US20230072761A1 - Immunoassay for Detecting Zika Virus Infection - Google Patents
Immunoassay for Detecting Zika Virus Infection Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
- G01N2333/185—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a method for detecting Zika virus infection in a biological sample from a subject.
- the biological sample is preferably blood, serum, plasma, cerebrospinal fluid, saliva or urine.
- Zika virus Zika virus
- Zika virus is a mosquito-borne flavivirus in the family Flaviviridae and is closely related to dengue, yellow fever, Japanese encephalitis, and West Nile viruses. It is primarily transmitted by Aedes mosquitoes but can also be sexually transmitted. While most people infected with Zika virus do not have symptoms, Zika virus infection during pregnancy has been linked to microcephaly and other neurological complications. Detection of IgM antibodies to Zika virus provides an important tool for diagnosis and follow up care for an acute or recent Zika virus infection. Virus-specific IgM antibodies are typically present after the first week of illness and may be detectable for up to 12 weeks.
- Zika virus is a membrane-enveloped virus with an icosahedral capsid structure and a single positive-sense RNA genome.
- the RNA genome encodes three structural proteins and seven nonstructural (NS) proteins.
- One of the structural proteins is the envelope (E) protein that encapsulates the virus and is the main target of neutralizing antibodies.
- the nonstructural protein 1 (NS1) is secreted by infected cells as a hexameric lipoprotein particle and is involved in immune evasion and pathogenesis.
- NS1 is the only nonstructural protein secreted from infected cells and is another target of the immune response.
- Zika virus serological tests There exist several significant challenges in developing a diagnostic test for Zika virus IgM antibodies.
- the most common problem of Zika virus serological tests is the extensive cross-reactivity of antibodies elicited by this virus with other flaviviruses, such as dengue and West Nile virus, which share a high degree of sequence homology with Zika virus.
- the E glycoprotein is the most immunogenic viral antigen and it has been widely used in diagnostic tests for the detection of flavivirus-specific human IgM antibodies in order to maximize diagnostic sensitivity.
- the Centers for Disease Control and Prevention have developed an antibody capture enzyme-linked immunosorbent assay (Zika MAC-ELISA) for the qualitative detection of Zika virus IgM antibodies in serum or cerebrospinal fluid.
- Zika MAC-ELISA antibody capture enzyme-linked immunosorbent assay
- the Zika MAC-ELISA involves the use of anti-human IgM antibodies to bind the corresponding IgM antibodies of the sample.
- the detection of Zika-specific IgM is achieved by means of Zika E antigen along with an enzyme-conjugated anti-viral antibody.
- the InBios “ZIKV DetectTM IgM Capture ELISA” makes use of the Zika virus envelope glycoproteins in a reverse capture format to detect circulating IgM antibodies against these viral proteins.
- PRNT plaque-reduction neutralization testing
- the discrimination between positive and negative specimens involves a complex confirmative test, which includes comparing the Zika virus IgM values with sample reactivity measured versus non-specific antigens such as a Flavivirus common antigen and a mammalian cell common antigen.
- Antibodies directed against the viral NS1 protein show a reduced degree of cross-reactivity between different flaviviruses. Indeed, the comparison of NS1 protein from dengue, West Nile and Zika virus revealed diverse electrostatic characteristics in the loop surface interface, which may result in altered binding properties (Song et al. Nat Struct Mol Biol. 2016;23:456-458). Additionally, antibodies to NS1 from Zika virus infected patients were shown to be specific for Zika virus while antibodies against the E protein are highly cross-reactive to Dengue virus NS1 (Stettler et al. Science. 2016 Aug. 19;353(6301):823-6).
- Anti-Zika virus ELISA provided by Euroimmun a recombinant Zika NS1 protein is used as antigen component in an indirect IgM capture ELISA.
- serological tests for Zika virus face the challenge of detecting IgM antibodies in patients with previous flavivirus exposure, as these subjects often exhibit a weak IgM response to Zika virus infection even in the acute phase. Seroconversion rates of IgM and IgG are different between primary and secondary dengue virus infections. During a secondary infection, patients often exhibit a weaker and shorter IgM response along with a stronger IgG response than during a primary dengue infection. A similar response occurs in patients with a Zika virus infection who have had previous flavivirus exposure.
- ZIKV Zika virus
- the detection method of the invention makes use of a diagnostic procedure that takes advantage of the strength of the IgM response to the more specific NS1 protein in primary infections and the strength of the IgG response in secondary infections. Further features and advantages of the method of the invention are defined in the dependent claims.
- the present invention relates to an in vitro method for the detection of Zika virus (ZIKV) infection in a biological sample from a subject, comprising the steps of:
- the method of the invention was designed to improve diagnostic accuracy of a recent Zika virus infection by combining the use of the more specific NS1 protein with a unique assay procedure.
- the method of the present invention relies on two distinct (separate) antibody capture immunoassays which are preferably carried out on the same biological fluid sample and aim at detecting Zika virus IgM and IgG antibodies, respectively, directed against the NS1 viral protein.
- the assessment of the presence or absence of Zika virus infection is determined according to the combined results provided by the Zika IgM and Zika IgG immunoassays.
- IgM-class and/or IgG-class antibodies present in the biological fluid sample are captured on solid surfaces coated with IgM binding molecules and IgG binding molecules, respectively.
- Antibodies against viral NS1 proteins are known to be present at low levels in serum after flavivirus infections, probably due to a relative abundance of antibodies to the premembrane (prM) and envelope (E) structural proteins of flaviviruses, thus resulting in lower sensitivity of NS1 isotype immunoassays.
- anti-NS1 IgM antibodies are in the minority population compared to anti-virus like particles (VLP) IgM antibodies, presumably 3- to 12-fold lower than anti-VLP antibodies levels in the human serum, depending on the timing of serum collection.
- VLP anti-virus like particles
- the anti-NS1 IgM antibodies do not have a sufficiently high titer to compete with anti-VLP IgM antibodies in IgM-capture assays.
