Patent Publication Number: US-11662350-B2

Title: Method and kit for detection of anti-Zika virus antibodies

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 16/435,787, filed on Jun. 10, 2019, which is a continuation of U.S. Pat. No. 10,317,413, filed on Nov. 14, 2017, and claims priority to U.S. provisional application No. 62/421,694, filed Nov. 14, 2016, and to U.S. provisional application No. 62/438,681, filed Dec. 23, 2016, the contents of which applications are incorporated by reference in their entireties into the present disclosure. 
    
    
     FIELD OF THE INVENTION 
     The present disclosure relates to, inter alia, methods for detection of Zika virus infection and compositions of matter and kits for use in performing said method. More particularly, methods for detection of anti-Zika virus antibodies is provided. More particularly still, a method for detecting the presence of anti-Zika virus antibodies, and distinguished from the presence of antibodies to other flaviviruses. Also disclosed are kits for the performance of such methods. 
     BACKGROUND OF THE INVENTION 
     Zika virus (ZIKV) belongs to the genus of flavivirus within the family Flaviviridae. Many flaviviruses are significant human pathogens, including ZIKV, yellow fever (YFV), dengue virus (DENV serotypes 1 to 4), Japanese encephalitis virus (JEV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV). ZIKV is predominantly transmitted by  Aedes  spp. mosquitoes, which also transmit DENV and YFV, as well as chikungunya virus (an emerging alphavirus). Besides mosquitoes, ZIKV can also be transmitted through maternofetal route, sex, blood transfusion, and organ transplantation. Approximately 80% of the ZIKV infections are asymptomatic. Disease symptoms associated with ZIKV infection include headaches, fever, lethargy, rash, conjunctivitis, myalgia, and arthralgia. Severe diseases of ZIKV infection include neurotropic Guillain-Barre syndrome and congenital microcephaly. The flavivirus genome is a single-strand, positive-sense RNA of approximately 11,000 nucleotides. It contains a 5′ untranslated region (UTR), an open-reading frame (ORF), and a 3′ UTR. The single ORF encodes a long polyprotein which is processed into ten viral proteins, including three structural proteins—capsid (C), precursor membrane (prM), and envelope (E)—and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). 
     Diagnosis of ZIKV infection is performed through detection of viral components (e.g., viral RNA, viral proteins, or virus isolation) and detection of host immune response (e.g., antibodies against viral proteins). For viral component-based diagnosis, RT-PCR, immunoassay, and virus isolation detect ZIKV RNA, viral proteins, and live virus, respectively (Lanciotti et al., 2008); among them, RT-PCR is the most popular assay because of its sensitivity and specificity. The viremic phase of ZIKV infection usually lasts for about one week, yet occasionally persists beyond two weeks. Due to the short duration of the viremic phase, the diagnostic window for detection of viral components is narrow. Therefore, host immune response-based assays play an important role, among which enzyme-linked immunosorbent assays (ELISA), such as IgM-capture ELISA (MAC-ELISA), and plaque reduction neutralization test (PRNT) are the two most commonly used serologic assays in ZIKV diagnosis. Conventionally, serologic diagnosis of Zika virus (ZIKV) infection relies mainly upon IgM-capture ELISA which is confounded with the flaw of cross-reactivity among different flaviviruses. Unfortunately, the interpretation of conventional IgM-capture ELISA assays for ZIKV and other flaviviruses are challenging due to the cross-reactive nature of anti-flaviviral antibodies conventionally used in such tests, leading to equivocal diagnostic results. This challenge is confounding Zika diagnosis because (i) many flaviviruses (e.g., ZIKV and DENV) produce similar disease symptoms and (ii) antibodies from patients infected with ZIKV cross-react with other flaviviruses. Consequently, ZIKV IgM-capture ELISA results typically require neutralization tests for confirmation. Furthermore, PRNT is time-consuming, labor-intensive, slow, and low-throughput, and cost-ineffective, impairing attempts at rapid diagnosis to halt or slow spread if infection. Moreover, PRNT still relies upon both virus-specific and cross-reactive epitopes of viral E protein such that the results may be inconclusive with respect to flavivirus infections (Shan et al., 2016a). There is therefore a need to improve the accuracy and speed of serologic diagnosis for flaviviruses, ZIKV in particular. 
     SUMMARY OF THE INVENTION 
     The present disclosure relates to, inter alia, a method of detecting the presence of an anti-Zika virus (ZIKV) antibody in a sample, including contacting a sample with a suspension having a plurality of microspheres wherein individual microspheres are conjugated to a peptide and the peptide includes a ZIKV peptide selected from the group including ZIKV NS1, ZIKV NS5, and ZIKV envelope protein, forming a first incubated suspension wherein forming includes incubating said sample with said suspension to permit binding of anti-ZIKV antibodies present in the sample to said microspheres, forming a second incubated suspension wherein forming includes contacting said first incubated suspension with an anti-ZIKV antibody detecting-reagent to permit binding of the anti-ZIKV antibody detecting reagent to said microspheres, removing from the second incubated suspension anti-ZIKV antibody detecting-reagent molecules that are not bound to said microspheres, and detecting the presence of anti-ZIKV antibody detecting-reagent molecules in the second incubated suspension. 
     In some embodiments, the method includes individual microspheres conjugated to ZIKV peptides that differ from ZIKV peptides to which other individual microspheres are conjugated, and the ZIKV peptides are selected from ZIKV NS1, ZIKV NS5, and ZIKV envelope protein, and any two of the foregoing. 
     In other embodiments, different ZIKV peptides are ZIKV NS1 and ZIKV NS5. In some embodiments, individual microspheres are conjugated to ZIKV NS1, and in other embodiments individual microspheres are conjugated to ZIKV NS5. In some embodiments, some microspheres are conjugated to ZIKV Envelope protein, some microspheres are conjugated to ZIKV NS1, and some microspheres are conjugated to ZIKV NS5. In further embodiments, some microspheres are conjugated to ZIKV Envelope protein and some microspheres are conjugated to ZIKV NS1. 
     In other embodiments, the sample includes a biological sample from a subject and the biological sample is selected from the group including bodily fluid, blood, serum, plasma, saliva, tears, feces, semen, mucous, tissue, tissue homogenate, cellular extract, spinal fluid, and any combination of two or more of the foregoing. In other embodiments, the subject is a human. 
     In other embodiments, detecting includes measuring an autofluorescence signal emitted by an anti-ZIKV antibody-detecting reagent, an autofluorescence signal emitted by a microsphere, or an autofluorescence signal emitted by an anti-ZIKV antibody-detecting reagent and an autofluorescence signal emitted by a microsphere. 
     Some embodiments further include detecting the presence of an antibody to a Dengue virus (DENV) protein in a sample, wherein the protein comprises NS1 and the DENV comprises DENV1, DENV2, DENV3, DENV4, or any combination of two or more of the foregoing. For example, some microspheres may be conjugated to DENV1 NS1, some microspheres may be conjugated to DENV2 NS1, some microspheres may be conjugated to DENV3 NS1, and some microspheres may be conjugated to DENV4 NS1. In further embodiments, some microspheres may be conjugated to ZIKV Envelope protein, some microspheres may be conjugated to ZIKV NS1, some microspheres may be conjugated to ZIKV NS5, some microspheres may be conjugated to DENV1 NS1, some microspheres may be conjugated to DENV2 NS1, some microspheres may be conjugated to DENV3 NS1, and some microspheres may be conjugated to DENV4 NS1. In still further embodiments, some microspheres may be conjugated to ZIKV Envelope protein, some microspheres may be conjugated to ZIKV NS1, some microspheres may be conjugated to DENV1 NS1, some microspheres may be conjugated to DENV2 NS1, some microspheres may be conjugated to DENV3 NS1, and some microspheres may be conjugated to DENV4 NS1. 
     In another aspect, provided is a kit for detecting the presence of an anti-Zika virus (ZIKV) antibody in a sample, including a plurality of microspheres wherein individual microspheres are conjugated to a peptide and the peptide comprises a ZIKV peptide selected from the group consisting of ZIKV NS1, ZIKV NS5, and ZIKV envelope protein. In some embodiments, the kit includes individual microspheres conjugated to one of two or three different ZIKV peptides which differ from ZIKV peptides to which other microspheres are conjugated to, and the different ZIKV peptides are ZIKV NS1, ZIKV NS5, and ZIKV envelope protein, or any two of the foregoing. In some embodiments, the different ZIKV peptides are ZIKV NS1 and ZIKV NS5. In other embodiments, the different ZIKV peptides are ZIKV envelope protein, ZIKV NS1, and ZIKV NS5. In further embodiments, the different ZIKV peptides are ZIKV envelope protein and ZIKV NS1. In still further embodiments, the kit may further include individual microspheres conjugated to one of two or more different DENV peptides which differ from DENV peptides to which other microspheres are conjugated to, and the different DENV peptides are DENV1 NS1, DENV2 NS1, DENV3, NS1, or DENV4 NS1. For example, a kit may include microspheres with the following peptides conjugated thereto: ZIKV NS1, ZIKV NS5, ZIKV Envelope protein, DENV1 NS1, DENV2 NS2, DENV3 NS3, and DENV4 NS4. In some examples, a kit may contain a subset of the foregoing. For example, a kit may contain microspheres with the following peptides conjugated thereto: ZIKV NS1, ZIKV Envelope protein, DENV1 NS1, DENV2 NS2, DENV3 NS3, and DENV4 NS1. In yet other embodiments, a kit may contain any combination of any two or more of the foregoing microspheres. For example, in some embodiments, the different ZIKV peptides are ZIKV NS1 and ZIKV NS5. In other embodiments, the different ZIKV peptides are ZIKV envelope protein, ZIKV NS1, and ZIKV NS5. In further embodiments, the different ZIKV peptides are ZIKV Envelope protein and ZIKV NS1. 
     In other embodiments, individual microspheres are conjugated to ZIKV NS1. In still other embodiments, individual microspheres are conjugated to ZIKV NS5. In still other embodiments, the kit includes an anti-ZIKV antibody-detecting reagent. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       For the purpose of illustrating aspects of the present invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings. Further, as provided, like reference numerals contained in the drawings are meant to identify similar or identical elements. The foregoing and other objects, features, and advantages of the invention are apparent from the following detailed description taken in conjunction with the accompanying drawings in which: 
         FIG.  1    shows SDS-PAGE analysis of recombinant ZIKV NS5 protein. Full-length NS5 of ZIKV was expressed in an  E. coli  system, and purified through affinity followed by size-exclusion chromatography (see details in Materials and Methods). The recombinant protein was analyzed on a 12% Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio-rad). 
         FIG.  2    shows a Table (Table 5) of a summary of PRNT, IgM-capture ELISA, and multiplex MIA diagnosis. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Aspects of an invention disclosed herein and certain features, advantages, and details thereof, are explained more fully below with reference to the non-limiting embodiments illustrated in the accompanying drawings. Descriptions of well-known materials, fabrication tools, processing techniques, etc., are omitted so as to not unnecessarily obscure the invention in detail. It should be understood, however, that the detailed description and the specific examples, while indicating some embodiments, are given by way of illustration only, and are not by way of limitation. Various substitutions, modifications, additions and/or arrangements within the spirit and/or scope of the underlying inventive concepts will be apparent to those skilled in the art from this disclosure. 
     Disclosed herein is a method for detecting the presence of anti-ZIKV antibodies in a sample, overcoming shortcomings of the prior art. Conventionally, detection of anti-ZIKV antibodies in a sample may require a high volume of sample, may take longer than several hours to perform, may be of low sensitivity resulting in a failure to detect a presence of anti-ZIKV antibodies in a sample, and low selectivity resulting in evidence of a presence of anti-ZIKV antibodies in a sample when no such antibodies are present. Such shortcomings may pose difficulties in accurate detection of the presence of anti-ZIKV antibodies in a sample. According to the present disclosure, small volumes of sample may be used to generate high-probability detection of anti ZIKV-antibodies in a sample. 
     In one aspect, disclosed is a method for detecting the presence of anti-Zika virus antibodies, such as in a sample from a subject. Such detection may indicate current or prior infection with ZIKV. In particular, the presence of antibodies to the NS5 protein of ZIKV may be detected. The presence of different antibody types, including IgG, IgM, or both, may be accomplished in accordance with the present disclosure. As would be understood by skilled artisans, the presence of different antibody types following a subject&#39;s exposure to ZIKV or other flaviviruses follows different time frames, with IgM antibodies to particular antigens being produced sooner after infection than IgG antibodies to said antigens. In various embodiments, detection of one, the other, both, and/or other types of antibodies to ZIKV-derived antigens may provide an indication of a time frame of a subject&#39;s exposure to ZIKV relative to when a sample was obtained from the subject. For example, presence of IgM anti-ZIKV antibodies may signify more recent exposure while presence of IgG anti-ZIKV antibodies may signify less recent exposure. 
