Patent Publication Number: US-2023152321-A1

Title: Methods and reagents for zika virus immunoassays

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 17/250,857 filed on Mar. 12, 2021 which is a 35 U.S.C. 371 national application of international application no. PCT/US2019/051630 filed Sep. 18, 2019, which claims priority of U.S. Provisional application No. 62/732,964 filed Sep. 18, 2018, the contents of which are fully incorporated herein by reference. 
    
    
     TECHNICAL FIELD 
     Disclosed herein are methods of detecting an anti-Zika virus IgM antibody in a biological sample from a human subject and methods of diagnosing Zika virus infection in the subject. 
     BACKGROUND 
     Zika virus is a single-stranded, positive-sense RNA virus (Musso D, Gubler D J. Zika virus. Clin Microbiol Rev. 2016 July; 29(3):487-524) belonging to the family Flaviviridae which includes closely related dengue, West Nile, Japanese encephalitis, and yellow fever viruses (Rabe I B, Staples J E, Villanueva J, et al. Interim guidance for Interpretation of Zika virus antibody test results. MMWR. 2016; 65(21):543-546). Zika virus was first isolated from rhesus monkey in the Zika forest of Uganda in 1947 (Musso et al., 2016). Zika virus infection was first reported in humans in Nigeria in 1954, and the first epidemic was reported on the Western Pacific Island of Yap in 2007 and later in French Polynesia in 2013 and 2014. Zika virus outbreak first emerged in the Americas in Brazil in March 2015, and spread to several countries and territories by March 2016 (Petersen L R, Jamieson D J, Powers A M, Honein M A. Zika virus. N Engl J Med. 2016; 374 (16): 1552-1563). 
     Zika virus infection is mainly transmitted by bite of an infected mosquito ( Aedes aegypti ). However, transmission from mother to fetus during pregnancy and through sexual contact with infected partners has been reported (id.). Potential transmission through blood transfusion has been documented (Musso et al., 2016). Most people infected with Zika virus exhibit mild symptoms or are asymptomatic (do not develop symptoms) (Zika: The basics of the virus and how to protect against it. CDC&#39;s response to Zika. Centers for Disease Control and Prevention website www_cdc_gov7zika/pdfs/fs-zika-basics_pdf. Published Jun. 27, 2017. Accessed: Aug. 12, 2016). Common symptoms include fever, rash, joint pain and red eyes and these symptoms can be observed for up to a week (Petersen et al., 2016). Zika virus infection in pregnant women may cause microcephaly in the fetus, a major public health concern. The association of Zika virus infection with Guillain-Barre syndrome, a neurological illness that can cause temporary paralysis, has also been reported (Musso et al., 2016; Petersen et al., 2016). 
     During Zika virus infection, viremia is expected for one week after onset of symptoms, and Zika virus-specific IgM antibodies are reported to develop during the first week after development of symptoms and are expected to persist up to 12 weeks (Rabe et al, 2016). The detection of Zika virus-specific IgM antibodies is used for diagnosis and appropriate clinical management of the suspected Zika virus infected patient (id.). 
     The emergence of Zika virus infection outbreak in the Americas in 2015-2016 required urgent development of a diagnostic test to detect Zika-virus specific IgM antibodies in individuals recently infected with Zika virus for their appropriate clinical management. 
     Immunoassays have been developed for the detection of anti-Zika IgM antibodies in biological samples from human subjects, but available immunoassays suffer from various deficiencies including: 1) non-specific reactivity with serum/plasma from normal donors and pregnant women, causing poor specificity in the Zika non-endemic population; 2) cross-reactivity to Zika IgG antibodies in the sample from the human subject, resulting in poor specificity in the Zika endemic population; 3) the need to test single samples with up to 3 different antigens in the same assay, increasing the time and cost to produce a result; and/or 4) the need to test each sample with at least two different assays, also increasing time and cost to produce a result. Therefore, a need persists for immunoassay methods and reagents for reliably and efficiently detecting anti-Zika virus IgM antibody in a biological sample from a subject. 
     SUMMARY 
     Disclosed herein are methods of detecting an anti-Zika virus IgM antibody in a biological sample from a human subject, the methods comprising a first immunoassay and, optionally, a second immunoassay, wherein the first immunoassay comprises: a) incubating the biological sample with an anti-human IgG Fc antibody, a labeled Zika virus antigen, and a solid support comprising an anti-human IgM antibody, wherein, in the presence of an anti-Zika virus IgG antibody, an anti-Zika virus IgM antibody, or an anti-Zika virus IgG antibody and an anti-Zika virus IgM antibody in the biological sample, a complex I is formed, the complex I comprising (i) the anti-Zika virus IgG antibody, the anti-Zika virus IgM antibody, or the anti-Zika virus IgG antibody and the anti-Zika virus IgM antibody, (ii) the labeled Zika virus antigen, and (iii) the solid support comprising the anti-human IgM antibody; and b) detecting the complex I, the presence of which indicates the presence of the anti-Zika virus IgM antibody, the anti-Zika virus IgG antibody, or the anti-Zika virus IgG antibody and the anti-Zika virus IgM antibody in the biological sample, and if the complex I is detected, performing the second immunoassay, comprising: c) incubating the biological sample with a solid support comprising an anti-human IgM antibody, and a labeled Zika virus antigen, wherein, in the presence of an anti-Zika virus IgM antibody in the biological sample, a complex II is formed, the complex II comprising (i) the solid support comprising the anti-human IgM antibody, (ii) the anti-Zika virus IgM antibody, and (iii) the labeled Zika virus antigen; and d) detecting the complex II, the presence of which indicates the presence of the anti-Zika virus IgM antibody in the biological sample. 
     Also disclosed are methods of detecting antibodies to Zika virus in a subject, the methods comprising performing a first immunoassay comprising:
         a) incubating a biological sample from the subject with:
           a solid support comprising an anti-human IgM antibody,   an anti-human IgG Fc antibody, and   a labeled Zika virus antigen,   wherein, in the presence of an anti-Zika virus IgG antibody, an anti-Zika virus IgM antibody, or an anti-Zika virus IgG antibody and an anti-Zika virus IgM antibody in the biological sample, a complex I is formed, the complex I comprising (i) the solid support comprising the anti-human IgM antibody, (ii) the anti-Zika virus IgG antibody, the anti-Zika virus IgM antibody, or the anti-Zika virus IgG antibody and the anti-Zika virus IgM antibody, and (iii) the labeled Zika virus antigen;   b) detecting the complex I, and   b i ) if the complex I is not detected, determining that the subject is negative for antibodies to Zika virus; or   b ii ) if the complex I is detected, determining that the subject is positive for anti-Zika virus antibodies.   
               