- anti-Dengue IgM assays such a problem was addressed by including the depletion of anti-prM/E antibodies and a significant increase of the assay positive/negative (P/N) ratio value was achieved.
- the present inventors found that a IgM- or IgG capture surface on which antibody binding molecules are immobilized in an amount corresponding essentially to the amount of total IgM and IgG antibodies in the tested sample enable the detection of specific anti-NS1 IgM- or IgG antibodies also when present in very small amounts, thereby significantly improving the sensitivity of the diagnostic assay.
- the use of such a solid surface for capturing total IgM antibodies present in the tested sample avoids the need for any enrichment step of the fraction of specific anti-NS1 IgM antibodies, for example by depleting anti-VLP IgM antibodies as in the prior art assays.
- the use of the IgG capture surface in the method of the invention allows to capture all the tested IgG, thus being able to detect peak levels of Zika virus NS1-specific IgG antibodies, as it would only occur during a more recent infection.
- Zika virus NS1-specific IgM and IgG antibodies are captured on a solid surface by contacting a biological sample with IgM binding molecules and/or IgG binding molecules immobilized on the solid surface in an amount which corresponds substantially to the amount of total IgM and IgG antibodies contained in the tested biological sample.
- biological sample is intended to mean a pre-determined volume of the biological fluid used in the method of the invention.
- the biological fluid sample undergoes a dilution step before incubation with the IgG antibody capture surface.
- the sample dilution factor is comprised between 10-fold and 500-fold, preferably between 50-fold and 200-fold, more preferably it is of about 90-fold. The use of such a sample pre-dilution step ensures the detection of peak levels of IgG antibodies to Zika virus.
- a solid surface is used for capturing IgM- and/or IgG antibodies.
- suitable solid supports are the wells of a microtitre plate, the surface of a microparticle such as a latex, polystyrene, silica, chelating sepharose or magnetic beads, membranes, strips or chips.
- the solid support is a bead, more preferably a paramagnetic micro-particle (PMP).
- PMP paramagnetic micro-particle
- the IgM- and/or IgG-binding molecule may be covalently bound to the solid surface, or non-covalently attached through nonspecific bonding.
- the antibody binding protein immobilized on the solid surface is an anti-IgG antibody, an anti-IgM antibody, an immunoglobulin-binding peptide or a peptidomimetics.
- anti-IgM antibodies and anti-IgG antibodies are especially preferred, even more preferably monoclonal anti-IgM and anti-IgG antibodies.
- the biological fluid sample is preferably selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, urine or saliva.
- the biological fluid sample may optionally include further components, such as for example: diluents, preservatives, stabilizing agents and/or buffers. If needed, dilutions of the biological fluid sample are prepared using any suitable diluent buffer known in the art.
- the detection of Zika virus NS1-specific IgM and/or IgG antibodies captured on the solid surface is performed by employing the NS1 antigen, epitope or a fragment thereof.
- the NS1 protein used as the detection reagent is the whole recombinant protein; alternatively, it is an epitope or an immunogenic fragment of the Zika virus NS1 protein.
- the NS1 antigen is labeled with means capable of providing directly or indirectly a detectable signal.
- Suitable labels for use in the present invention include for example fluorescent compounds, chemiluminescent compounds, radio-active compounds, enzymes and enzyme substrates, molecules suitable for colorimetric detection, binding proteins, epitopes, enzymes or substrates.
- any signal molecule or label known in the art may be used in the method of the present invention.
- the means capable of indirectly providing a detectable signal comprise an antibody that binds to the antigen, for example an anti-NS1 antibody. Such antibody may be provided with direct or indirect label means as described above for the antigen.
- the Zika IgM and Zika IgG immunoassays comprise i) contacting the biological sample with IgM binding molecules coated on a solid surface in an amount corresponding substantially to the amount of IgM antibodies in said sample, thereby capturing the IgM antibodies in the sample, ii) contacting the biological sample with IgG binding molecules coated on a solid surface in an amount corresponding substantially to the amount of IgG antibodies in said sample, thereby capturing the IgG antibodies in the sample, and iii) detecting captured ZIKV IgM antibodies and ZIKV IgG antibodies specific to ZIKV NS1 protein employing a labeled ZIKV NS1 antigen, epitope or fragment thereof.
- the diagnostic assessment of the patient's sample relies upon the intensity of the respective signals obtained from the ZIKV IgM- and ZIKV IgG-assays.
- an automatic analyzer is used which extrapolates the signal intensities on calibration standard curves to obtain a IgM index and a IgG index. Then, the analyzer combines the two index values to produce a single result.
- the calibration standard curve may be generated, for example, by using human serum or defribinated plasma samples containing known amounts of Zika virus IgM or Zika virus IgG.
- the Zika IgM test consists of both a high and a low cut-off value (threshold) for the IgM index. Specimens below the low cut-off value are considered Zika IgM negative and specimens above the high cut-off value are considered Zika IgM positive. Specimens between the two cut-off values are resolved by the result of the Zika IgG test. Stated another way, diagnostic sensitivity is achieved through the use of a low cut-off value for the Zika IgM test only in the presence of detectable Zika IgG antibodies. In the absence of detectable Zika IgG antibodies, a higher cut-off value for the Zika IgM test is used thereby maximizing specificity in non-infected individuals.
- the Zika IgG test helps to differentiate a true negative specimen from a specimen exhibiting a weak IgM response due to a secondary flavivirus infection.
- the diagnostic assessment procedure maximizes diagnostic sensitivity and specificity. This approach is particularly effective in endemic regions because of the use of an IgG assay that only detects peak levels of IgG during a recent infection.
- Subjects without prior exposure to flaviviruses exhibit a strong IgM response following infection with Zika virus and are detected using the higher cut-off value for the Zika IgM test regardless of the Zika IgG result.
- Subjects who have had previous exposure to another flavivirus may exhibit a weak IgM response but a strong IgG response and are therefore detected through the use of the low cut-off value.
