifuge tube to ensure the full volume of Conjugate Concentrate is in bottom of tube prior to opening. Add one volume of Conjugate Concentrate to 99 volumes (i.e. 1:100 dilution) of required Conjugate Diluent to get the required working strength Conjugate Solution. Additive A One bottle containing 400 l of stabiliser solution with no excess Antigen present. Additive B One bottle containing 300 l Zika NS1 Antigen diluted 1:1 in stabiliser solution at a final concentration of 250 ug/ml Additive C One bottle containing 300 l Dengue3 NS1 Antigen diluted 1:1 in stabiliser solution at a final concentration of 250 ug/ml TMB Substrate- One bottle containing 10 ml of 3,3,5,5-tetramethylbenzidine and Ready to Use: stabilisers in a colourless solution (TMB Substrate-ready to use) Keep substrate away from sunlight. The Substrate Solution should be colourless; if it is purple before being used, it should be discarded and fresh Substrate Solution used. Once opened, the bottle of TMB Substrate is stable refrigerated (2-8 C.) for 30 days, but must be discarded if crystals have formed. Stop Solution (2M One bottle containing 5 ml of 2M sulphuric acid (H.sub.2SO.sub.4) ready to H.sub.2SO.sub.4): use. Wash Fluid: One bottle containing 100 ml of 10 times concentrated strength Wash Buffer Solution. Add one volume of Wash Fluid Concentrate to 9 volumes of distilled or deionised water to give the required volume or dilute the entire contents of one bottle of Wash Fluid to a final volume of 1000 ml. Store the working strength Wash Fluid at 18- 30 C. in a closed vessel under which conditions it will remain fit for purpose for one month.

[0204] Additional Material and Instruments Required (not part of kit): [0205] Good quality deionised or distilled water [0206] Clean vessels for wash solution preparation [0207] Microtitre plate cover [0208] Micropipettes and disposable tips capable of delivering 200 L, 100 L, 20 L and 1-5 l volumes. [0209] Waste discard container with disinfectant [0210] EIA plate reader capable of reading optical density at 450 nm (and 620-650 nm). [0211] Incubator, 37 C.

Specimen Collection & Preparation

[0212] Currently serum and plasma (EDTA, citrated or heparinised) samples are suitable specimens for the test and should be obtained from whole blood using standard laboratory procedure.

[0213] Either fresh serum or plasma samples can be used for this assay. If not used immediately, they can be stored at 2-8 C. for one week. Care should be taken to ensure that the serum samples are clear and not contaminated by microorganisms. Plasma samples collected into EDTA, sodium citrate or heparin may be tested, but highly lipaemic, icteric or heavily haemolysed samples should not be used as they can give false results in the assay. For optimal results, do not heat inactive samples.

[0214] All samples for testing are optimally diluted 1:200 in Transport Medium prior to testing, see below.

Assay Procedure

Step 1: Reagents Preparation

[0215] Allow all reagents to reach room temperature (18-25 C.) prior to use.

[0216] Check the Wash Buffer concentrate for the presence of salt crystals. If crystals have formed in the solution, re-suspend by warming at 37 C. until the crystals have dissolved. Dilute the stock bottle concentrated wash fluid buffer 10 times with distilled or deionised water. Use a clean vessel to dilute the buffer. It is recommended that working strength buffer be prepared as required on the day of use. Remaining Wash Buffer Concentrate stock should be re-stored at 2-8 C. if not used.