     The disclosed methods can further comprise performing a second immunoassay comprising:
         c) incubating the biological sample with:
           a solid support comprising an anti-human IgM antibody, and   a labeled Zika virus antigen,   wherein, in the presence of an anti-Zika virus IgM antibody in the biological sample, a complex II is formed, the complex II comprising (i) the solid support comprising the anti-human IgM antibody, (ii) the anti-Zika virus IgM antibody, and (iii) the labeled Zika virus antigen;   
           d) detecting the complex II, and
           d i ) if the complex II is not detected, determining that the human subject is negative for anti-Zika virus IgM antibodies.   
               

     The disclosed methods can further comprise:
         d ii ) if the complex II is detected, repeating steps c) and d) at least in duplicate, and   e) if the complex II is detected in the equivalent of at least 2 of 3 replicates, determining that the human subject is positive for anti-Zika virus IgM antibodies.       

     Also disclosed are kits comprising: a solid support, an anti-human IgM antibody, an anti-human IgG Fc antibody, and a labeled Zika virus antigen. The kits can comprise reagents for a first immunoassay and reagents for a second immunoassay, wherein the reagents for the first immunoassay comprise: a solid support, an anti-human IgM antibody, an anti-human IgG Fc antibody, and a labeled Zika virus antigen; and wherein the reagents for the second immunoassay comprise: a solid support, an anti-human IgM antibody, and a labeled Zika virus antigen. The kits can further comprise instructions for performing the first and the second immunoassays, wherein the instructions direct a user to perform the first immunoassay to determine the presence or absence of anti-Zika virus antibodies in a biological sample from a human subject, and wherein the instructions further direct the user to perform the second immunoassay only if it is determined in the first immunoassay that the biological sample is positive for anti-Zika virus antibodies. 
     Also disclosed herein are algorithms for performing the disclosed methods and methods of diagnosing Zika virus infection in a subject using the disclosed methods and algorithms. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed methods and kits, there are shown in the drawings exemplary embodiments of the methods and kits; however, the methods and kits are not limited to the specific embodiments disclosed. In the drawings: 
         FIG.  1    is a schematic representation of an embodiment of the “Zika Test” algorithm, whereby a result of Negative for Antibodies to Zika Virus or Presumptive Zika-Positive is determined for a given biological sample from a subject. 
     