- MyOne paramagnetic-particles were coated at 0.5% with 30 ⁇ g/mg of ⁇ -human IgM antibody or ⁇ -human IgG antibody using a sodium sulfate coating buffer (1.6 mM Sodium Phosphate Monobasic, 18.3 mM Sodium Phosphate Dibasic and 9% Sodium Sulfate at pH 8.2).
- the PMPs were blocked with blocking buffer containing 0.5% BSA, and diluted to a final bead concentration of 3.75 mg/ml (0.375% particles) in storage buffer(1.8 mM Potassium Phosphate Monobasic, 10 mM Sodium Phosphate Dibasic, 114 mM Sodium Chloride, 2.7 mM Potassium Chloride, 0.05% Tween 20, 0.1% Bovine Serum Albumin, and 0.09% Sodium Azide at pH 7.4).
- a full length recombinant Zika virus NS1 antigen was buffer exchanged and conjugated at a target of 1X molar excess with cyclic N-(4-aminobutyl)-N-ethylisoluminol (ABEI) in PBS buffer pH 7.4.
- the conjugated antigen was then purified,diluted at a target concentration of 12.5 ⁇ g/ml in conjugate diluent (100 mM NaCl, 0.1% Tween-20, 2 ml/L ProClin, 1% BSA, 3% D-Mannitol, pH 7.4) and lyophilized.
- conjugate diluent 100 mM NaCl, 0.1% Tween-20, 2 ml/L ProClin, 1% BSA, 3% D-Mannitol, pH 7.4
- the lyophilized conjugate was reconstituted in diH 2 O and used on the ancillary bay of the analyzer.
- the assay aims at the detection of IgM antibodies against Zika virus NS1 present in serum samples, calibrators and controls.
- the calibrators and controls were composed of anti-Zika IgM positive serum diluted with defibrinated/delipidated serum.
- the assay was carried out on the DiaSorin LIAISON® XL analyzer.
- 20 ⁇ l of anti-IgM coated magnetic particles were dispensed into a reaction cuvette and incubated with a 10 ⁇ l serum sample or control sample for 36 cycles (equivalent to a total of 12 minutes and 36 seconds). Unbound material was then removed by a washing step with Wash/System liquid.
- a total of 40 ⁇ l of cABEI-conjugate NS1 antigen and 250 ⁇ l assay buffer (100 mM NaCl, 0.1% Tween 20, 3.0% Bovine Serum Albumin, and 0.2% ProClin 300 at pH 7.4) were dispensed in the reaction cuvette, and a second incubation and washing step were carried out as above-described. After this second wash, Starter Reagents were added and a flash chemiluminescence reaction was induced. The light signal, and hence the amount of isoluminol-antigen conjugate, was measured by a photomultiplier as relative light units (RLU) in the analyzer reading chamber. Measured values were indicative of the presence of IgM antibodies to Zika virus NS1 in patient sera, calibrators, or controls.
- RLU relative light units
- the above-described assay steps were carried out on an automated instrument.
- the assay was calibrated using two point calibrators to obtain an instrument working curve based upon the assay master curve. Sample RLU values were then automatically converted to an Index value based on the instrument working curve.
- the assay aims at the detection of IgG antibodies against Zika virus NS1 present in serum samples, calibrators and controls.
- the calibrators and controls were composed of anti-Zika IgG positive serum diluted with defibrinated/delipidated serum.
- the assay was carried out on the DiaSorin LIAISON® XL analyzer.
- the DiaSorin LIAISON® XL analyzer automatically calculates an Index value for both the ZIKV-M and ZIKV-C reagent packs based on each individual calibration. The analyzer then automatically combines the two Index values to produce a single result. Table 1 below shows the results obtained from examples 3 and 4, expressed in terms of M Index and C Index values, and corresponding diagnostic assessment.
- the diagnostic specificity of the Zika Capture IgM assay of the invention was assessed by evaluating negative agreement using serum samples from 218 apparently healthy donors and 32 pregnant donors collected in the United States and presumed negative for Zika virus infection. The results of the evaluation are reported in Table 4 below. Negative percent agreement was found to be 99.6%.
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Abstract
The present invention relates to a method for detecting Zika vims (ZIKV) infection in a biological sample from a subject. The method comprises testing the sample for IgM- and IgG-ZIKV NSI antibodies and determining the ZIKV IgM and ZIKV IgG signal intensities; and scoring the sample as positive or negative for ZIKV infection based on the combined results of such determinations. The biological sample is preferably blood, serum, plasma, cerebrospinal fluid, saliva or urine.
Description
- This application is a continuation application of, and claims priority to U.S. patent application Ser. No. 16/494,913 (filed on Sep. 17, 2019; pending), which application is a § 371 National Stage Application of PCT/EP2018/056990 (filed on Mar. 20, 2018; now expired), which application claims benefit of U.S. Patent Application Ser. No. 62/473,548 (filed on Mar. 20, 2017; now expired). Each of these applications is hereby incorporated by reference in its entirety.
- The present invention relates to a method for detecting Zika virus infection in a biological sample from a subject. The biological sample is preferably blood, serum, plasma, cerebrospinal fluid, saliva or urine.
- Zika virus (ZIKV) is a mosquito-borne flavivirus in the family Flaviviridae and is closely related to dengue, yellow fever, Japanese encephalitis, and West Nile viruses. It is primarily transmitted by Aedes mosquitoes but can also be sexually transmitted. While most people infected with Zika virus do not have symptoms, Zika virus infection during pregnancy has been linked to microcephaly and other neurological complications. Detection of IgM antibodies to Zika virus provides an important tool for diagnosis and follow up care for an acute or recent Zika virus infection. Virus-specific IgM antibodies are typically present after the first week of illness and may be detectable for up to 12 weeks.
- Similar to other flaviviruses, Zika virus is a membrane-enveloped virus with an icosahedral capsid structure and a single positive-sense RNA genome. The RNA genome encodes three structural proteins and seven nonstructural (NS) proteins. One of the structural proteins is the envelope (E) protein that encapsulates the virus and is the main target of neutralizing antibodies. The nonstructural protein 1 (NS1) is secreted by infected cells as a hexameric lipoprotein particle and is involved in immune evasion and pathogenesis. NS1 is the only nonstructural protein secreted from infected cells and is another target of the immune response.