     In another aspect, presence of antibodies to ZIKV NS5 may be performed. ZIKV NS5 protein is encoded for by nucleotides 7668-10376 of the ZIKV genome (GenBank number KU955593.1; Shan et al., 2016, Cell Host &amp; Microbe 19:891-900). This sequence, and the peptide product thereof, ZIKV NS5 peptide, differ from the genetic and peptide sequences of NS5 for other viruses, including flaviviruses. For example, NS5 of DENV1-4 differ from ZIKV NS5. As disclosed herein, affinity of antibodies to ZIKV NS5 produced by a subject subsequent to exposure to ZIKV may differ from the affinity of said antibodies to NS5 protein of other viruses, such as DENV or other flaviviruses. The detection of the presence of an anti-ZIKV NS5 antibody in a subject on the basis of whether said antibody binds to ZIKV NS5 may therefore signify that the subject was exposed to ZIKV as distinguished from having been exposed to DENV. Specifically, identifying antibodies in a subject that bind ZIKV NS5 in accordance with the present disclosure indicates that the subject was exposed to ZIKV and may be distinguished from an indication that said subject was exposed to DENV, or other viruses such as other flaviviruses. 
     In another aspect, presence of antibodies to ZIKV NS1 may be detected. ZIKV NS1 protein is encoded for by nucleotides 2490-3545 of the ZIKV genome (see, e.g., GenBank accession number KU955593.1; Shan et al., 2016, Cell Host &amp; Microbe 19:891-900). This sequence, and the peptide product thereof, ZIKV NS1 peptide, differ from the genetic and peptide sequences of NS1 for other viruses, including flaviviruses. For example, NS1 of DENV1-4 differ from ZIKV NS1. Gene and peptide sequence of NS1 for each of DENV1-4 also differ from each other. The complete genomes of DENV1, DENV2, DENV3, and DENV4 are known (see, e.g., GenBank accession numbers NC_001477.1, NC_001474.2, NC_001475.2, and NC_002640.1, respectively). As disclosed herein, affinity of antibodies to ZIKV NS1 produced by a subject subsequent to exposure to ZIKV differs from the affinity of said antibodies to NS1 protein of other viruses, such as DENV1-4 or other flaviviruses. Nucleotide sequences for DENV1-4 NS1, and the amino acid sequences of such proteins, is known (see, e.g., GenBank accession numbers NP_722461.1, NP_739584.2, YP_001531169.2, and NP_740318.1 for DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1, respectively). 
     In another aspect, presence of antibodies to a flavivirus envelope protein may be detected. ZIKV envelope peptide amino acid sequence is amino acids 291-792 of ZIKV polyprotein (GenBank accession number AAV34151). Envelope protein of other strains of ZIKV may also be used, as may Envelope proteins from other flaviviruses, such as DENV1 envelope protein, DENV2 envelope protein, DENV3 envelope protein, or DENV1 envelope protein, given the highly conserved amino acid sequences of envelope protein among these viruses and strains thereof. 
     As further disclosed herein, affinity of antibodies to DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4 NS1 produced by a subject subsequent to exposure to DENV1-4, respectively, may differ from the affinity of said antibodies to NS1 protein of other viruses, such as DENV serotypes to which the subject was not exposed, or other flaviviruses. The detection of the presence of an anti-ZIKV NS1 antibody in a subject on the basis of whether said antibody binds to ZIKV NS1 may therefore signify that the subject was exposed to ZIKV as distinguished from having been exposed to DENV1-4. Furthermore, the detection of the presence of an anti-DENV1 NS1, -DENV2 NS1, -DENV3 NS1, or -DENV4 NS1 antibody in a subject on the basis of whether said antibody binds to DENV1 NS1, -DENV2 NS1, -DENV3 NS1, or -DENV4 NS1, respectively, may therefore signify that the subject was exposed to DENV1, DENV2, DENV3, or DENV4, respectively, as distinguished from having been exposed to another serotype of DENV, ZIKV, or to another flavivirus. Specifically, as disclosed herein, identifying antibodies in a subject that bind ZIKV NS1 in accordance with the present disclosure indicates that the subject was exposed to ZIKV and may be distinguished from an indication that said subject was exposed to DENV, or other viruses such as other flaviviruses. And, as disclosed herein, identifying antibodies in a subject that bind to DENV1 NS1, -DENV2 NS1, -DENV3 NS1, or -DENV4 NS1 in accordance with the present disclosure indicates that the subject was exposed to DENV1, DENV2, DENV3, or DENV4, respectively, and may be distinguished from an indication that said subject was exposed to another serotype of DENV, or to ZIKV or another flavivirus. 
     Antibodies produced in response to ZIKV exposure that have affinity for ZIKV Envelope protein (E) may also have affinity for E protein of other flaviviruses, such as DENV or other types of flaviviruses. Similarities of the peptide sequences and/or conformations of E protein of different flaviviruses may result in the production of antibodies that are cross-reactive to various flaviviruses. If a subject has been exposed to ZIKV, the subject may produce antibodies that have affinity not only for ZIKV E protein but also for E protein of other flaviviruses, including DENV. Likewise, a subject who has been exposed to DENV1-4 may produce antibodies with affinity for E protein of other flaviviruses. Detection in a subject of an antibody with affinity for ZIKV E protein may signify that the subject was exposed to ZIKV, DENV1, DENV2, DENV3, DENV4, or another flavivirus. And, detection in a subject of an antibody with affinity for DENV1 E, DENV2 E, DENV3 E, or DENV4 E may signify that the subject was exposed to ZIKV, DENV1, DENV2, DENV3, DENV4, or another flavivirus. 
     As further disclosed herein, in one embodiment, detection of multiple different antibodies may be performed on a given sample or samples from a given subject or subjects. In some embodiments, a sample from a subject may be tested for the presence of an antibody with affinity for ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, DENV4 NS1, or any combination of two or more of the foregoing. In one non-limiting example, a sample from a subject may be tested for the presence of antibodies to ZIKV NS5, ZIKV NS1, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1. In another non-limiting example, a sample from a subject may be tested for the presence of antibodies to ZIKV NS1, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1. The presence of IgG and IgM antibodies to the foregoing antigens may be tested. If a subject has been exposed to ZIKV but not to other flaviviruses, IgG antibodies, IgM antibodies, or both, to ZIKV NS1, ZIKV NS5, and ZIKV E may be detected. If the subject has been exposed to DENV1-4, antibodies to DENV1-4 NS1 may be detected, as well as to ZIKV E owing to the similarities between ZIKV E and DENV E and cross-reactivity of antibodies thereto. If a subject has been exposed to both ZIKV and DENV, antibodies to ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1 may all be detected in a sample or samples from said subject. 
     In a particular embodiment, presence or absence of anti-ZIKV NS5 antibodies may be detected in a sample from a subject. As disclosed herein, high selectivity of anti-ZIKV NS5 antibodies for ZIKV NS5 relative to NS5 or other proteins from other flaviviruses, such as DENV, corresponds to low cross-reactivity of anti-ZIKV NS5 antibodies for other flaviviruses. Similarly, as disclosed herein, low cross-reactivity of antibodies produced in response to exposure to flaviviruses other than ZIKV, such as DENV, to ZIKV NS5 results from selectivity of antibodies produced in response to expose to said other viruses relative to ZIKV NS5. Detection of the presence of an anti-ZIKV NS5 antibody in a sample from a subject as disclosed herein indicates that the subject has been exposed to ZIKV. The absence of anti-ZIKV NS5 antibodies in a sample from a subject may signify that the subject was not exposed to NS5, even if the subject was exposed to another virus, such as another flavivirus such as DENV. 
     In another particular embodiment, presence or absence of anti-ZIKV NS1 antibodies may be detected in a sample from a subject. As disclosed herein, high selectivity of anti-ZIKV NS1 antibodies for ZIKV NS1 relative to NS1 or other proteins from other flaviviruses, such as DENV, corresponds to low cross-reactivity of anti-ZIKV NS1 antibodies for other flaviviruses. Similarly, as disclosed herein, low cross-reactivity of antibodies produced in response to exposure to flaviviruses other than ZIKV, such as DENV, to ZIKV NS1 results from selectivity of antibodies produced in response to expose to said other viruses relative to ZIKV NS1. Detection of the presence of an anti-ZIKV NS1 antibody in a sample from a subject as disclosed herein indicates that the subject has been exposed to ZIKV. The absence of anti-ZIKV NS1 antibodies in a sample from a subject may signify that the subject was not exposed to NS1, even if the subject was exposed to another virus, such as another flavivirus such as DENV. 
     In another particular embodiment, presence or absence of anti-ZIKV envelope protein antibodies may be detected in a sample from a subject. In particular examples, presence or absence of anti-ZIKV NS1, anti-ZIKV NS5, anti-ZIKV envelope protein, anti-DENV1 NS1, anti-DENV2 NS1, anti-DENV3 NS1, and anti-DENV4 NS1 may all be detected in a sample from a subject. In another example, presence or absence of anti-ZIKV NS1, anti-ZIKV envelope protein, anti-DENV1 NS1, anti-DENV2 NS1, anti-DENV3 NS1, and anti-DENV4 NS1 may all be detected in a sample from a subject. 
     In some embodiments, for each and every of the anti-flaviviral protein antibodies disclosed herein, an assay for detection thereof may generate some level of signal indicating presence of reactive antibody in a sample from a subject known not to have been exposed to a or the flaviviruses of interest. Such background signal is known by those skilled in this field to occur and is attributable to some baseline reactivity of sample constituents with a test antigen used as disclosed herein for identifying presence of an anti-flaviviral antibody. Conventionally, an average level of such background may be determined for a given assay against which detection results for a tested subject may be compared to determine whether a given antibody is present. For example, a number of subjects known or believed not to have been exposed to ZIKV or DENV may be tested for the presence of anti-ZIKV and anti-DENV protein antibodies as disclosed herein and an average level of background reactivity of assays as disclosed herein may be determined for a given assay. 
     For subsequent testing of a subject who may or may not express anti-ZIKV or anti-DENV protein antibodies, a sample taken from such subject may be tested in an assay as disclosed herein and results compared to an average from tests of subjects known or believed not to have been exposed to such flaviviruses. In some instances, a level of detection signal some degree or amount above an average background level of expression may be selected as minimum level qualifying for evincing presence of a given anti-flaviviral protein antibody in a sample from a subject. If a subject is tested for the presence of antibodies against one or more of the foregoing flaviviral proteins and the level of detection signifying presence of any one or more of such antibodies falls below, or above, such cutoff value, then such antibodies may be determined not to have been detected, or to have been detected, respectively, in such sample. In some examples, a cutoff value may be a certain degree above an average level of background signal detected in subjects known or believed not to have been exposed to the flaviviruses in question. For example, a cutoff may be a number of standard deviations above such mean background. A cutoff value may be one, two, or three standard deviations above mean background expression, meaning that antibody to a given flaviviral protein is determined to have been detected for a sample when an assay yields a given level of signal signifying such presence. Cutoff levels may be determined on an anti-flaviviral protein antibody by anti-flaviviral protein antibody basis, as well as on an assay-by-assay basis. 
     In some embodiments, if a test detects presence of anti-flaviviral proteins, a test may be repeated at a later date and comparing indications that such antibodies were detected at different tests performed. For example, presence or absence of an IgG antibody, or an IgM antibody, or both types of antibodies, to one or more of the flaviviral proteins disclosed herein, may be detected. For example, IgM antibodies to ZIKV envelope and ZIKV NS1, may be detected. Such results may indicate current or recent infection with ZIKV, such as in approximately the past 21 days, as would detection of IgM antibodies to ZIKV NS5. In another example, IgG antibodies to ZIKV envelope and ZIKV NS1, may be detected. Such results may indicate past infection with ZIKV, such as more than 21 days ago, as would detection of IgG antibodies to ZIKV NS5. In still another example, IgM and IgG antibodies to ZIKV envelope and ZIKV NS1, may be detected. Such results may indicate current or recent infection with ZIKV, such as in approximately the past 21 days, as well as past infection, such as more than approximately 21 days ago, as would detection of IgM and IgG antibodies to ZIKV NS5. 
     In still further embodiments, an avidity assay may be performed to determine whether an anti-flaviviral protein antibody, such as anti-ZIKV envelope protein, anti-ZIKV-NS1, anti-ZIKV NS5, anti-DENV1 NS1, anti-DENV2 NS2, anti-DENV3 NS1, or anti-DENV4 NS1 antibody, or any combination of two or more of the foregoing antibodies, are present and if so, whether they bind to ZIKV envelope protein, ZIKV-NS1, ZIKV NS5, DENV1 NS1, DENV2 NS2, DENV3 NS1, or DENV4 NS1, respectively, with high or low avidity. Detection of high-avidity-binding antibodies that bind to a flaviviral protein with relatively higher avidity (e.g., anti-ZIKV envelope/ZIKV envelope, anti-ZIKV NS1/ZIKV/NS1, anti-ZIKV NS5/ZIKV NS5, anti-DENV1 NS1/DENV1 NS1, anti-DENV2 NS1/DENV2 NSI, anti-DENV3 NS1/DENV3 NS1, or anti-DENV4 NS1/DENV4 binding) may indicate a subject was infected with a given flavivirus as disclosed herein longer ago. Detection of low-avidity-binding antibodies that bind to a flaviviral protein with relatively low avidity (e.g., anti-ZIKV envelope/ZIKV envelope, anti-ZIKV NS1/ZIKV/NS1, anti-ZIKV NS5/ZIKV NS5, anti-DENV1 NS1/DENV1 NS1, anti-DENV2 NS1/DENV2 NSI, anti-DENV3 NS1/DENV3 NS1, or anti-DENV4 NS1/DENV4 binding) may indicate a subject was infected with a given flavivirus more recently, or is currently infected with a given flavivirus. High avidity binding is binding that persists or is still detectable after incubation of a solution of a flaviviral protein or composition such as a polyplex microsphere bound to such antigen as disclosed herein with urea. Urea is known to disrupt low-avidity antigen-antibody association. When a sample is incubated with urea (e.g., 6 M, 8 M, or 10 M urea for 10 minutes) yet presence of anti-flaviviral antibody as disclosed herein is still detected, then high-avidity binding antibodies that bind to a given flaviviral protein have been detected. Detection of absence of antibody after urea incubation indicates absence of high-avidity antibodies. Such avidity testing may be performed for any one, all or any combination of assays for presence of anti-flaviviral protein antibodies. 