[0217] Prepare the working strength Conjugate Solution (see Materials Provided section). In this example twenty four reactive samples, (72 wells in total), can be interrogated, together with the additional plate strip containing the six control wells as follows Prepare 3 separate tubes of working strength Conjugate solution with the addition of Additive A, B or C as follows: [0218] Conjugate A Tubedispense 3.15 ml of working strength Conjugate solution followed by the addition of 350 l of Additive A (no antigens) to give a total volume of 3.5 ml (volume required for both plate controls and samples). Mix well before Use. [0219] Conjugate B Tubedispense 2.25 ml of working strength Conjugate solution followed by the addition of 250 l of Additive B to give a total volume of 2.5 ml containing a molar excess of cold Zika NS1 Antigen (volume required for samples only). Mix well before Use [0220] Conjugate C Tubedispense 2.25 ml of working strength Conjugate solution followed by the addition of 250 l of Additive C to give a total volume of 2.5 ml containing a molar excess of cold Dengue3 NS1 Antigen (volume required for samples only). Mix well before Use

[0221] All other kit reagents are provided ready-to-use.

[0222] Dilute the serum or plasma samples 1:200 in Transport Medium by dispensing 2 l of sample into a labelled tube and adding 400 l of Transport Medium and mix.

Step 2: Numbering Wells

[0223] Remove and assemble the required number of anti-human IgG antibody coated microwell strips to perform the test. A minimum of 6 wells is optimal for the controls (2 positive controls and 4 negative controls) which are optimally included in each test run. Set the strips needed into strip-holder and only use the required number of strips for the test. (Use the separately provided ZIKA G-Capture ELISA Front sheet Template to record the sample identity numbers to be tested and procedural information).

Step 3: Adding Controls and Samples

[0224] Following the G-Capture ELISA Frontsheet Template as a guide, pipette 100 l of the Positive Control into the respective well positions (1A-1B). Pipette 100 l of the Negative Control into the respective well positions (1D-1F).

[0225] Set up each sample in triplicate to allow the addition of Conjugate A to one well, Conjugate B to a second well and Conjugate C to a third well.

[0226] Pipette 100 l of each Sample (pre-diluted 1:200 in Transport Medium) into three separate well positions as designated on the G-Capture ELISA Frontsheet Template (only testing the number of samples in a single test run that can be dispensed into assigned wells within ten minutesthe assay reagent volumes supplied will allow the testing of a total of 24 samples in triplicate).

[0227] Note: use a separate disposal pipette tip for each sample, negative control and positive control to avoid cross-contamination.

Step 4: Incubation

[0228] Cover the plate with a plate sealer and mix gently by tapping the side of the plate strip holder. Incubate at 372 C. in a moist chamber or dry incubator for 602 minutes (If a dry incubator is used do not open the door frequently).

Step 5: Washing

[0229] After the end of the incubation, remove and discard the plate sealer. Wash each well 5 times with diluted wash buffer (see reagent preparation). The wash cycle is carried out as follows: aspirate the contents of the well and dispense at least 300 l per well of wash buffer to form a meniscus. Allow the microwells to soak for 30-60 seconds and then aspirate. Repeat the wash cycle a further four times. Alternatively an automatic plate washer may be used. After the final washing cycle turn down the strips plate onto blotting paper or clean towel and tap the plate to remove any remaining wash buffer.

Step 6: Conjugate

[0230] Dispense 100 l of Conjugate A into the 2 Positive control and 4 Negative control well positions (1A-1F)

[0231] For each sample tested in triplicate: [0232] Dispense 100 l of Conjugate A into each sample well one, [0233] Dispense 100 l of Conjugate B into each sample well two, [0234] Dispense 100 l of Conjugate C into each sample well three.

[0235] Cover the plate with a plate sealer and incubate at 372 C. in a moist chamber or dry incubator for 302 minutes.

Step 7: Washing

[0236] After the end of the incubation, remove and discard the plate sealer. Wash each well 5 times as in Step 5. After the final washing cycle turn down the strips plate onto blotting paper or clean towel and tap the plate to remove any remaining wash buffer.

Step 8: Substrate

[0237] Dispense 100 l of ready to use TMB Substrate into each well. This is best performed using a multi-channel pipette.

[0238] Cover the plate with a plate sealer and mix gently by tapping the side of the plate strip holder. Incubate the plate at 372 C. in a moist chamber or dry incubator for 302 minutes, avoiding light.