- There exist several significant challenges in developing a diagnostic test for Zika virus IgM antibodies. The most common problem of Zika virus serological tests is the extensive cross-reactivity of antibodies elicited by this virus with other flaviviruses, such as dengue and West Nile virus, which share a high degree of sequence homology with Zika virus.
- Commercially available assays for the serological diagnosis of Zika virus infections typically rely on the use of ZIKV envelope glycoproteins, such as the E glycoprotein, or NS1 protein as antigen reagents.
- The E glycoprotein is the most immunogenic viral antigen and it has been widely used in diagnostic tests for the detection of flavivirus-specific human IgM antibodies in order to maximize diagnostic sensitivity.
- The Centers for Disease Control and Prevention (CDC) have developed an antibody capture enzyme-linked immunosorbent assay (Zika MAC-ELISA) for the qualitative detection of Zika virus IgM antibodies in serum or cerebrospinal fluid. The Zika MAC-ELISA involves the use of anti-human IgM antibodies to bind the corresponding IgM antibodies of the sample. The detection of Zika-specific IgM is achieved by means of Zika E antigen along with an enzyme-conjugated anti-viral antibody.
- Also the InBios “ZIKV Detect™ IgM Capture ELISA” makes use of the Zika virus envelope glycoproteins in a reverse capture format to detect circulating IgM antibodies against these viral proteins.
- However, human antibodies against the E glycoprotein are cross reactive among flavivirus species. Assays which employ such protein as antigen component reportedly exhibit a high level of cross reactivity to other flaviviruses and usually require several additional calculations and laborious and expensive confirmatory retesting for the final reporting.
- To minimize the likelihood of false-positive results, presumed positive or equivocal assay results obtained with the CDC “Zika MAC-ELISA” assay require confirmation by plaque-reduction neutralization testing (PRNT). PRNT is a labor-intensive procedure and not readily amenable to high throughput, making it difficult to use for large-scale surveillance and vaccine trials.
- In the Inbios assay, the discrimination between positive and negative specimens involves a complex confirmative test, which includes comparing the Zika virus IgM values with sample reactivity measured versus non-specific antigens such as a Flavivirus common antigen and a mammalian cell common antigen.
- Antibodies directed against the viral NS1 protein show a reduced degree of cross-reactivity between different flaviviruses. Indeed, the comparison of NS1 protein from dengue, West Nile and Zika virus revealed diverse electrostatic characteristics in the loop surface interface, which may result in altered binding properties (Song et al. Nat Struct Mol Biol. 2016;23:456-458). Additionally, antibodies to NS1 from Zika virus infected patients were shown to be specific for Zika virus while antibodies against the E protein are highly cross-reactive to Dengue virus NS1 (Stettler et al. Science. 2016 Aug. 19;353(6301):823-6).
- In the “Anti-Zika virus ELISA” provided by Euroimmun a recombinant Zika NS1 protein is used as antigen component in an indirect IgM capture ELISA.
- However, immunoassay methods which make use of the NS1 protein as antigen reagent have demonstrated poor diagnostic sensitivity, due to a significant prevalence of false-negative results, leading to risks and uncertainties.
- Besides the risk of either cross-reactivity or low sensitivity, serological tests for Zika virus face the challenge of detecting IgM antibodies in patients with previous flavivirus exposure, as these subjects often exhibit a weak IgM response to Zika virus infection even in the acute phase. Seroconversion rates of IgM and IgG are different between primary and secondary dengue virus infections. During a secondary infection, patients often exhibit a weaker and shorter IgM response along with a stronger IgG response than during a primary dengue infection. A similar response occurs in patients with a Zika virus infection who have had previous flavivirus exposure. Patients without prior flavivirus exposure are expected to produce a typical seroconversion profile following Zika virus infection, while patients who have had previous flavivirus exposure exhibit a shorter and weaker IgM response. Correspondingly, these patients also exhibit a much stronger IgG response. This is particularly relevant as Zika virus is widely circulating in areas endemic for other flaviviruses thereby posing a significant challenge for serodiagnostics.
- Thus, there exists a need in the art to develop a method for the diagnosis of Zika virus infection which overcomes the drawbacks and limitations of the prior art.
- In particular, there is a need for an immunological method which would enable the specific and reliable detection of Zika virus infection without exhibiting substantial cross-reactivity with antibodies triggered by different flavivirus infections.
- There is also a need for an immunological method with improved diagnostic sensitivity of Zika virus infections in subjects who are likely to have a high background exposure to other related flaviviruses.
- These and other needs are met by an in vitro method for the detection of Zika virus (ZIKV) infection as defined in appended claim 1.
- The detection method of the invention makes use of a diagnostic procedure that takes advantage of the strength of the IgM response to the more specific NS1 protein in primary infections and the strength of the IgG response in secondary infections. Further features and advantages of the method of the invention are defined in the dependent claims.
- The present invention relates to an in vitro method for the detection of Zika virus (ZIKV) infection in a biological sample from a subject, comprising the steps of:
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- a) testing the sample for IgM antibodies specific to ZIKV NS1 protein or an epitope or immunogenic fragment thereof and determining the ZIKV IgM signal intensity;
- b) testing the sample for IgG antibodies to ZIKV NS1 protein or an epitope or immunogenic fragment thereof and determining the ZIKV IgG signal intensity;
- c) establishing a first ZIKV IgM signal intensity threshold and a second ZIKV IgM signal intensity threshold, wherein the second ZIKV IgM signal intensity threshold is higher than the first ZIKV IgM signal intensity threshold;
- d) establishing at least a third ZIKV IgG signal intensity threshold;
- e) detecting the presence or absence of ZIKV infection in the sample according to the following criteria:
- i) a ZIKV IgM signal intensity lower than the first ZIKV IgM signal intensity threshold is indicative of the absence of ZIKA infection;
- ii) a ZIKV IgM signal intensity higher than the second ZIKV IgM signal intensity threshold is indicative of the presence of ZIKA infection;
- iii) when the ZIKV IgM signal intensity is higher than the first ZIKV IgM signal intensity threshold and lower than or equal to the second ZIKV IgM signal intensity threshold:
- iiia) a ZIKV IgG signal intensity lower than the at least third ZIKV IgG signal intensity threshold is indicative of the absence of a ZIKV infection; and
- iiib) a ZIKV IgG signal intensity higher than the at least third ZIKV IgG signal intensity threshold is indicative of the presence of a ZIKV infection.