     In some embodiments, presence or absence of a given anti-flaviviral protein antibody or antibodies may indicate that a subject from which a tested sample was taken was previously exposed to or infected with or whose body presently or formerly contained a given flavivirus. For example, if anti-ZIKV envelope protein antibodies are detected in a sample, a subject may be determined to have had or presently have a flavivirus. Given cross-reactivity of an anti-ZIKV envelope protein antibody with envelope proteins of other flaviviruses, such as DENV1, DENV2, DENV3, or DENV4, for example, apparent detection of an anti-ZIKV envelope antibody may signify that the subject has had or has ZIKV, DENV1, DENV2, DENV3, or DENV4. By contrast, as disclosed herein, given selectivity of antibodies to ZIKV NS1 to binding to ZIKV NS1 relative to binding to other flaviviral proteins, such as DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4 NS1, which is low, detection of anti-ZIKV NS1 antibodies as disclosed herein signifies that a subject has or has had ZIKV in particular. Similarly, as disclosed herein, given selectivity of antibodies to ZIKV NS5 to binding to ZIKV NS5 relative to binding to other flaviviral proteins, such as DENV1 NS5, DENV2 NS5, DENV3 NS5, or DENV4 NS5, which is low, detection of anti-ZIKV NS5 antibodies as disclosed herein signifies that a subject has or has had ZIKV. 
     As further disclosed herein, detection of presence multiple different anti-flaviviral protein antibodies combined with detection of absence of e multiple different anti-flaviviral protein antibodies in combination permits possible discriminative determination of presence, or prior presence, of ZIKV, DENV, or both, in a subject. For example, a subject may have had or may have ZIKV but not DENV of any serotype, in which case presence of anti-ZIKV NS1, anti-ZIKV-NS5, and anti-ZIKV envelope protein antibodies may be determined to be present in a sample from such subject, whereas anti-DENV NS1, anti-DENV2 NS1, anti-DENV3 NS1, and anti-DENV4 NS1 antibodies may be determined not to be present in samples from such patients. In another example, a subject may have had or may have a flavivirus of a DENV serotype but not ZIKV, in which case absence of anti-ZIKV NS1 and anti-ZIKV-NS5 antibodies may be detected, and presence of anti-ZIKV envelope protein antibodies, and anti-DENV NS1, anti-DENV2 NS1, anti-DENV3 NS1, and anti-DENV4 NS1 antibodies (depending on which DENV serotype the subject had or has), may be determined to be present in samples from such patients. 
     The presence of an antibody to a given antigen as disclosed herein may be performed by any of a number of different known methods. In one embodiment, a microsphere immunoassay (MIA) may be used for the detection of an antibody to a given antigen. As would be appreciated by skilled artisans, rapid detection of multiple assays can be performed on low volumes of specimen taken from a subject. In some embodiments, presence of antibodies to one or more of ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4 may be identified in a sample from a subject. In accordance with above-identified differential responsiveness to such testing on the basis of prior viral exposure, different combinations of positive results for particular antibodies may indicate exposure of the subject to ZIKV, DENV1-4, or another flavivirus. 
     Several options for MIA are available. In some embodiments, flaviviral antigens may be covalently coupled to microbeads and contacted to a sample from a subject, whereupon antibodies to the given antigen present in the sample bind to the bead via the antigen. Subsequent identification of antibody bound to the bead indicates that antibodies to the antigen were present in the sample. In other embodiments, antigen may be coupled to microbeads that have an anti-antigen antibody, or other molecule designed to bind to the antigen such as an affimer or aptamer or other designer ligand, fused thereto via binding to said antibody. Subsequent contacting of the antigen-coupled microbeads to sample from a subject may then be performed as described. 
     Different sources of viral antigen may be used in accordance with the present disclosure. For example, recombinant peptides whose sequence correspond to the sequence of viral peptide of interest (i.e., ZIKV NS1, ZIKV NS5, ZIKV E, or NS1 proteins of any of DENV1-4) may be synthesized by well-known methods and purified. Recombinant peptides may differ in sequence from native of wild-type flaviviral protein, provided it retains structural, sequential, or conformational properties sufficient to permit recognition by antibodies generated in a subject subsequent to exposure to the corresponding flavivirus. flaviviral protein from which the to which the recombinant Different assays may detect whether antibodies to flaviviral antigen in a sample are IgG antibodies or IgM antibodies, by known methods (e.g., IgG- or IgM-specific methods of detection of anti-flaviviral antigen antibody bound to microbeads, depleting IgG or IgM antibodies from a sample before contacting the sample with antigen-coupled beads, or selecting out IgG or IgM antibodies from a sample and contacting the selected-out antibodies with antigen-coupled microbeads. 
     In another embodiment, ELISA could be used to detect the presence of antibodies to one or more of ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4 in a sample. For example, anti-IgM antibodies can be coated onto a plate, such as a 96-well or other multi-well plate, and contacted with sample from a subject. IgM antibodies from the sample would become bound to the anti-IgM-coated plate. Following well-known protocols, the IgM-bound plate could then be contacted with antiviral protein of interest, such as ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4, followed by contact with a visualizable antibody to the viral protein. In this way, antiviral protein of interest would, via binding to any antibodies thereto present in the sample that had become bound to the anti-IgM antibody on the plate, be bound to plates that had been exposed to samples from subject that had been exposed to the flavivirus. In turn, such samples would be visualizable, via binding of visualizable anti-flaviviral protein, such as through conjugation with antibodies, fluorophores, or other well-known visualizable or detectable markers. Upon processing to detect presence of the visualizable marker, plates that had been contacted with a sample from a subject that had been exposed to the corresponding flavivirus can be identified and distinguished from those that have not. By performing MAC-ELISA on a sample for antibodies to several different flaviviral proteins (for example, any combination of two or more of ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4), it can be determined whether the subject had been exposed to ZIKV, DENV, and/or another flavivirus. 
     In another embodiment, a dot blot may be performed to detect the presence of antibodies to one or more of ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4. In dot blotting, a spot or dot of antigen is applied to a membrane, such as nitrocellulose or polyvinylidine fluoride (PVDF). The membrane may then be exposed to a sample from a subject. If the subject has been exposed to a virus that contains the antigen applied to the membrane, such antibodies will couple to a region of the membrane to which the antigen has been applied. The membrane can then be contacted by secondary antibodies (e.g., anti-IgM or anti-IgG) coupled to a visualizable marker (e.g., radiolabel, fluorescent tag, magnetic tag, enzyme, etc.). Visualizable marker is thereby coupled to any region of the membrane to which an antigen recognized by antibodies thereto present in the sample. Upon detection of said marker, presence of antibodies to said antigen in the sample may be determined. In some embodiments, a dot blot analysis may be performed to detect the presence of antibodies to any one or more of ZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4, and any combination of two or more of the foregoing. As described above, different permutations of positive and negative results for different anti-flaviviral antigen antibodies suggests that the subject from which the sample was taken was exposed to different flavivirus(es). 
     In yet another embodiment, immunochromatography may be used to identify the presence of one or more flaviviral antibody in a subject. As one nonlimiting example, and immunochromatographic strip test (ICST) may be used. In such assays, a small strip comprising capillary beds may be exposed to a sample from a subject. Embedded in the strip may be a viral antigen to which antibodies are to be detected. When an aqueous sample is drawn into contact with such antigens following contact with the capillary structure of the strip, the antigens are bound to any antibodies thereto that are present in the sample. The antigen may carry visualizable markers, such as colored latex, gold nanoparticles, or fluorescent or magnetic particles which can be visualized with appropriate readers. Further along the strip, a capture portion containing antibodies to the antigen immobilized to the strip bed may be present, such as in a concentrated region, stripe, or area. As sample continues to migrate along the capillary bed, it encounters the immobilized antibodies of the capture portion. If antibody to the particular antigen were present in the sample, their migration across the capillary bed would have carried bound, visualizable antigen with them, which, in turn, would be immobilized on the capture portion owing to the anti-antigen antibodies immobilized therein. As more antibody accumulates that the capture portion, the visualizable markers become detectable and, when detected, signify the presence of antibody to the antigen of interest. 
     In some examples, multiple ICST strips, each designed to permit the detection of antibodies to a different flaviviral antigen (e.g., ZIKV NS1, ZIKV NS5, ZIKV E, or NS1 proteins of any of DENV1-4) may be used to test the presence of antibodies to more than one such antigen in a sample. As described above, different permutations of positive and negative results for different anti-flaviviral antigen antibodies suggests that the subject from which the sample was taken was exposed to different flavivirus(es). 
     As would be appreciated by skilled artisans, modifications of the foregoing methods may be performed in keeping the with present disclosure. Other methods where ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, DENV4 NS1, or combinations of any two or more of the foregoing, are used to bind antibodies present in a sample, followed by methods used to visualize, detect, quantify, bind, identify, or otherwise record the presence and/or quantity thereof in the sample may be performed, all of which are explicitly intended as embodiments of the present disclosure, of which the foregoing examples are but non-limiting representations of the method disclosed herein. Kits containing reagents used in performing any such method, including reagents, antigens, labeling reagents or labeled antigens or antibodies, microspheres, binding membranes such as nitrocellulose, PVDF, or the like, lateral flow strips, with or without reagents needed for performing the disclosed method included therein, any tubes, vials, needles, or other tools needed for obtaining and/or storing samples, etc., are explicitly included in and considered embodiments of an invention disclosed herein. 
     As would be understood by skilled artisans, antigens used in any of the foregoing methods may be modified in accordance with standard molecular biology techniques. For example, antigens may be produced with identifiable tags appended thereto to facilitate isolation, identification, concentration, collection, application, or other uses thereof. As one non-limiting example, antigens with histidine tags may be produced to facilitate separation of recombinant antigens from other constituents employed during synthesis thereof, and subsequent concentration of said antigens in a usable form, solvent, diluent, or concentration. Although histidine tags are mentioned here as one non-limiting example, skilled artisans would appreciate that any of a wide variety of such tools could be adapted to and employed with the above-disclosed methods and compositions, without departing from, and hereby explicitly recounted as included within, the method as disclosed herein. 
     Although non-limiting examples discussed herein include using full-length recombinant flaviviral peptides as antigens, skilled artisans would appreciate that full-length peptides would not necessarily be required for use in all embodiments of the method and compositions disclosed herein. For example, a specific or portions less than full-length viral proteins may be used rather than full-length protein. A portion less than full-length protein to which antibodies produced in response to a subject&#39;s exposure to a given flavivirus will bind may be used rather than full-length protein, for example. In some embodiments, a portion less than full-length flaviviral protein to which antibodies produced in response to a subject&#39;s exposure to a given flavivirus, but not another flavivirus, will bind may be used. In other examples, portions less than full length protein that bind to antibodies produced in response to either of two or more different flaviviruses may be used. 