[0239] The enzymatic reaction between the substrate and conjugate will produce a blue colour in the positive control and any anti-ZIKA positive sample wells.

Step 9: Stopping the Reaction

[0240] After the end of the incubation, remove and discard the plate sealer. Add 50 l of Stop Solution into each well. This is best performed using a multi-channel pipette and the stop solution should be added using the same timing and sequence that was used to add the substrate solution. Mix gently by tapping the side of the plate strip holder. Following addition of stop solution, an intensive yellow colour will develop in the positive control and any anti-ZIKA positive sample wells.

Step 10: Measure the Absorbance

[0241] Calibrate the plate reader with the blank well and read the absorbance at 450 nm within 10 minutes after stopping the reaction (if a dual filter instrument is used, set the reference wavelength at 630 or between 620 and 650 nm on the spectrophotometric plate reader). Calculate the cut-off value and evaluate the results.

Interpretation of Results & Quality Control

[0242] Each microplate is optimally considered separately when calculating and interpreting results of the assay, regardless of the number of plates concurrently processed. The results are calculated by relating each sample optical density (OD) value to the cut-off value (CO) of the plate.

Calculation of Cut-Off Value:

[0243]
Cut-off value (CO)=NC.sub.mean+0.1

NC.sub.mean=the mean absorbance value for 4 negative controls

[0244] If one of the negative control values does not meet the Quality Control range specifications, it should be discarded and the mean value calculated again using the remaining values.

Quality Control Range

[0245] Each sample absorbance test OD result (S) is valid if the Quality Control criteria are verified as below:

[0246] The absorbance value OD of each negative control must be less than 0.100

[0247] The absorbance value OD of each positive control must be greater than 0.800

Interpretation of Results

[0248] Negative results (S/CO1): Samples giving an absorbance less or equal to the cut-off value are considered negative, that is, no NS1 antibodies to ZIKA Virus have been detected using this ELISA kit.

[0249] Positive results (S/CO >1): Samples giving absorbance greater than the cut-off value are positive for this assay, that is, NS1 antibodies to ZIKA Virus have been detected with this ELISA kit.

[0250] Samples containing antibodies specific to Zika NS1 Antigen will be blocked by the addition of Conjugate B, thus will show an absorbance less than the sample control well (wells with the addition of Conjugate A.)

[0251] Samples containing antibodies specific to Dengue3 NS1 Antigen will be blocked by the addition of Conjugate C, thus will show an absorbance less than the sample control well (wells with the addition of Conjugate A).

Example 1

[0252] Double Antigen Bridging Assay (DABA) with the Addition of Cold Zika NS1 and Dengue3 NS1 Antigen for the Identification and Blocking of Heterologous Antibodies to Zika Virus NS1 Glycoprotein

[0253] The experiment carried out in this example followed the DABA methods provided above. [0254] MyBiosource Recombinant Antigen Zika NS1 Coated Plate @ 2 g/ml [0255] Zika NS1 Ag Conjugate (i.e. labelled antigen) to be used at WORKING DILUTION OF 1:40K in Diasorin Conjugate Diluent [0256] Conjugate ONEstored in Stabiliser at 1:10 Dilution (once removed from 20 C. kept at 4 C.) [0257] Make 1 ml of 1:40K (e.g. 1:40,000) conjugate for strip ONE and 2 ml of 1:20K for strips 2-3 [0258] ZIKA NS1 Ag diluted to 1:68 in Diasorin Conjugate Diluent=50 g/ml (unlabelled antigen) [0259] Dengue 3 NS1 Ag diluted to 1:10 in Diasorin Conjugate Diluent=49 g/ml (unlabelled antigen) [0260] Positive Control=ZKA35 diluted 1:10K in NHP (0.356 g/ml) [0261] Negative Control=Normal human plasma samples (English blood donors)