- As illustrated below, the method of the invention was designed to improve diagnostic accuracy of a recent Zika virus infection by combining the use of the more specific NS1 protein with a unique assay procedure.
- The inventors found that the use of the NS1 protein as the detection reagent confers Zika virus specificity against other flaviviruses, while the assay procedure maximizes diagnostic sensitivity in subjects with previous flavivirus exposure and improves specificity in non-infected individuals.
- In one embodiment, the method of the present invention relies on two distinct (separate) antibody capture immunoassays which are preferably carried out on the same biological fluid sample and aim at detecting Zika virus IgM and IgG antibodies, respectively, directed against the NS1 viral protein. The assessment of the presence or absence of Zika virus infection is determined according to the combined results provided by the Zika IgM and Zika IgG immunoassays.
- In a preferred embodiment of the method of the invention, IgM-class and/or IgG-class antibodies present in the biological fluid sample are captured on solid surfaces coated with IgM binding molecules and IgG binding molecules, respectively.
- Antibodies against viral NS1 proteins are known to be present at low levels in serum after flavivirus infections, probably due to a relative abundance of antibodies to the premembrane (prM) and envelope (E) structural proteins of flaviviruses, thus resulting in lower sensitivity of NS1 isotype immunoassays. For instance, in acute dengue serum specimens, anti-NS1 IgM antibodies are in the minority population compared to anti-virus like particles (VLP) IgM antibodies, presumably 3- to 12-fold lower than anti-VLP antibodies levels in the human serum, depending on the timing of serum collection. Consequently, the anti-NS1 IgM antibodies do not have a sufficiently high titer to compete with anti-VLP IgM antibodies in IgM-capture assays. In prior art anti-Dengue IgM assays, such a problem was addressed by including the depletion of anti-prM/E antibodies and a significant increase of the assay positive/negative (P/N) ratio value was achieved.
- The present inventors found that a IgM- or IgG capture surface on which antibody binding molecules are immobilized in an amount corresponding essentially to the amount of total IgM and IgG antibodies in the tested sample enable the detection of specific anti-NS1 IgM- or IgG antibodies also when present in very small amounts, thereby significantly improving the sensitivity of the diagnostic assay. As an additional advantage, the use of such a solid surface for capturing total IgM antibodies present in the tested sample avoids the need for any enrichment step of the fraction of specific anti-NS1 IgM antibodies, for example by depleting anti-VLP IgM antibodies as in the prior art assays. At the same time, the use of the IgG capture surface in the method of the invention allows to capture all the tested IgG, thus being able to detect peak levels of Zika virus NS1-specific IgG antibodies, as it would only occur during a more recent infection.
- Therefore, in a preferred embodiment of the method of the invention, Zika virus NS1-specific IgM and IgG antibodies are captured on a solid surface by contacting a biological sample with IgM binding molecules and/or IgG binding molecules immobilized on the solid surface in an amount which corresponds substantially to the amount of total IgM and IgG antibodies contained in the tested biological sample.
- In the context of the present disclosure, the expression “biological sample” is intended to mean a pre-determined volume of the biological fluid used in the method of the invention.
- In a more preferred embodiment, the biological fluid sample undergoes a dilution step before incubation with the IgG antibody capture surface. Preferably, the sample dilution factor is comprised between 10-fold and 500-fold, preferably between 50-fold and 200-fold, more preferably it is of about 90-fold. The use of such a sample pre-dilution step ensures the detection of peak levels of IgG antibodies to Zika virus.
- In the method of the invention, a solid surface is used for capturing IgM- and/or IgG antibodies. Non limiting examples of suitable solid supports are the wells of a microtitre plate, the surface of a microparticle such as a latex, polystyrene, silica, chelating sepharose or magnetic beads, membranes, strips or chips. Preferably, the solid support is a bead, more preferably a paramagnetic micro-particle (PMP). The IgM- and/or IgG-binding molecule may be covalently bound to the solid surface, or non-covalently attached through nonspecific bonding.
- According to an embodiment of the invention, the antibody binding protein immobilized on the solid surface is an anti-IgG antibody, an anti-IgM antibody, an immunoglobulin-binding peptide or a peptidomimetics. Especially preferred are anti-IgM antibodies and anti-IgG antibodies, even more preferably monoclonal anti-IgM and anti-IgG antibodies.
- In the context of the present invention, the biological fluid sample is preferably selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, urine or saliva. The biological fluid sample may optionally include further components, such as for example: diluents, preservatives, stabilizing agents and/or buffers. If needed, dilutions of the biological fluid sample are prepared using any suitable diluent buffer known in the art.
- According to the method of the present invention, the detection of Zika virus NS1-specific IgM and/or IgG antibodies captured on the solid surface is performed by employing the NS1 antigen, epitope or a fragment thereof.
- In a preferred embodiment of the invention, the NS1 protein used as the detection reagent is the whole recombinant protein; alternatively, it is an epitope or an immunogenic fragment of the Zika virus NS1 protein.
- In one embodiment of the invention, the NS1 antigen is labeled with means capable of providing directly or indirectly a detectable signal. Suitable labels for use in the present invention include for example fluorescent compounds, chemiluminescent compounds, radio-active compounds, enzymes and enzyme substrates, molecules suitable for colorimetric detection, binding proteins, epitopes, enzymes or substrates. In practice, any signal molecule or label known in the art may be used in the method of the present invention. In another embodiment, the means capable of indirectly providing a detectable signal comprise an antibody that binds to the antigen, for example an anti-NS1 antibody. Such antibody may be provided with direct or indirect label means as described above for the antigen.