     In yet other embodiments, an antigen with less than total homology to a flaviviral protein, such as ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4 NS1 may be used, as may a combination of any two or more of the foregoing. For example, a recombinant flaviviral protein with an amino acid sequence that corresponds to some but not all of the amino acids in the flaviviral protein to which it corresponds may be used. In some examples, a recombinant flaviviral antigen with 75% or more homology to a corresponding naturally occurring flaviviral protein may be used. In some examples, a flaviviral antigen with between 75%-80% homology, 80%-85% homology, 85%-90% homology, 90%-95% homology, 95%-100% homology, 90%-91% homology, 91%-92% homology, 92%-93% homology, 93%-94% homology, 94%-95% homology, 95%-96% homology, 96%-97% homology, 97%-98% homology, or 99%-100% homology may be used. Several strains of ZIKV have been identified with high levels of homology between their sequences of ZIKV envelope protein, between their sequences of ZIKV NS1, and between their sequences of ZIKV NS5. ZIKV protein sequences homologous to such known strains may be used, within homology ranges as disclosed herein, in accordance with the present disclosure. A recombinant flaviviral antigen with less than 100% homology to a corresponding flaviviral protein to which antibodies produced in response to a subject&#39;s exposure to a given flavivirus will bind may be used rather than a recombinant protein with 100% homology. In some embodiments, a recombinant flaviviral protein of less than 100% homology to flaviviral protein to which antibodies produced in response to a subject&#39;s exposure to a given flavivirus, but not another flavivirus, will bind may be used. In other examples, a recombinant flaviviral protein of less than 100% homology to flaviviral protein to which antibodies produced in response to a subject&#39;s exposure to two or more given flaviviruses may be used. 
     In other embodiments, an antigen with an amino acid sequence that corresponds to only a portion of a full-length flaviviral protein, such as ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4 NS1 may be used, as may a combination of any two or more of the foregoing. For example, a truncated portion of ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4 NS1, which is recognized by antibodies generated in response to flaviviral infection, may be used. In some embodiments, an antigen may include amino acids not present in naturally occurring flaviviral proteins. For example, as would be understood by skilled artisans, for purposes of antigen synthesis and purification, an antigen may contain an amino acid tag, such as on its C-terminus or its N-terminus, without interfering with the binding thereto of an antibody produced by an individual as a result of flaviviral infection. Many possible such tags ae well-known, including a poly-histidine tag, a myc-tag, a FLAG-tag, an HA-tag, or many others, all of which could be included as part of an antigen and be within the scope of methods and kits as disclosed herein. 
     A sample may be a biological sample, such as a bodily fluid, blood, serum, plasma, saliva, tears, feces, semen, mucous, tissue, tissue homogenate, cellular extract, or spinal fluid, containing anti-flavivirus antigen antibodies, such as IgG anti-NS5, IgM anti-NS5, IgG anti-E, or IgM anti-E. A subject may be any vertebrate, such as a horse, pig, cow, dog, cat, bat, primate, including human, goat, sheep, deer, rabbit, mouse, rat, chicken or other avian species, or other animal. 
     EXAMPLES 
     The following examples are presented to further describe techniques in accordance with the method disclosed herein, but should not be read as limiting, because variations still within the scope of embodiments of the present invention will be apparent to those skilled in the art. 
     Reagents. Wash buffer and phosphate buffered saline pH 7.4, 0.05% sodium azide (PBS-TN) were purchased from Sigma (Sigma Aldrich, St. Louis, Mo.). Chemicals, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), were supplied by Pierce Chemicals (Pierce, Rockford, Ill.). Microspheres, calibration microspheres, and sheath fluid were obtained from Luminex Corporation (Luminex Corp., Austin, Tex.). 
     Serum samples. Studies were performed on serum from de-identified clinical specimens submitted to New York State Department of Health for ZIKV IgM-capture ELISA and Arbovirus MIA testing. 
     Positive and negative serum controls. ZIKV positive control sera were defined as positive titer from a Plaque Reduction Neutralization Test of 90% inhibition (PRNT 90 ) against ZIKV, but negative PRNT 90  titer against DENV. Similarly, DENV positive control sera were defined as positive PRNT 90  titer against DENV, but negative PRNT 90  titer against ZIKV. Negative control sera were defined as no ZIKV PRNT 90  titer as well as negative Arbovirus MIA result using WNV E protein as the diagnostic antigen (Wong et al., 2003). PBN (consisting of PBS, 1% BSA, 0.05% Sodium Azide, pH 7.4) was used as a blank control. 
     Expression and purification of recombinant ZIKA NS5 protein. The cDNA fragment encoding the full-length NS5 of ZIKV was amplified from an infectious clone pFLZIKV (Shan et al., 2016b), fused with a C-terminal (His)6-tag, and cloned into vector pNIC28-Bsa4 (GenBank accession EF198106), resulting in plasmid construct pNIC28-ZIKA-NS5. ZIKA NS5 protein was expressed in  E. coli  Rosetta 2 pLysS  E. coli  (Stratagene) and purified using a method as previously described (Zhao et al., 2015) with some modifications. Briefly, transformed  E. coli  cells was induced by 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the cell density reached OD600 of 0.6-0.8. After incubation at 18° C. for 16 h, the cells were harvested, re-suspended in buffer A (20 mM Tris-HCl, pH 8.5, 550 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 10 mM imidazole, and 0.5×EDTA-free protease inhibitor cocktail) by sonication. The lysate was clarified by centrifugation at 40,000 g for 30 min at 4° C. The resulting supernatant was loaded onto a HisTrap Fast Flow column (GE Healthcare). The protein was eluted using a linear gradient of imidazole concentration from 40 to 500 mM. The fractions containing ZIKA NS5-(His)6 protein were pooled, concentrated, and further purified by gel filtration using a HiLoad Superdex 200 16/60 column (GE Healthcare) in buffer B (20 mM Na-Hepes, pH 8.2, 500 mM NaCl, 10% glycerol, and 5 mM DTT). The peak fractions containing ZIKA NS5-(His)6 protein were pooled and concentrated to approximately 1-2 mg/ml before storage at −80° C. 
     Recombinant ZIKV E, NS1, and DENV NS1 proteins. Recombinant ZIKV E, NS1, and DENV-1 to DENV-4 NS1 proteins were purchased from Meridian (Meridian Life Science, Inc., Memphis, Tenn.). All Meridian recombinant proteins were produced in insect cells and purified by affinity chromatography method. Purified proteins were analyzed by 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and stored in PBS pH 7.4. 
     Conjugation of protein antigens to microsphere Luminex beads. Recombinant proteins were covalently coupled to Luminex MicroPlex Microspheres carboxylated polystyrene microparticles following a previously reported protocol (Wong et al., 2003). Briefly, 50 μg of purified protein was used to couple to the surface of 6.25×106 microspheres in a two-step carbodiimide process. (i) Activation of microspheres. Microspheres were activated with 10 l of N-hydroxysuccinimide (sulfo-NHS) (50 mg/ml) followed by 10 l of 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide-HCl (50 mg/ml). Microspheres were then incubated for 20 min at room temperature with gentle vortexing at 10-min intervals. (ii) Coupling of recombinant proteins. Each recombinant protein was added to the activated microspheres with distinct fluorescence. Protein-microsphere mixtures were incubated for 3 h in the dark on a LabTech tube rotator (Barstead/Thermolyne, Dubuque, Iowa). The microspheres were then washed twice by centrifugation and resuspended in 1.0 ml PBS-TN [phosphate buffered saline pH 7.4, 0.05% sodium azide, 1% bovine serum albumin (BSA)]. The protein coupled microsphere were then stored at 4° C. 
     Multiplex reagent preparation and microsphere immunofluorescence assay (MIA) procedure. All reagent dilutions and assays were carried out in PBS-TN (phosphate buffered saline pH 7.4, 0.05% sodium azide, 1% BSA) (Sigma Aldrich, St. Louis, Mo.). The bead mixture consisted of seven beads, each coupled with a different flavivirus recombinant protein. Three bead sets contained ZIKV E (Meridian), NS1 (Meridian), and NS5 (described above). Four other bead sets contained NS1 proteins from DENV-1 to -4. The bead sets were stored at 4° C. in the dark and diluted 1:100 directly in PBS-TN before use. Biotin conjugated goat anti-human IgG/A/M affinity purified secondary antibody (Life Technologies, Grand Island, N.Y.) was diluted 1:8000 in PBS-TN directly before use. Streptavidin-R-phycoerythrin (1 mg/ml SA-PE, Life Technologies, Grand Island, N.Y.) was diluted 1:100 in PBS-TN before use. All serum samples were stored at −80° C. Samples were thawed and diluted 1:100 in PBS-TN right before use. Diluted samples were used within 1 h post dilution. 
     A 96-well MultiScreenHTS BV 1.2 μm Filter Plate (Millipore Billerica, Mass.) was wetted with 100 μl PBS-TN and washed once with washing buffer (PBS, 0.05% Tween 20, pH 7.4; Sigma Aldrich, St. Louis, Mo.). Samples (50 μl) were dispensed in each well to which 50 μl bead mixture was added. The plate was incubated in the dark on a shaker for 30 min and washed three times with 190 μl washing buffer. After addition of 50 μl conjugate antibody, samples were incubated in the dark on a shaker for 30 min and washed three times with 190 μl wash buffer. After adding detection reagent SA-PE (50 μl), samples were incubated in the dark on a shaker for 30 min, washed twice with 190 μl wash buffer, and transferred to a flat bottom 96-well plate (Corning Incorporated, Kennebunk, Me.). Analysis was performed using a Luminex 100 Analyzer configured to count 100 beads per bead class and a 100 μl sample size. 
     For avidity assays, urea at a final concentration of 8 M was added to a sample during incubation of the sample with microspheres bound to a given antigen and urea incubation was for 10 min. Another microsphere-sample was incubated in the absence of urea in parallel. Median fluorescence intensity of each sample was then determined. An Avidity Index was identified, which is a ratio of median fluorescence intensity of urea-treated sample divided by median fluorescence intensity of non-urea treated sample, times 100. An IgG Avidity Index of less than or equal to 30% indicates infection occurred within the past 65 days, or early acute infection. An IgG Avidity Index of from 31% to 60% indicates infection occurred between 65 and 190 days ago, or late acute infection. An avidity index of greater than or equal to 60% indicates infection occurred more than 190 days ago. In samples in which anti-Zika NS1 or anti-Zika NS5 antibodies are detected in addition to anti-DENV antigen antibodies, avidity testing assists in determining whether one infection preceded the other, or whether one or the other infection is current or more acute than the other. For example, a subject may have a high Avidity Index for anti-DENV antigens and a low Avidity Index for anti-ZIKV antigens, indicating recent or current ZIKV infection and past DENV infection. 
     Results 
     Rationale of assay platform and antigen selection. The MIA platform for assay development was used because of (i) its multiplex capability to simultaneously detect antibodies against a number of viral proteins, (ii) rapid assay turnaround time in &lt;4 h, and (iii) low specimen volume requirement (10 μl serum). This is in contrast with the well-established IgM-capture ELISA platform that requires &gt;2 days of the assay turnaround time. For antigen selection, three recombinant ZIKV proteins were employed for the multiplex assay: E, NS1, and NS5. ZIKV E protein was included to sensitively detect viral infection; however, due to the cross-reactive nature of E antibodies among flaviviruses, an E-positive signal does not confirm ZIKV infection. ZIKV NS1 and NS5 proteins were included to improve assay specificity through detection of virus-type specific antibodies. In addition, recombinant DENV NS1 proteins from each of the four serotypes were included for testing virus-type specificity. All antigens, except ZIKV NS5, were commercially purchased. ZIKV NS5 was cloned, expressed, and purified to &gt;95% homogeneity. See  FIG.  1   . SDS-PAGE analysis of recombinant ZIKV NS5 protein. Full-length NS5 of ZIKV was expressed in an  E. coli  system, and purified through affinity followed by size-exclusion chromatography (see details in Materials and Methods). The recombinant protein was analyzed on a 12% Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio-rad). 
     Establishment of multiplex MIA. Seven recombinant antigens described above (ZIKV E, NS1, NS5, and DENV-1 to -4 NS1) were individually conjugated to microsphere beads, each with a distinct fluorescent signature. A mixture of seven antigen-conjugated beads were reacted with patient serum and quantified by anti-human immunoglobulins (reactive with IgG, IgM, and IgA) with a red fluorescent phycoerythrin. To establish the cutoff level for each antigen, we assayed 20 presumed human sera from healthy individuals in the context of multiplex MIA. The results revealed cutoff values (defined as mean plus three times standard deviations) to be 1363, 284, 1905, 746, 549, 339, and 655 for ZIKV E, NS1, NS5, and DENV-1, -2, -3, and -4 NS1, respectively. These cutoff values were used to determine positive (&gt;cutoff) and negative (&lt;cutoff) when diagnosing patient specimens. 
     Stratification of patient sera. A well-defined set of patient specimens may be used to develop and verify the multiplex assay. A total of 154 patient sera with known ZIKV and DENV PRNT results were selected for assay development. Based on the PRNT results, patient sera were categorized into four distinct groups. Group I specimens (7 patients; Table 1) were both ZIKV- and DENV-negative, as defined by PRNTs&lt;10 for both viruses. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Type I specimens with neither ZIKV nor DENV infection defined by PRNT* 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 ZIKV IgM- 
                   