[0262] Protocol: [0263] Add 70 l of Sample Diluent followed by 30 l of Sample to relevant wells [0264] Incubate for 1 hour at 37 C. [0265] Wash 5 in CLIN TECH Wash Buffer [0266] Add 100 l of 1:40K Conjugate to strip 1 [0267] Add 50 l of 1:20KConjugate+50 l of various dilutions of NS1 Antigen OR Dengue 3 Antigen to relevant strips [0268] Incubate for 2 hours at 37 C. [0269] Wash 5 in CLIN TECH Wash Buffer [0270] Add 100 l of Ready to Use TMB Substrate [0271] Incubate for 30 minutes at 37 C. [0272] Add 50 l CLIN TECH stop solution

[0273] The plate was arranged as follows:

TABLE-US-00004 ZIKA NS1-50 l of D3 NS1-50 l of Antigen NONE 50 ug/ml 49 ug/ml Addition 100 l of 1:40K 50 l of 1:20K 50 l of 1:20K Conjugate 1 2 3 A ZK35 (0.356 ZK35 (0.356 ZK35 (0.356 g/ml) (positive g/ml) g/ml) control) (positive control) (positive control) B ZK35 (0.356 g/ml) (positive ZK35 (0.356 g/ml) ZK35 (0.356 g/ml) control) (positive control) (positive control) C NHP (negative NHP (negative NHP (negative control) control) control) D NHP (negative NHP (negative NHP (negative control) control) control) E RS 1700 0093 RS 1700 0093 RS 1700 0093 F RS 1600 5394 RS 1600 5394 RS 1600 5394 G RS 1600 2063 RS 1600 2063 RS 1600 2063 H RS 1600 2616 RS 1600 2616 RS 1600 2616

[0274] Samples E-H are from individuals who have previously been diagnosed to have active ZIKA infection through the demonstration of ZIKA PCR reactive plasma.

[0275] The OD measurements at 450/630 nm are presented in Table 1 below:

TABLE-US-00005 Raw OD read at 450/630 nm 1 2 3 A 1.158 0.018 1.024 B 1.004 0.026 0.906 C 0.048 0.022 0.043 D 0.050 0.024 0.049 E 0.960 0.060 0.478 F 3.520 0.160 3.367 G 2.698 0.088 2.571 H 1.068 0.044 0.957 Pos CTL Mean 1.081 0.022 0.965 Neg CTL Mean 0.049 0.023 0.046 Cut-Off 0.149 0.123 0.146 (positive results are underlined and italicised)

[0276] The % inhibition was calculated and is presented in Table 2 below:

TABLE-US-00006 Inhibition Percentage by Excess Antigen (50 g/ml) Zika NS1 Dengue 3 NS1 Positive Control 98% 11% Negative Control N/A N/A N/A N/A RS 1700 0093 94% 50% RS 1600 5394 95% 4% RS 1600 2063 97% 5% RS 1600 2547 96% 10%

[0277] In this table are shown the percentage of the signal reductions displayed in the assay when the quench of each sample's reactivity is either ZIKA NS1 antigen or dengue 3 NS1 antigen. As can be seen there is virtually complete ablation of the signal by the ZIKA antigen indicating that all the moieties that are causing the labelled ZIKA NS1 antigen to bind to the solid-phase are blocked by ZIKA. In comparison in only one of the four convalescent plasmas, RS 1700 0093 does the dengue antigen cause reduction of the signal, in this case by only 50% which indicates that the response to the ZIKA virus infection in this particular individual comprises ZIKA specific antibody of which a component is also present on dengue 3 NS1. A response following ZIKA infection in a patient who has also previously been infected by dengue 3 will comprise specific ZIKA antibody, a component of which will also be displayed on the dengue 3 NS1 antigen.