    
    
     DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 
     The disclosed methods and kits may be understood more readily by reference to the following detailed description taken in connection with the accompanying FIGURES, which form a part of this disclosure. It is to be understood that the disclosed methods and kits are not limited to the specific methods and kits described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods and kits. 
     Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed methods and kits are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement. 
     Throughout this text, the descriptions refer to methods of detecting an antibody and methods of diagnosing Zika virus infection. Where the disclosure describes or claims a feature or embodiment associated with a method of detecting an antibody, such a feature or embodiment is equally applicable to the methods of diagnosing Zika virus infection. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of diagnosing Zika virus infection, such a feature or embodiment is equally applicable to the methods of detecting an antibody. 
     Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges were explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the invention as described herein. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value. It is not intended that the scope of the invention be limited to the specific values recited when defining a range. All ranges are inclusive and combinable. 
     When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. 
     It is to be appreciated that certain features of the disclosed methods and kits which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods and kits that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. 
     As used herein, the singular forms “a,” “an,” and “the” include the plural. 
     Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein. 
     The term “comprising” is intended to include examples encompassed by the terms “consisting essentially of” and “consisting of”; similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.” 
     Disclosed herein are immunoassays and methods for detecting anti-Zika virus IgM antibody in a biological sample from a human subject and/or diagnosing Zika virus infection in a human subject. 
     The methods disclosed herein can comprise incubating the biological sample with an anti-human IgG Fc antibody, a labeled Zika virus antigen, and a solid support comprising an anti-human IgM antibody. In the presence of an anti-Zika virus IgG antibody, an anti-Zika virus IgM antibody, or an anti-Zika virus IgG antibody and an anti-Zika virus IgM antibody in the biological sample, a complex I is formed, the complex I comprising (i) the anti-Zika virus IgG antibody, the anti-Zika virus IgM antibody, or the anti-Zika virus IgG antibody and the anti-Zika virus IgM antibody, (ii) the labeled Zika virus antigen, and (iii) the solid support comprising the anti-human IgM antibody. The method can further comprise detecting the complex I, the presence of which indicates the presence of the anti-Zika virus IgM antibody, the anti-Zika virus IgG antibody, or the anti-Zika virus IgG antibody and the anti-Zika virus IgM antibody in the biological sample. If the complex I is detected, the method can further comprise performing the second immunoassay, comprising incubating the biological sample with a solid support comprising an anti-human IgM antibody, and a labeled Zika virus antigen. In the presence of an anti-Zika virus IgM antibody in the biological sample, a complex II is formed, the complex II comprising (i) the solid support comprising the anti-human IgM antibody, (ii) the anti-Zika virus IgM antibody, and (iii) the labeled Zika virus antigen. The method can further comprise detecting the complex II, the presence of which indicates the presence of the anti-Zika virus IgM antibody in the biological sample. 
     In some embodiments, the Zika virus antigen is Zika virus NS1 antigen, or an immunogenic fragment thereof. The Zika virus NS1 antigen or immunogenic fragment thereof can be recombinant. 
     In some embodiments, the anti-human IgG Fc antibody can immunospecifically bind to an Fc region of a human IgG antibody. For example, the anti-human IgG Fc antibody can be a monoclonal or a polyclonal antibody raised against a human IgG Fc antigen. In some embodiments, the anti-human IgG Fc antibody is a goat-anti-human IgG Fc antibody. That is, the anti-human IgG Fc antibody can be a goat antibody to human IgG Fc. 
     Without wishing to be bound by theory, it is predicted that the anti-human IgG Fc antibody can bind to or otherwise couple with the solid support in the incubation reaction. For example, the anti-human IgG Fc can non-covalently bind to the solid support. Accordingly, in some embodiments, the anti-human IgG Fc antibody is indirectly linked to the solid support. In some embodiments, the anti-human IgG Fc antibody is a component of complex I. 