                   
                   
                   
                   
                   
                   
               
               
                 ZIKV 
                 DENV 
                 capture 
                   
                 ZIKV 
                 ZIKV 
                 DENV-1 
                 DENV-2 
                 DENV-3 
                 DENV-4 
               
               
                 PRNT 
                 PRNT 
                 ELISA 
                 ZIKV E 
                 NS1 
                 NS5 
                 NS1 
                 NS1 
                 NS1 
                 NS1 
               
               
                 Cutoff 
                 Cutoff 
                 Cutoff 2 &amp; 
                 Cutoff 
                 Cutoff  
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
               
               
                 10 
                 10 
                 3 
                 1363 
                 284 
                 1905 
                 746 
                 549 
                 339 
                 655 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 &lt;10 
                 &lt;10 
                 0.2 
                 1189 
                 167 
                 573 
                 1200 
                 935 
                 100 
                 450 
               
               
                 &lt;10 
                 &lt;10 
                 0.5 
                 1415 
                 155 
                 308 
                 220 
                 259 
                 61 
                 96 
               
               
                 &lt;10 
                 &lt;10 
                 0.9 
                 195 
                 54 
                 1128 
                 87 
                 51 
                 230 
                 175 
               
               
                 &lt;10 
                 &lt;10 
                 1.9 
                 366 
                 91 
                 1900 
                 306 
                 216 
                 168 
                 136 
               
               
                 &lt;10 
                 &lt;10 
                 1.9 
                 7742 
                 4619 
                 4267 
                 127 
                 105 
                 113 
                 275 
               
               
                 &lt;10 
                 &lt;10 
                 3.8 
                 178 
                 69 
                 534 
                 235 
                 141 
                 113 
                 211 
               
               
                 &lt;10 
                 &lt;10 
                 11.6 
                 1001 
                 53 
                 211 
                 418 
                 509 
                 75 
                 125 
               
               
                   
               
               
                 *The table is sorted with increasing value of IgM-capture ELISA result. 
               
            
           
         
       
     
     Group II specimens (9 patients; Table 2) were ZIKV-negative and DENV-positive, as defined by ZIKV PRNT&lt;10 and DENY PRNT&gt;10. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Type II specimens with DENV only infection defined by PRNT* 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 ZIKV IgM- 
                   
                   
                   
                   
                   
                   
                   
               
               
                 ZIKV 
                 DENV 
                 capture 
                   
                 ZIKV 
                 ZIKV 
                 DENV-1 
                 DENV-2 
                 DENV-3 
                 DENV-4 
               
               
                 PRNT 
                 PRNT 
                 ELISA 
                 ZIKV E 
                 NS1 
                 NS5 
                 NS1 
                 NS1 
                 NS1 
                 NS1 
               
               
                 Cutoff 
                 Cutoff 
                 Cutoff 2 &amp; 
                 Cutoff 
                 Cutoff  
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
               
               
                 10 
                 10 
                 3 
                 1363 
                 284 
                 1905 
                 746 
                 549 
                 339 
                 655 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 &lt;10 
                 20 
                 0.19 
                  9131 
                 234 
                 170 
                 5375 
                 1579 
                 3091 
                 1442 
               
               
                 &lt;10 
                 40 
                 0.60 
                  7145 
                 211 
                 331 
                 9101 
                 2831 
                 5415 
                 3740 
               
               
                 &lt;10 
                 80 
                 1.5 
                 11139 
                 2514 
                 249 
                 9145 
                 6658 
                 10609 
                 14941 
               
               
                 &lt;10 
                 160 
                 1.5 
                  9596 
                 1806 
                 177 
                 7486 
                 5198 
                 8241 
                 11867 
               
               
                 &lt;10 
                 640 
                 1.5 
                  5381 
                 46 
                 227 
                 5323 
                 860 
                 2235 
                 1352 
               
               
                 &lt;10 
                 80 
                 1.6 
                  8071 
                 67 
                 554 
                 6862 
                 510 
                 1765 
                 658 
               
               
                 &lt;10 
                 40 
                 5.1 
                  6017 
                 51 
                 1473  
                  98 
                 50 
                 99 
                 165 
               
               
                 &lt;10 
                 160 
                 5.8 
                  7756 
                 38 
                 968 
                 4259 
                 179 
                 582 
                 305 
               
               
                 &lt;10 
                 40 
                 77 
                  8703 
                 106 
                 1009  
                 8805 
                 1511 
                 3165 
                 1558 
               
               
                   
               
               
                 *The table is sorted with increasing value of IgM-capture ELISA result. 
               