Example 2

[0278] Immunoglobulin G (IgG) Capture Enzyme Immunoassay with the Addition of Cold Zika NS1 and Dengue3 NS1 Antigen for the Identification and Blocking of Heterologous Antibodies to Zika Virus NS1 Glycoprotein

[0279] The experiment carried out in this example followed the IgG Capture Enzyme Immunoassay methods provided above. Specifically, the following were used: [0280] AffiniPure Rabbit anti human IgG Coated Plate @ 5 g/ml [0281] MyBiosource Zika NS1 Antigen Conjugate to be used at a working dilution of 1:4K (e.g. 1:1000) in Transport Medium (labelled antigen) [0282] Conjugate ONEstored in Stabiliser (CLIN TECH) at 1:10 Dilution (once removed from 20 C. kept at 4 C.) [0283] Positive Control=ZKA35 mAb to be used at 1:1K (e.g. 1:1000) dilution in NHP (3.56 g/ml) [0284] Negative Control=NHP [0285] ZIKA NS1 Ag diluted to 1:68 in Transport Medium=50 g/ml (unlabelled antigen) [0286] Dengue 3 NS1 Ag diluted to 1:10 in Transport Medium=49 g/ml (unlabelled antigen)

[0287] Protocol: [0288] Add 100 l of Samples and Controlsdiluted 1:200 in Transport Medium prior to addition to wells [0289] Incubate for 1 hour at 37 C. [0290] Wash 5 in CLIN TECH Wash Buffer [0291] Add 100 l of 1:4K Conjugate to strip 1 [0292] Add 50 l of ZIKA NS1 Antigen followed by 50 l of 1:2KConjugate to strip 2 [0293] Add 50 l of DENGUE 3 NS1 Antigen followed by 50 l of 1:2KConjugate to strip 3 [0294] Incubate for 30 minutes at 37 C. [0295] Wash 5 in CLIN TECH Wash Buffer [0296] Add 100 l of Ready to Use TMB Substrate [0297] Incubate for 30 minutes at 37 C. [0298] Add 50 l CLIN TECH stop solution

[0299] The plate was arranged as follows:

TABLE-US-00007 Zika NS1-50 l of D3 NS1-50 l of Antigen NONE 50 ug/ml 49 ug/ml Addition 100 l of 1:4K 50 l of 1:2K 50 l of 1:2K Conjugate 1 2 3 A ZK35 1:1K ZK35 1:1K ZK35 1:1K (3.56 g/ml) (3.56 g/ml) (3.56 g/ml) (positive control) (positive control) (positive control) B ZK35 1:1K ZK35 1:1K ZK35 1:1K (3.56 g/ml) (3.56 g/ml) (3.56 g/ml) (positive control) (positive control) (positive control) C NHP (negative NHP (negative NHP (negative control) control) control) D NHP (negative NHP (negative NHP (negative control) control) control) E RS 1700 0093 RS 1700 0093 RS 1700 0093 F RS 1600 5394 RS 1600 5394 RS 1600 5394 G RS 1600 2063 RS 1600 2063 RS 1600 2063 H RS 1600 2547 RS 1600 2547 RS 1600 2547

[0300] Samples E-H are from individuals who have previously been diagnosed to have active ZIKA PG-8T infection through the demonstration of ZIKA PCR reactive plasma.

[0301] The OD measurements at 450/630 nm are presented in Table 3 below:

TABLE-US-00008 Raw OD read at 450/630 nm 1 2 3 A 1.588 0.054 1.560 B 1.615 0.051 1.555 C 0.064 0.050 0.048 D 0.058 0.042 0.048 E 0.280 0.056 0.106 F 1.671 0.111 0.871 G 0.384 0.051 0.351 H 0.541 0.061 0.496 Pos CTL Mean 1.602 0.053 1.558 Neg CTL Mean 0.061 0.046 0.048 Cut-Off 0.161 0.146 0.148 (positive results above the cut-off value are underlined and italicised)

[0302] The % inhibition was calculated and is presented in Table 4 below:

TABLE-US-00009 Inhibition Percentage by Excess Antigen (50 g/ml) Zika NS1 Dengue 3 NS1 Positive Control 97% 3% Negative Control N/A N/A N/A N/A RS 1700 0093 80% 62% RS 1600 5394 93% 48% RS 1600 2063 87% 9% RS 1600 2547 89% 8%

[0303] These data demonstrate that the addition of the ZIKA NS1 antigen in the blocking step quenches the signal generated by convalescent plasma in the G capture assay and that a component of the signal is also carried on dengue 3 NS1 antigen but that the inhibition is best achieved by the ZIKA NS1 antigen.