- In still yet another embodiment, the Zika IgM and Zika IgG immunoassays comprise i) contacting the biological sample with IgM binding molecules coated on a solid surface in an amount corresponding substantially to the amount of IgM antibodies in said sample, thereby capturing the IgM antibodies in the sample, ii) contacting the biological sample with IgG binding molecules coated on a solid surface in an amount corresponding substantially to the amount of IgG antibodies in said sample, thereby capturing the IgG antibodies in the sample, and iii) detecting captured ZIKV IgM antibodies and ZIKV IgG antibodies specific to ZIKV NS1 protein employing a labeled ZIKV NS1 antigen, epitope or fragment thereof.
- The diagnostic assessment of the patient's sample relies upon the intensity of the respective signals obtained from the ZIKV IgM- and ZIKV IgG-assays. In a specific embodiment, an automatic analyzer is used which extrapolates the signal intensities on calibration standard curves to obtain a IgM index and a IgG index. Then, the analyzer combines the two index values to produce a single result. In the method of the invention, the calibration standard curve may be generated, for example, by using human serum or defribinated plasma samples containing known amounts of Zika virus IgM or Zika virus IgG.
- The Zika IgM test consists of both a high and a low cut-off value (threshold) for the IgM index. Specimens below the low cut-off value are considered Zika IgM negative and specimens above the high cut-off value are considered Zika IgM positive. Specimens between the two cut-off values are resolved by the result of the Zika IgG test. Stated another way, diagnostic sensitivity is achieved through the use of a low cut-off value for the Zika IgM test only in the presence of detectable Zika IgG antibodies. In the absence of detectable Zika IgG antibodies, a higher cut-off value for the Zika IgM test is used thereby maximizing specificity in non-infected individuals. In this way, the Zika IgG test helps to differentiate a true negative specimen from a specimen exhibiting a weak IgM response due to a secondary flavivirus infection. As such, the diagnostic assessment procedure maximizes diagnostic sensitivity and specificity. This approach is particularly effective in endemic regions because of the use of an IgG assay that only detects peak levels of IgG during a recent infection.
- Subjects without prior exposure to flaviviruses exhibit a strong IgM response following infection with Zika virus and are detected using the higher cut-off value for the Zika IgM test regardless of the Zika IgG result. Subjects who have had previous exposure to another flavivirus may exhibit a weak IgM response but a strong IgG response and are therefore detected through the use of the low cut-off value.
- According to a specific embodiment of the invention, the following diagnostic assessment procedure is used:
-
- (i) IgM and IgG tests are run at the same time on a sample;
- (ii) samples with an IgM result below a first lower threshold are scored as “negative”;
- (iii) samples with an IgM result above a second higher threshold value are scored as “positive”;
- (iv) for samples with an IgM result between the lower threshold and higher threshold, the IgG result is examined and those samples with an IgG result above a third IgG threshold are scored as “positive”, while those with an IgG result below the third IgG threshold are scored as “negative”.
- The following alternative embodiments of the diagnostic assessment procedure are also within the scope of the invention, in that they are the same in principal and effect to the procedure described above:
-
- 1. A multilevel IgG procedure, in which the procedure is similar to that described above, but more than one IgG cutoff is applied to the IgM result, leading to additional levels of diagnosis in addition to “negative” and “positive” (for example: negative, possible positive, presumptive positive, positive).
- 2. A reflex procedure, by which IgM is run, and IgM results that are in the equivocal region (or gray zone or reflex zone) are reflexed to an IgG assay for “confirmation” (High IgG=positive confirmation, Low IgG=negative or unconfirmed.)
- 3. A retest-reflex procedure, by which IgM is run, equivocals are retested on IgM, repeat equivocals are reflexed to IgG for “confirmation” or “differentiation”.
- 4. A reverse order procedure, by which an IgG assay is run first, and the result of the IgG test determines to which of two possible IgM tests the sample is reflexed. A “negative”, or low, IgG result reflexes to a sensitive (low cut-off) IgM test, and a “positive”, or high, IgG result reflexes to an insensitive (high cut-off) IgM test.
- 5. A triple test, wherein IgA are tested in addition to IgM and IgG. The following examples are provided by way of illustration only and are not intended to limit the scope of the invention as defined in the appended claims.
- MyOne paramagnetic-particles (PMPs) were coated at 0.5% with 30 μg/mg of α-human IgM antibody or α-human IgG antibody using a sodium sulfate coating buffer (1.6 mM Sodium Phosphate Monobasic, 18.3 mM Sodium Phosphate Dibasic and 9% Sodium Sulfate at pH 8.2). The PMPs were blocked with blocking buffer containing 0.5% BSA, and diluted to a final bead concentration of 3.75 mg/ml (0.375% particles) in storage buffer(1.8 mM Potassium Phosphate Monobasic, 10 mM Sodium Phosphate Dibasic, 114 mM Sodium Chloride, 2.7 mM Potassium Chloride, 0.05% Tween 20, 0.1% Bovine Serum Albumin, and 0.09% Sodium Azide at pH 7.4).
- A full length recombinant Zika virus NS1 antigen was buffer exchanged and conjugated at a target of 1X molar excess with cyclic N-(4-aminobutyl)-N-ethylisoluminol (ABEI) in PBS buffer pH 7.4. The conjugated antigen was then purified,diluted at a target concentration of 12.5 μg/ml in conjugate diluent (100 mM NaCl, 0.1% Tween-20, 2 ml/L ProClin, 1% BSA, 3% D-Mannitol, pH 7.4) and lyophilized. Before use in the method of the invention, the lyophilized conjugate was reconstituted in diH2O and used on the ancillary bay of the analyzer.