            
           
         
       
     
     Group III specimens (43 patients; Table 3) were ZIKV-positive and DENV-negative, as defined by ZIKV PRNT&gt;10 and DENV PRNT&lt;10. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Type III specimens with ZIKV only infection defined by PRNT * 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 ZIKV IgM- 
                   
                   
                   
                   
                   
                   
                   
               
               
                 ZIKV 
                 DENV 
                 capture 
                   
                 ZIKV 
                 ZIKV 
                 DENV-1 
                 DENV-2 
                 DENV-3 
                 DENV-4 
               
               
                 PRNT 
                 PRNT 
                 ELISA 
                 ZIKV E 
                 NS1 
                 NS5 
                 NS1 
                 NS1 
                 NS1 
                 NS1 
               
               
                 Cutoff 
                 Cutoff 
                 Cutoff 2 &amp; 
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
               
               
                 10 
                 10 
                 3 
                 1363 
                 284 
                 1905 
                 746 
                 549 
                 339 
                 655 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 80 
                 &lt;10 
                 0.6 
                 9960 
                 5207 
                 2624 
                 1346 
                 1172 
                 454 
                 744 
               
               
                 320 
                 &lt;10 
                 0.99 
                 10692 
                 6173 
                 1676 
                 409 
                 531 
                 660 
                 631 
               
               
                 2560 
                 &lt;10 
                 1.8 
                 16318 
                 9407 
                 10423 
                 548 
                 737 
                 802 
                 492 
               
               
                 320 
                 &lt;10 
                 1.9 
                 12377 
                 6550 
                 21510 
                 391 
                 644 
                 927 
                 422 
               
               
                 40 
                 &lt;10 
                 2.4 
                 858 
                 617 
                 1401 
                 349 
                 272 
                 249 
                 374 
               
               
                 10 
                 &lt;10 
                 2.75 
                 2185 
                 297 
                 1777 
                 795 
                 729 
                 189 
                 285 
               
               
                 80 
                 &lt;10 
                 3.04 
                 800 
                 481 
                 227 
                 163 
                 213 
                 118 
                 252 
               
               
                 160 
                 &lt;10 
                 3.80 
                 9088 
                 7010 
                 3298 
                 438 
                 588 
                 782 
                 459 
               
               
                 320 
                 &lt;10 
                 3.97 
                 11272 
                 4849 
                 2129 
                 1556 
                 1268 
                 250 
                 838 
               
               
                 320 
                 &lt;10 
                 4 
                 11358 
                 4933 
                 2179 
                 1503 
                 1212 
                 212 
                 735 
               
               
                 320 
                 &lt;10 
                 4.5 
                 8592 
                 5500 
                 4362 
                 311 
                 455 
                 452 
                 220 
               
               
                 2560 
                 &lt;10 
                 4.5 
                 8592 
                 5500 
                 4362 
                 311 
                 455 
                 452 
                 220 
               
               
                 160 
                 &lt;10 
                 6.2 
                 3896 
                 3152 
                 2524 
                 574 
                 477 
                 203 
                 371 
               
               
                 160 
                 &lt;10 
                 7.88 
                 5782 
                 701 
                 574 
                 340 
                 260 
                 125 
                 217 
               
               
                 1280 
                 &lt;10 
                 7.9 
                 3233 
                 5196 
                 5865 
                 231 
                 263 
                 315 
                 246 
               
               
                 1280 
                 &lt;10 
                 7.9 
                 4777 
                 6771 
                 8260 
                 364 
                 417 
                 508 
                 381 
               
               
                 320 
                 &lt;10 
                 8.1 
                 8842 
                 4811 
                 15599 
                 214 
                 349 
                 561 
                 276 
               
               
                 320 
                 &lt;10 
                 8.1 
                 9768 
                 5001 
                 18463 
                 253 
                 405 
                 532 
                 294 
               
               
                 160 
                 &lt;10 
                 8.29 
                 457 
                 540 
                 930 
                 464 
                 258 
                 204 
                 168 
               
               
                 1280 
                 &lt;10 
                 10.9 
                 496 
                 1010 
                 3139 
                 102 
                 84 
                 74 
                 108 
               
               
                 160 
                 &lt;10 
                 13.5 
                 12420 
                 7611 
                 1809 
                 141 
                 195 
                 218 
                 401 
               
               
                 160 
                 &lt;10 
                 13.5 
                 14292 
                 8757 
                 2491 
                 180 
                 243 
                 265 
                 548 
               
               
                 160 
                 &lt;10 
                 13.7 
                 7432 
                 6664 
                 2876 
                 90 
                 159 
                 337 
                 149 
               
               
                 40 
                 &lt;10 
                 17 
                 536 
                 1554 
                 2612 
                 153 
                 202 
                 122 
                 153 
               
               
                 640 
                 &lt;10 
                 17.70 
                 6381 
                 7713 
                 18520 
                 1095 
                 1082 
                 628 
                 419 
               
               
                 640 
                 &lt;10 
                 20.1 
                 2474 
                 2312 
                 2038 
                 197 
                 205 
                 67 
                 82 
               
               
                 640 
                 &lt;10 
                 20.18 
                 3703 
                 1566 
                 4269 
                 75 
                 69 
                 314 
                 54 
               
               
                 160 
                 &lt;10 
                 22 
                 530 
                 428 
                 679 
                 151 
                 116 
                 110 
                 119 
               
               
                 160 
                 &lt;10 
                 22 
                 999 
                 5442 
                 3802 
                 217 
                 234 
                 263 
                 86 
               
               
                 320 
                 &lt;10 
                 25 
                 7905 
                 6814 
                 5828 
                 244 
                 350 
                 242 
                 166 
               
               
                 320 
                 &lt;10 
                 25.5 
                 8518 
                 6914 
                 6650 
                 261 
                 378 
                 303 
                 228 
               
               
                 1280 
                 &lt;10 
                 27 
                 8494 
                 5334 
                 3631 
                 102 
                 107 
                 103 
                 274 
               
               
                 1280 
                 &lt;10 
                 27.18 
                 10443 
                 6998 
                 4530 
                 157 
                 163 
                 158 
                 407 
               
               
                 640 
                 &lt;10 
                 28 
                 3488 
                 11213 
                 4269 
                 290 
                 452 
                 579 
                 62 
               
               
                 640 
                 &lt;10 
                 28.8 
                 8741 
                 7471 
                 3820 
                 278 
                 492 
                 662 
                 147 
               
               
                 640 
                 &lt;10 
                 29.9 
                 6212 
                 4915 
                 5969 
                 193 
                 221 
                 288 
                 115 
               
               
                 640 
                 &lt;10 
                 30 
                 3998 
                 3187 
                 3867 
                 133 
                 141 
                 158 
                 73 
               
               
                 1280 
                 &lt;10 
                 32 
                 11745 
                 7674 
                 4459 
                 174 
                 49 
                 242 
                 250 
               
               
                 1280 
                 &lt;10 
                 32.3 
                 13259 
                 8154 
                 4726 
                 227 
                 309 
                 304 
                 309 
               
               
                 640 
                 &lt;10 
                 42.1 
                 9721 
                 7456 
                 658 
                 143 
                 276 
                 230 
                 139 
               
               
                 2560 
                 &lt;10 
                 47.2 
                 16346 
                 4928 
                 3714 
                 149 
                 158 
                 320 
                 60 
               
               
                 320 
                 &lt;10 
                 50.4 
                 8250 
                 7160 
                 638 
                 134 
                 251 
                 215 
                 122 
               
               
                 320 
                 &lt;10 
                 65.6 
                 3645 
                 2785 
                 1229 
                 52 
                 74 
                 77 
                 79 
               
               
                   
               
               
                 *The table is sorted with increasing value of IgM-capture ELISA result. 
               
            
           
         
       
     
     Group IV specimens (95 patients; Table 4) were both ZIKV- and DENV-positive, as defined by PRNTs&gt;10 for both viruses. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Type IV specimens with ZIKV and/or DENV infection(s) defined by PRNT* 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 ZIKV IgM- 
                   
                   
                   
                   
                   
                   
                   
               
               
                 ZIKV 
                 DENV 
                 capture 
                   
                 ZIKV 
                 ZIKV 
                 DENV-1 
                 DENV-2 
                 DENV-3 
                 DENV-4 
               
               
                 PRNT 
                 PRNT 
                 ELISA 
                 ZIKV E 
                 NS1 
                 NS5 
                 NS1 
                 NS1 
                 NS1 
                 NS1 
               
               
                 Cutoff 
                 Cutoff 
                 Cutoff 2 &amp; 
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
                 Cutoff 
               
               
                 10 
                 10 
                 3 
                 1363 
                 284 
                 1905 
                 746 
                 549 
                 339 
                 655 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 2560 
                 320 
                 0.3 
                 12411 
                 3002 
                 6118 
                 7992 
                 5677 
                 11340 
                 10357 
               
               
                 10 
                 40 
                 0.40 
                 7446 
                 285 
                 790 
                 7714 
                 2875 
                 5725 
                 4420 
               
               
                 320 
                 2560 
                 0.4 
                 15916 
                 4164 
                 11951 
                 10070 
                 8345 
                 14047 
                 13683 
               
               
                 20 
                 1280 
                 0.41 
                 18428 
                 802 
                 461 
                 7969 
                 5627 
                 10631 
                 9581 
               
               
                 80 
                 640 
                 0.45 
                 17887 
                 2203 
                 665 
                 5073 
                 3700 
                 5778 
                 9545 
               
               
                 160 
                 640 
                 0.45 
                 18357 
                 2550 
                 796 
                 5886 
                 4181 
                 6677 
                 10700 
               
               
                 20 
                 640 
                 0.45 
                 17361 
                 874 
                 551 
                 9206 
                 6339 
                 11251 
                 11171 
               