Example 3

[0304] Demonstration of Suppression of Antigenic Binding Cross-Reactivity Towards Dengue Virus Antigen in Samples Derived from ZIKA Patients

[0305] This example relates to the performance of an assay of the invention on a set of samples from subjects undergoing acute ZIKA infection. Said subjects were confirmed to have a ZIKA infection by PCR and their subsequent follow-up. In this experiment, conducted early in the field, the reactivity of the subject samples (sera) was measured in the DABA capture assay in two formats(i) using the unblocked conjugate format where the conjugate contained only HRPO-labelled ZIKA (i.e. absent a quenching step) or (ii) using the blocked format where the immobilised antibodies having cross-resistance to dengue are blocked/quenched (i.e. using the conjugate modified by addition of quenching dengue virus NS1 antigen)see FIG. 1.

[0306] For comparative purposes, the same samples were testing in an IgG capture assay in both the unblocked conjugate and blocked formatssee FIG. 2.

[0307] For both the DABA and IgG capture assays, a significant decrease in the reactivity of samples containing cross-reactive antibodies was observed in the blocked format. This demonstrates successful application of the invention in the suppression of antigenic binding cross-reactivity towards dengue virus antigen in samples derived from ZIKA patients.

Example 4

Demonstration of Prevention of False-Positive ZIKA Virus Detection in Patient Samples

[0308] A set of 20 patient samples (sera) was assembled, which were demonstrated previously to have strong reactivity for anti-dengue virus antibody, and that were also reactive in both the DABA and IgG capture assays for the detection of anti-ZIKA antibodies.

[0309] Panel samples were tested in an IgG capture assay as before (for detection of ZIKA) in both the unblocked conjugate format (containing only HRPO labelled ZIKA NS1 antigen) or the blocked format (containing a quenching mixture of Dengue types 1-4 inclusive NS1 antigen)see FIG. 3.

[0310] For comparative purposes, the same samples were testing in a DABA assay in both the unblocked conjugate and blocked formatssee FIG. 4.

[0311] In both assays, control samples were included from patients recovering from proven ZIKA infection (circled).

[0312] For both the DABA and IgG capture, a significant decrease in the reactivity of the samples was observed in the blocked format. This demonstrates successful application of the invention in preventing false-positive detection of ZIKA samples.

[0313] Importantly, the detection of genuine ZIKA infection was not impeded, demonstrating the utility of the invention in the detection of ZIKA while preventing false-positive detection.

[0314] One of the principal concerns has been whether the blocking of spurious reactivity in a conjugate can have an impact on the sensitivity of an assay for detecting antibody to ZIKA. In both the DABA and the IgG capture assay the addition of ZIKA antigen reveals that some sera from patients undergoing PCR proven ZIKA virus infection clearly have pre-existing dengue Ab, not surprising given the co-circulation of these viruses in endemic countries. Data in both FIG. 1 and FIG. 2 demonstrate that there are sera whose reactivity is particularly reduced by the addition of blocking dengue antigen. Given that antibody to dengue is very common it is not a surprising observation.

[0315] In a subsequent analysis, using a quadrivalent dengue antigen pool to provide the blocking activity it can be seen that in the IgG capture assay the reactivity of all but two of the cross-reactive false positive samples is entirely quenched (FIG. 3). Interestingly the low level control positive ZIKA antibody-containing samples remain clearly detectable. In the DABA assay the false reactivity of all 20 samples is quenched with the control positive samples clearly visible.

[0316] These data support the use of a quenching component in the blocked conjugate which preserves the ability to detect ZIKA specific antibody whilst preventing spurious cross reactivity.

[0317] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.