- The assay aims at the detection of IgM antibodies against Zika virus NS1 present in serum samples, calibrators and controls. The calibrators and controls were composed of anti-Zika IgM positive serum diluted with defibrinated/delipidated serum. The assay was carried out on the DiaSorin LIAISON® XL analyzer. As an initial step, 20 μl of anti-IgM coated magnetic particles were dispensed into a reaction cuvette and incubated with a 10 μl serum sample or control sample for 36 cycles (equivalent to a total of 12 minutes and 36 seconds). Unbound material was then removed by a washing step with Wash/System liquid. Subsequently, a total of 40 μl of cABEI-conjugate NS1 antigen and 250 μl assay buffer (100 mM NaCl, 0.1% Tween 20, 3.0% Bovine Serum Albumin, and 0.2% ProClin 300 at pH 7.4) were dispensed in the reaction cuvette, and a second incubation and washing step were carried out as above-described. After this second wash, Starter Reagents were added and a flash chemiluminescence reaction was induced. The light signal, and hence the amount of isoluminol-antigen conjugate, was measured by a photomultiplier as relative light units (RLU) in the analyzer reading chamber. Measured values were indicative of the presence of IgM antibodies to Zika virus NS1 in patient sera, calibrators, or controls.
- The above-described assay steps were carried out on an automated instrument. The assay was calibrated using two point calibrators to obtain an instrument working curve based upon the assay master curve. Sample RLU values were then automatically converted to an Index value based on the instrument working curve.
- The assay aims at the detection of IgG antibodies against Zika virus NS1 present in serum samples, calibrators and controls. The calibrators and controls were composed of anti-Zika IgG positive serum diluted with defibrinated/delipidated serum. The assay was carried out on the DiaSorin LIAISON® XL analyzer. First, 15 μl of serum sample, calibrator or control were diluted in 130 μl specimen diluent (6.47 mM Sodium Phosphate dibasic, 1.18 mM Potassium Phosphate monobasic, 110 mM Sodium Chloride, 2.1 mM Potassium Chloride, 2.4 mM EDTA, 0.22% Tween 20, 0.28% Triton X705, 3.2% Bovine Serum Albumin and 0.2% Proclin 300 at pH 6.5). Next, 15 μl of diluted sample were diluted again in 130 μl specimen diluent. Then, 200 μl of specimen diluent along with 20 μl of anti-IgG coated magnetic particles were dispensed into a reaction cuvette, followed by the addition of 8 μl of diluted sample, calibrator or control. The mixture was subjected to a first incubation for 36 cycles (equivalent to a total of 12 minutes and 36 seconds). After a first washing step with Wash/System liquid in order to remove unbound material, 40 μl of cABEI-conjugate NS1 antigen and 250 μl assay buffer (100 mM NaCl, 0.1% Tween 20, 3.0% Bovine Serum Albumin, and 0.2% ProClin 300 at pH 7.4) were dispensed in the reaction cuvette, and the incubation and wash step were carried out again as above-described. After the second wash, Starter Reagents were added and a flash chemiluminescence reaction was induced. The light signal, and hence the amount of isoluminol-antigen conjugate, was measured by a photomultiplier as relative light units (RLU) in the analyzer reading chamber. Measured values were indicative of the presence of IgG antibodies to Zika virus NS1 in patient sera, calibrators, or controls.
- The above-described assay steps were carried out on an automated instrument (DiaSorin LIAISON® XL analyzer). Each reagent pack was calibrated using two point calibrators to obtain an instrument working curve based upon the assay master curve. Sample RLU values were then automatically converted to an Index value based on the instrument working curve for each reagent pack.
- The DiaSorin LIAISON® XL analyzer automatically calculates an Index value for both the ZIKV-M and ZIKV-C reagent packs based on each individual calibration. The analyzer then automatically combines the two Index values to produce a single result. Table 1 below shows the results obtained from examples 3 and 4, expressed in terms of M Index and C Index values, and corresponding diagnostic assessment.
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TABLE 1 ZIKV-M (IgM) Index ZIKV-C (IgG) Index Result <1.0 Any Value Negative ≥1.0 to <2.2 <4.0 ≥4.0 Positive ≥2.2 Any Value - In order to assess the sensitivity of the Zika Capture IgM assay of the invention, positive agreement was evaluated using serially collected serum samples from 58 symptomatic subjects (including 15 pregnant women) from the Dominican Republic found to be initially PCR positive for Zika virus. Multiple serial collections were obtained for 39 of the subjects and a single collection between 8 and 14 days post-symptom onset was obtained for 19 subjects.
- As shown in Table 2 below, all 58 positive subjects were reported as Zika IgM positive in the Zika Capture IgM assay. For serial collections, a positive Zika IgM result was obtained by the first draw following 8 days post onset of symptoms.
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TABLE 2 Positive Agreement* LIAISON ® XL Zika Capture IgM Population Positive Negative Total Positive Agreement Non- 43 0 43 100% Pregnant Pregnant 15 0 15 100% TOTAL: 58 0 58 100% *For serial collections, only the first positive result was included. Positive Percent Agreement: 100% (58/58, 95% CI: 93.8-100%) - The time course of Zika IgM detection in the Zika Capture IgM assay was assessed using all 188 specimens collected from the 58 subjects (Table 3).
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TABLE 3 Time Course of IgM Detection in Positive Subjects LIAISON ® XL Zika Capture IgM Days Post % Zika IgM Symptom Onset Positive Negative Total Detection ≤3 0 10 10 0% 4-7 5 24 29 17% 8-14 48 1* 49 98% 15-21 31 0 31 100% 22-28 21 1 22 95% 29-42 23 2 25 92% 43-84 16 6 22 73% *Specimen RT-PCR positive at 8 days post symptom onset - The diagnostic specificity of the Zika Capture IgM assay of the invention was assessed by evaluating negative agreement using serum samples from 218 apparently healthy donors and 32 pregnant donors collected in the United States and presumed negative for Zika virus infection. The results of the evaluation are reported in Table 4 below. Negative percent agreement was found to be 99.6%.
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TABLE 4 Negative Agreement LIAISON ® XL Zika Capture IgM Negative Population Positive Negative Total Agreement Pregnant Donors 0 32 32 100.0% Normal Donors 1 217 218 99.5% TOTAL: 1 249 250 99.6% Negative Percent Agreement: 99.6% (249/250, 95% CI: 97.4-99.7%) - In order to assess possible cross-reactivity of the Zika Capture IgM assay of the invention, specimens were analyzed which are seropositive for other closely related viruses or micro-organisms. The results of the cross-reactivity analysis are summarized in Table 5 below.