               
                 10 
                 80 
                 0.49 
                 16355 
                 1950 
                 570 
                 10920 
                 8275 
                 15354 
                 11740 
               
               
                 1280 
                 2560 
                 0.53 
                 18809 
                 6682 
                 650 
                 16472 
                 10777 
                 18272 
                 19435 
               
               
                 10 
                 160 
                 0.53 
                 8131 
                 844 
                 325 
                 5142 
                 3838 
                 7463 
                 6020 
               
               
                 1280 
                 2560 
                 0.56 
                 20863 
                 6548 
                 602 
                 16500 
                 10369 
                 19309 
                 19308 
               
               
                 160 
                 2560 
                 0.62 
                 17472 
                 3453 
                 2503 
                 12655 
                 2806 
                 11109 
                 16787 
               
               
                 160 
                 2560 
                 0.69 
                 18220 
                 3461 
                 2384 
                 13241 
                 2871 
                 12265 
                 17857 
               
               
                 2560 
                 640 
                 0.8 
                 18354 
                 8434 
                 2396 
                 382 
                 426 
                 556 
                 309 
               
               
                 640 
                 1280 
                 1.09 
                 16382 
                 4065 
                 3303 
                 11394 
                 7116 
                 13885 
                 7695 
               
               
                 320 
                 2560 
                 1.3 
                 17322 
                 4085 
                 875 
                 15507 
                 11175 
                 17637 
                 16700 
               
               
                 2560 
                 320 
                 1.41 
                 19995 
                 6926 
                 8456 
                 12524 
                 11268 
                 17951 
                 16740 
               
               
                 640 
                 1280 
                 1.53 
                 21501 
                 7952 
                 3037 
                 16148 
                 12733 
                 19792 
                 22445 
               
               
                 320 
                 160 
                 1.63 
                 14009 
                 6104 
                 1374 
                 11801 
                 11004 
                 17719 
                 4475 
               
               
                 320 
                 2560 
                 1.7 
                 19788 
                 8615 
                 3494 
                 16160 
                 13589 
                 19342 
                 20432 
               
               
                 160 
                 2560 
                 1.8 
                 16928 
                 3607 
                 551 
                 14462 
                 10561 
                 17158 
                 15002 
               
               
                 1280 
                 1280 
                 1.81 
                 17896 
                 7868 
                 2698 
                 14294 
                 11672 
                 17853 
                 21180 
               
               
                 320 
                 2560 
                 1.9 
                 20374 
                 8309 
                 2755 
                 16466 
                 13975 
                 19997 
                 20689 
               
               
                 640 
                 2560 
                 2.1 
                 21021 
                 12154 
                 15783 
                 18993 
                 14796 
                 21073 
                 22419 
               
               
                 640 
                 2560 
                 2.16 
                 21035 
                 11444 
                 13536 
                 18240 
                 15675 
                 21260 
                 22412 
               
               
                 5160 
                 5120 
                 2.3 
                 18554 
                 6707 
                 553 
                 12751 
                 10464 
                 17362 
                 20153 
               
               
                 5120 
                 1280 
                 2.3 
                 19606 
                 11677 
                 11105 
                 14953 
                 11886 
                 18231 
                 21051 
               
               
                 5120 
                 1280 
                 2.32 
                 18697 
                 9335 
                 7705 
                 12257 
                 9981 
                 16633 
                 17776 
               
               
                 640 
                 1280 
                 2.45 
                 20559 
                 10939 
                 21381 
                 14675 
                 10696 
                 18537 
                 17415 
               
               
                 2560 
                 1280 
                 2.5 
                 19945 
                 9641 
                 13280 
                 15589 
                 13022 
                 19484 
                 15561 
               
               
                 2560 
                 1280 
                 2.52 
                 18959 
                 8035 
                 11647 
                 14771 
                 12827 
                 19260 
                 13601 
               
               
                 320 
                 40 
                 2.57 
                 9760 
                 1309 
                 386 
                 5967 
                 1610 
                 2724 
                 3719 
               
               
                 160 
                 40 
                 2.59 
                 10893 
                 859 
                 180 
                 3100 
                 914 
                 1643 
                 6687 
               
               
                 160 
                 40 
                 2.72 
                 13602 
                 1082 
                 205 
                 4179 
                 1260 
                 2191 
                 8609 
               
               
                 320 
                 40 
                 2.99 
                 10133 
                 1442 
                 417 
                 6469 
                 1696 
                 3018 
                 4257 
               
               
                 320 
                 5120 
                 3.13 
                 18591 
                 7977 
                 1050 
                 11995 
                 9617 
                 15868 
                 14587 
               
               
                 160 
                 320 
                 3.53 
                 19680 
                 5587 
                 1681 
                 317 
                 292 
                 543 
                 270 
               
               
                 2560 
                 2560 
                 3.89 
                 17268 
                 4501 
                 5464 
                 13748 
                 9869 
                 17527 
                 12169 
               
               
                 5120 
                 5120 
                 3.99 
                 21644 
                 10859 
                 21825 
                 12761 
                 9859 
                 17755 
                 20283 
               
               
                 160 
                 320 
                 4.02 
                 16822 
                 6103 
                 2070 
                 14021 
                 10298 
                 17324 
                 15517 
               
               
                 640 
                 1280 
                 4.42 
                 20593 
                 5338 
                 1739 
                 12752 
                 11158 
                 18128 
                 18042 
               
               
                 1280 
                 1280 
                 4.43 
                 19973 
                 8594 
                 11143 
                 12763 
                 11051 
                 17739 
                 16997 
               
               
                 1280 
                 320 
                 4.79 
                 19355 
                 10636 
                 9796 
                 12751 
                 10778 
                 18003 
                 18073 
               
               
                 640 
                 1280 
                 4.88 
                 16101 
                 4858 
                 776 
                 13093 
                 8198 
                 14431 
                 8430 
               
               
                 2560 
                 1280 
                 5.01 
                 20780 
                 12080 
                 17177 
                 16920 
                 13760 
                 20359 
                 17012 
               
               
                 160 
                 640 
                 5.1 
                 19275 
                 6311 
                 2744 
                 14623 
                 12984 
                 19646 
                 15016 
               
               
                 160 
                 640 
                 5.34 
                 16289 
                 5747 
                 2635 
                 15009 
                 11904 
                 18826 
                 16649 
               
               
                 10 
                 80 
                 5.47 
                 16794 
                 1307 
                 1328 
                 389 
                 200 
                 346 
                 281 
               
               
                 1280 
                 640 
                 6.05 
                 16809 
                 6987 
                 3429 
                 13666 
                 12416 
                 18579 
                 5716 
               
               
                 1280 
                 80 
                 6.52 
                 2111 
                 3626 
                 1100 
                 209 
                 289 
                 243 
                 291 
               
               
                 320 
                 160 
                 7.04 
                 19585 
                 2905 
                 3390 
                 10849 
                 5579 
                 10368 
                 11556 
               
               
                 320 
                 160 
                 7.04 
                 20334 
                 3419 
                 4461 
                 11917 
                 6275 
                 12136 
                 12769 
               
               
                 320 
                 80 
                 7.32 
                 19940 
                 2687 
                 2942 
                 10583 
                 5368 
                 9833 
                 10471 
               
               
                 1280 
                 10 
                 7.49 
                 13338 
                 9654 
                 6618 
                 462 
                 650 
                 1220 
                 700 
               
               
                 1280 
                 10 
                 7.5 
                 12624 
                 9195 
                 6717 
                 341 
                 478 
                 986 
                 514 
               
               
                 2560 
                 20480 
                 8.3 
                 20265 
                 9171 
                 2720 
                 16781 
                 12747 
                 18712 
                 20473 
               
               
                 640 
                 5120 
                 8.5 
                 20671 
                 9098 
                 7078 
                 17100 
                 14388 
                 18608 
                 22061 
               
               
                 640 
                 10240 
                 9.04 
                 21114 
                 11671 
                 11204 
                 19358 
                 15223 
                 21543 
                 22106 
               
               
                 2560 
                 320 
                 10.16 
                 18279 
                 9899 
                 7213 
                 4524 
                 3165 
                 6948 
                 1278 
               
               
                 160 
                 640 
                 10.90 
                 20183 
                 7660 
                 2078 
                 2723 
                 2628 
                 5909 
                 1384 
               
               
                 320 
                 640 
                 10.99 
                 14737 
                 4336 
                 15626 
                 11828 
                 9458 
                 15927 
                 6841 
               
               
                 320 
                 2560 
                 11.1 
                 20977 
                 10041 
                 2254 
                 17295 
                 13477 
                 19549 
                 10638 
               
               
                 640 
                 10240 
                 11.4 
                 20785 
                 12169 
                 11619 
                 18884 
                 15119 
                 20667 
                 21989 
               
               
                 1280 
                 2560 
                 12 
                 19968 
                 8988 
                 4259 
                 17044 
                 13238 
                 19273 
                 22230 
               
               
                 1280 
                 2560 
                 12.54 
                 20467 
                 8901 
                 3673 
                 16470 
                 13221 
                 19341 
                 22846 
               
               
                 320 
                 640 
                 13 
                 20546 
                 6045 
                 1818 
                 15929 
                 10974 
                 18017 
                 3192 
               
               
                 640 
                 1280 
                 14 
                 18531 
                 5202 
                 1896 
                 11359 
                 10329 
                 16770 
                 16440 
               
               
                 640 
                 640 
                 14.64 
                 20336 
                 7675 
                 1722 
                 13716 
                 12002 
                 18849 
                 20055 
               
               
                 320 
                 2560 
                 15 
                 21156 
                 10463 
                 3187 
                 18463 
                 14572 
                 20246 
                 22876 
               
               
                 640 
                 2560 
                 16 
                 20527 
                 9862 
                 5041 
                 16141 
                 12408 
                 19080 
                 21991 
               
               
                 640 
                 2560 
                 16.29 
                 19340 
                 9066 
                 5185 
                 15029 
                 11748 
                 17407 
                 20104 
               
               
                 2560 
                 20480 
                 17 
                 22317 
                 6665 
                 7447 
                 15855 
                 10725 
                 17403 
                 5509 
               
               
                 2560 
                 20480 
                 17.23 
                 17999 
                 5738 
                 7214 
                 13635 
                 8930 
                 14737 
                 5748 
               
               
                 160 
                 1280 
                 17.41 
                 19848 
                 8375 
                 6839 
                 15679 
                 13745 
                 18998 
                 22484 
               
               
                 160 
                 160 
                 20.73 
                 9391 
                 6074 
                 3340 
                 356 
                 679 
                 566 
                 309 
               
               
                 10240 
                 320 
                 22 
                 17752 
                 7270 
                 4264 
                 62 
                 156 
                 388 
                 96 
               
               
                 1280 
                 1280 
                 22.2 
                 20185 
                 8584 
                 16360 
                 16030 
                 12594 
                 20100 
                 10783 
               
               
                 1280 
                 20480 
                 23 
                 20703 
                 10096 
                 7859 
                 18086 
                 13614 
                 18507 
                 21267 
               
               
                 320 
                 20 
                 23.6 
                 9924 
                 8081 
                 9837 
                 113 
                 203 
                 469 
                 231 
               
               
                 1280 
                 5120 
                 23.69 
                 18836 
                 8498 
                 4119 
                 15799 
                 12910 
                 18325 
                 20333 
               
               
                 320 
                 10 
                 25 
                 1061 
                 3108 
                 2294 
                 88 
                 89 
                 83 
                 894 
               
               
                 640 
                 640 
                 25.23 
                 20066 
                 9245 
                 4529 
                 17030 
                 14304 
                 20372 
                 22431 
               
               
                 2560 
                 2560 
                 26.13 
                 21184 
                 9524 
                 2734 
                 15615 
                 13845 
                 19053 
                 22401 
               
               
                 640 
                 2560 
                 27.9 
                 19121 
                 3787 
                 3230 
                 3522 
                 3308 
                 7426 
                 3591 
               
               
                 1280 
                 10240 
                 29.15 
                 19711 
                 10327 
                 2437 
                 16973 
                 14262 
                 18898 
                 20341 
               
               
                 1280 
                 2560 
                 29.9 
                 21902 
                 8590 
                 3728 
                 16006 
                 13452 
                 20836 
                 22510 
               
               
                 640 
                 1280 
                 30.53 
                 18080 
                 8675 
                 17545 
                 16414 
                 13525 
                 18677 
                 14452 
               
               
                 2560 
                 640 
                 32 
                 20207 
                 6094 
                 11566 
                 10231 
                 7930 
                 14480 
                 11206 
               
               
                 640 
                 2560 
                 33.4 
                 20649 
                 10126 
                 6628 
                 16806 
                 13194 
                 18759 
                 22293 
               
               
                 320 
                 160 
                 35.88 
                 4640 
                 4268 
                 2211 
                 294 
                 263 
                 175 
                 169 
               
               
                 320 
                 10 
                 44.8 
                 9837 
                 7200 
                 2590 
                 284 
                 253 
                 308 
                 194 
               
               
                 2560 
                 2560 
                 46.1 
                 19943 
                 7114 
                 7170 
                 15766 
                 11664 
                 18454 
                 8917 
               
               
                 640 
                 640 
                 47.93 
                 2828 
                 5437 
                 7229 
                 334 
                 1185 
                 1407 
                 299 
               
               
                 1280 
                 20 
                 49.11 
                 14063 
                 6262 
                 6839 
                 359 
                 291 
                 193 
                 250 
               
               
                 1280 
                 640 
                 57 
                 19062 
                 6018 
                 11756 
                 12258 
                 10914 
                 17240 
                 17024 
               
               
                   
               
               
                 *The table is sorted with increasing value of IgM-capture ELISA result. 
               
            
           
         
       
     
     It should be noted that, due to cross-neutralization of antibodies among flaviviruses, group IV specimens could derive from patients who were (i) infected with both ZIKV and DENV, (ii) infected with ZIKV only but had antibodies cross-reactive to DENV, or (iii) infected with DENV only but with antibodies cross-reactive to ZIKV. 
     Multiplex MIA and IgM-capture ELISA diagnosis. Patient samples were subjected to multiplex MIA and the well-established IgM-capture ELISA (Martin et al., 2000). Tables S1 to S4 summarize the results for groups I to IV specimens, respectively. Each specimen is presented with results from PRNT, IgM-capture ELISA, and multiplex MIA for individual antigens. It should be pointed out that, for IgM-capture ELISA, P/N&lt;2 is defined as negative, P/N 2-3 as equivocal, and P/N&gt;3 as positive. Table 5 in  FIG.  2    summarizes the overall diagnostic results. For Table 5 in  FIG.  2   , results from Tables 1 to 4 are summarized for comparison of PRNT, IgM-capture ELISA, and multiplex MIA diagnosis. For each diagnostic parameter, the total number of samples that were diagnosed as “positive” (greater than cutoff line) or “negative” (less than cutoff line) is indicated, followed by its corresponding percentage of the total number of specimens from that specific specimen group. Percentage (%)=(number of positive or negative specimens/total number of specimen from the specific specimen group)×100%. 
     For group I specimens (neither ZIKV nor DENV infection), both ZIKV IgM-capture ELISA and E MIA showed 71% negative; the MIA results from ZIKV NS1, ZIKV NS5, and combined DENV-1 to -4 NS1 showed 86% negative. For group II specimens (DENV infection only), ZIKV IgM-capture ELISA showed 67% negative (i.e., 33% cross-reactivity with DENV); ZIKV E and combined DENV-1 to -4 NS1 MIA showed 100% and 89% positive, respectively; in contrast, ZIKV NS1 and NS5 MIA showed 78% and 100% negative, respectively. For group III specimens (ZIKV infection only), ZIKV IgM-capture ELISA showed 86% positive; ZIKV E, NS1, and NS5 MIA showed 84%, 100%, and 74% positive, respectively; whereas combined DENV-1 to -4 NS1 MIA showed 63% negative (i.e., 37% cross-reactivity with ZIKV). For group IV specimens (at least one infection from ZIKV and/or DENV), ZIKV IgM-capture ELISA, E, NS1, NS5, and combined DENV NS1 showed 63%, 99%, 100%, 73%, and 96% positive, respectively. These results enable the following analysis. 
     (I) Comparison of ZIKV IgM-capture ELISA and E MIA. Compared with IgM-capture ELISA, ZIKV E MIA alone showed equivalent accuracy when diagnosing group III specimens, with 86% and 84% of the samples tested positive from IgM-capture ELISA and E MIA, respectively. When diagnosing group IV specimens, the E MIA showed better sensitivity than IgM-capture ELISA, with 63% and 99% of specimens tested positive, respectively. Two factors may account for this improvement. (i) MIA measures IgG and IgA in addition to IgM, whereas IgM-capture ELISA does not capture IgG and IgA. (ii) The amount of IgM declines after the convalescent phase of ZIKV infection; therefore, specimens (taken long after convalescent phase) may have low levels of IgM and high levels of IgG, which is not detected by the IgM-capture ELISA. Taken together, the results indicate that E MIA alone has equivalent or better sensitivity than IgM-capture ELISA. 
     (II) Relative specificity of ZIKV E, NS1, and NS5 MIA. Comparison of the results from ZIKV E, NS1, and NS5 MIA demonstrates that antibody response to NS1 and NS5 antigens is more ZIKV-specific than that to E antigen. Specifically, ZIKV E MIA showed 100% cross-reactivity with specimens with DENV only infection from group II, confirming the cross-reactive nature of flavivirus E antibodies. In contrast, ZIKV NS1 MIA showed 14% and 22% false positive results when testing groups I and II specimens, but 100% positive accuracy when analyzing groups III and IV specimens. For ZIKV NS5 MIA, the assay exhibited 14% and 0% false positive results when testing groups I and II specimens, and 74% and 73% positive accuracy when analyzing groups III and IV specimens. The results clearly indicate that inclusion of ZIKV NS1 and NS5 in the MIA could improve the diagnostic accuracy when compared with the MIA that uses E protein alone. 
     (III) Cross reactivity between DENV/ZIKV NS1 proteins and their antibodies. Although antibody response to ZIKV NS1 is more virus-type specific than that to E protein (see above), we clearly observed cross reactivity between DENV and ZIKV NS1 proteins and their antibodies. Specifically, DENV NS1 MIA showed 89% and 96% positive accuracy when testing groups II and IV specimens, respectively; and 14% and 37% false positive results when testing groups I and III specimens, respectively. The 37% false positive result demonstrates that DENV NS1 cross-reacts to specimens with ZIKV-only-infection. Reciprocally, ZIKV NS1 MIA exhibited 22% false positive when testing specimens with DENV-only-infection from group II. Altogether, the results showed 22-37% cross reactivity between DENV and ZIKV NS1 proteins. The data are in agreement with the recent report that antibodies to NS1 are largely ZIKV-specific. 
     Conventionally, ZIKV serologic diagnosis is mainly based on IgM-capture ELISA with Emergency Use Authorization (EUA) approval from FDA. There is a need for improved methods for detection of anti-ZIKV antibodies in samples from a subject. As disclosed herein, the diagnostic power of viral envelope protein (that elicits robust, yet cross-reactive antibodies to other flaviviruses) is hereby combined with the differential power of viral nonstructural proteins NS1 and NS5 (that induce more virus-type specific antibodies). Disclosed herein is use of an assay using an MIA format that can shorten assay turnaround time. Over 10,000 sera have been tested by conventional, presently FDA emergency use authorized Zika MAC-ELISA testing. Any such samples presumptive as positive for Zika IgM according to such assay was further tested via PRNT. Approximately 80% of the time such assays resulted in identifying the sample as indicating “undifferentiated flavivirus” (meaning indicating that some flaviviral infection had been present, but not definitively ruling in or out ZIKV or DENV. Further testing on over 3600 of these samples permitted an identification of whether a subject had been infected with ZIKV, DENV, both, or neither, and whether in the past 65 days, past 65-190 days, or more than 190 days ago. As a result, as opposed to high rates of “undifferentiated flavivirus” results, the method disclosed herein permitted confirmatory identification of such specifics of infection in approximately 90% of cases where flaviviral infection was indicated. 
     Furthermore, an assay disclosed herein was performed on many Zika IgM nonreactive, PCR negative specimens from pregnant women with known dengue history. Nearly half of 199 samples from such pregnant women had evidence of a history of Zika infection at some time according to an assay disclosed herein, combining detection of anti ZIKV E, anti-ZIKV NS1, and anti-DENV1-4 NS1 antibodies. Some pregnant women who are IgM negative to Zika may still deliver an infant at risk of congenital Zika syndrome, indicating the importance of accurate testing such as disclosed herein. 
     Using over 3600 samples patient samples with known ZIKV and DENV PRNT results, the method was verified and improved ability to distinguish ZIKV infection from DENV infection according to methods and kits as disclosed herein. Embodiments of a method disclosed herein are distinct from the single antigen-based (either E or NS1) diagnostic assays, including the E-based IgM-captured ELISA from InBios (with EUA approval from FDA), NS1-based indirect ELISA from EuroImmun (approved for clinical use in Europe), and NS1-based IgM-capture ELISA from NovaTec (currently for investigational research use). 
     The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be understood that the terms “comprise”, “have”, “include”, and “contain” (and any related variants thereof) are open-ended linking verbs. As a result, a method, step, or device that “comprises”, “has”, “includes” or “contains” one or more steps or elements possesses those one or more steps or elements, but is not limited to possessing only those one or more steps or elements. The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims below, if any, are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed. The disclosure herein is illustrative and not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. Embodiments are described to best explain the principles of one or more aspects of the invention and the practical application, and to enable others of ordinary skill in the art to understand one or more aspects of the invention for various embodiments with various modifications as are suited to the particular use contemplated. Further details pertaining to the present disclosure can be found in Appendix A, attached hereto and incorporated herein in its entirety. 
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