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TABLE 5 LIAISON ® XL Zika Capture IgM % Cross Organism/Condition N Positive Negative Reactivity Flaviviruses Anti-Dengue virus (IgM) 10 0 10 0% Anti-West Nile Virus (IgM) 14 0 14 0% Yellow fever virus post- 10 0 10 0% immunization Other Anti-Chikungunya virus (IgM) 5 0 5 0% Viruses/diseases Anti-Cytomegalovirus (IgM) 3 0 3 0% Anti-Epstein Barr Virus (IgM) 3 0 3 0% Anti-Parvovirus B19 (IgM) 5 0 5 0% Anti-Varicella zoster virus (IgM) 5 0 5 0% Anti-nuclear Antibodies (ANA) 3 0 3 0% Anti-Malaria/anti-plasmodium 3 0 3 0% falciparum Anti-Hepatitis (C) virus (optional) 5 0 5 0% Enterovirus (optional) 3 0 3 0%
Claims (22)
1. An in vitro method for the detection of Zika virus (ZIKV) infection in a biological sample from a subject, comprising the steps of:
a) testing the sample for IgM antibodies specific to ZIKV NS 1 protein or an epitope or immunogenic fragment thereof and determining the ZIKV IgM signal intensity;
b) testing the sample for IgG antibodies to ZIKV NS1 protein or an epitope or immunogenic fragment thereof and determining the ZIKV IgG signal intensity;
c) establishing a first ZIKV IgM signal intensity threshold and a second ZIKV IgM signal intensity threshold, wherein the second ZIKV IgM signal intensity threshold is higher than the first ZIKV IgM signal intensity threshold;
d) establishing at least a third ZIKV IgG signal intensity threshold;
e) detecting the presence or absence of ZIKV infection in the sample according to the following criteria:
i) a ZIKV IgM signal intensity lower than the first ZIKV IgM signal intensity threshold is indicative of the absence of ZIKA infection;
ii) a ZIKV IgM signal intensity higher than the second ZIKV IgM signal intensity threshold is indicative of the presence of ZIKA infection;
iii) when the ZIKV IgM signal intensity is higher than the first ZIKV IgM signal intensity threshold and lower than or equal to the second ZIKV IgM signal intensity threshold:
iiia) a ZIKV IgG signal intensity lower than the at least third ZIKV IgG signal intensity threshold is indicative of the absence of a ZIKV infection; and
iiib) a ZIKV IgG signal intensity higher than the at least third ZIKV IgG signal intensity threshold is indicative of the presence of a ZIKV infection.
2. The method according to claim 1 , wherein step a) and/or step b) are repeated.
3. The method according to claim 1 , wherein step a) is carried out before step b).
4. The method according to claim 1 , wherein step a) is carried out after step b).
5. The method according to claim 1 , which also includes testing the sample for IgA antibodies specific to ZIKV NS1 protein or an epitope or immunogenic fragment thereof.
6. The method according to claim 1 , wherein one or both of said steps a) and b) comprises an immunoassay.
7. The method according to claim 1 , wherein the IgM antibodies to Zika virus NS1 protein or an epitope or immunogenic fragment thereof are captured on a solid surface by contacting the biological sample with IgM binding molecules coated on the solid surface, and are detected using the NS1 protein or epitope or immunogenic fragment thereof as the detection reagent.
8. The method according to claim 7 , wherein the IgM binding molecules are anti-human IgM antibodies.
9. The method according to claim 1 , wherein the IgG antibodies to Zika virus NS1 protein or an epitope or immunogenic fragment thereof are captured on a solid surface by contacting the biological sample with IgG binding molecules coated on the solid surface, and are detected using the NS1 protein or epitope or immunogenic fragment thereof as the detection reagent.
10. The method according to claim 9 , wherein the IgG binding molecules are anti-human IgG antibodies.
11. The method according to claim 7 , wherein the amount of IgM binding molecules coated on the solid surface which are contacted with the biological sample substantially corresponds to the amount of IgM antibodies which are present in the biological sample.
12. The method according to claim 7 , wherein the solid surfaces are magnetic beads.
13. The method according to claim 1 , wherein one or both of said steps a) and b) comprises a chemiluminescent immunoassay.
14. The method according to claim 13 , which employs an isoluminol derivative as a chemiluminescent label
15. The method according to claim 6 , wherein the immunoassay comprises:
i) contacting the biological sample with IgM binding molecules coated on a solid surface in an amount corresponding substantially to the amount of IgM antibodies in said sample, thereby capturing the IgM antibodies in the sample,
ii) contacting the biological sample with IgG binding molecules coated on a solid surface in an amount corresponding substantially to the amount of IgG antibodies in said sample, thereby capturing the IgG antibodies in the sample, and
iii) detecting captured ZIKV IgM antibodies and ZIKV IgG antibodies specific to ZIKV NS1 protein employing a labeled ZIKV NS1 antigen, epitope or fragment thereof.
16. The method according to claim 15 , wherein the biological sample, before contacting the IgG binding molecules coated on the solid surface, is diluted by a dilution factor comprised between 10-fold and 500-fold.
17. The method according to claim 1 , wherein ZIKV IgM and/or ZIKV IgG signal intensities are determined by reference to a calibration curve.
18. The method according to claim 1 , wherein the biological fluid is blood, plasma, serum, urine, cerebrospinal fluid or saliva.
19. The method according to claim 1 , wherein the infection is a secondary flavivirus infection.
20. The method according to claim 1 , wherein the subject is a human.
21. The method according to claim 9 , wherein the amount of IgG binding molecules coated on the solid surface which are contacted with the biological sample substantially corresponds to the amount of IgG antibodies which are present in the biological sample.
22. The method according to claim 9 , wherein the solid surfaces are magnetic beads.
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