     The anti-human IgM antibody can be directly or indirectly linked to the solid support. In some embodiments, the anti-human IgM antibody is biotinylated and the solid support comprises streptavidin. Thus, the anti-human IgM antibody can be indirectly linked to the solid support through a biotin-streptavidin interaction. In some embodiments, the anti-human IgG Fc antibody is indirectly linked to the solid support. Additionally, the anti-human IgG Fc antibody can be biotinylated and thereby also indirectly linked to the solid support comprising streptavidin. 
     The biological sample can be serum or plasma, and it can further comprise an anticoagulant, including, for example, EDTA or heparin. In some embodiments, the biological sample is from a human. The biological sample is obtained from a human subject at least 8 days after onset of symptoms of Zika virus infection or risk of exposure to Zika virus. In some embodiments, the biological sample must be obtained from a human subject at least 8 days after onset of symptoms of Zika virus infection or risk of exposure to Zika virus to ensure accurate detection of anti-Zika virus IgM antibodies in the biological sample. 
     In the first immunoassay, the “Zika Ab” assay, the anti-human IgM antibody and the solid support can be present in a buffer comprising tricine, sodium chloride, TWEEN® 20 (polyoxyethylene (20) sorbitan monolaurate) detergent, disodium EDTA, preservative, sulfhydryl-modified bovine serum albumin, and the anti-human IgG Fc antibody. In the second immunoassay, the “Zika M” assay, the IgM antibody and the solid support can be present in a buffer comprising tricine, sodium chloride, TWEEN® 20 detergent, disodium EDTA, preservative, sulfhydryl-modified bovine serum albumin, but without the anti-human IgG Fc antibody.— 
     The methods disclosed herein can further comprise determining a level of the anti-Zika virus IgG antibody, the anti-Zika virus IgM antibody, or the anti-Zika virus IgG antibody and the anti-Zika virus IgM antibody in the biological sample. In some embodiments, the level of the anti-Zika virus IgG antibody, the anti-Zika virus IgM antibody, or the anti-Zika virus IgG antibody and the anti-Zika virus IgM antibody in the biological sample is directly proportional to the level of the complex I or complex II detected. Detecting the signal, as used herein, can comprise measuring a signal from the label and comparing the signal to a control signal from a biological sample from a human known to be negative for anti-Zika virus antibodies. 
     In the event that the complex II is detected in the Zika M assay, the method can further include repeating the steps of the Zika M assay at least in duplicate and determining that the human subject is positive for anti-Zika virus IgM antibodies if the complex II is detected in the equivalent of at least 2 of 3 replicates. 
     Also disclosed herein are methods of diagnosing Zika virus infection in a subject comprising the disclosed methods of detecting anti-Zika virus IgM antibodies in a biological sample from a subject. 
     Further disclosed are algorithms for detecting Zika virus-specific IgM antibodies in a biological sample ( FIG.  1   ). The algorithms disclosed herein can guide a user or an automated system through the steps of detecting antibodies to Zika virus in a subject in a manner that increases efficiency and reliability of the methods disclosed herein. For example, the algorithms can be used to reduce the number of assays required for a particular sample to determine that it is, for example, negative for anti-Zika virus IgM antibodies. In some embodiments, the algorithms comprise a set of instructions to guide a user or an automated system through the methods of detecting antibodies to Zika virus in a biological sample. An example algorithm can instruct the user or system to perform a method of detecting antibodies to Zika virus in a subject comprising performing a first immunoassay comprising:
         a) incubating a biological sample from the subject with:
           a solid support comprising an anti-human IgM antibody,   an anti-human IgG Fc antibody, and   a labeled Zika virus antigen,   wherein, in the presence of an anti-Zika virus IgG antibody, an anti-Zika virus IgM antibody, or an anti-Zika virus IgG antibody and an anti-Zika virus IgM antibody in the biological sample, a complex I is formed, the complex I comprising (i) the solid support comprising the anti-human IgM antibody, (ii) the anti-Zika virus IgG antibody, the anti-Zika virus IgM antibody, or the anti-Zika virus IgG antibody and the anti-Zika virus IgM antibody, and (iii) the labeled Zika virus antigen;   
           b) detecting the complex I, and
           b i ) if the complex I is not detected, determining that the subject is negative for antibodies to Zika virus; or   b ii ) if the complex I is detected, determining that the subject is positive for anti-Zika virus antibodies.   
               

     The algorithms can further instruct the user or the system to terminate the assays if the complex I is not detected in step b i , thereby reducing the number of assays, the amount of reagents, and the time required to detect anti-Zika virus antibodies in a biological sample. However, if in step b) a result is obtained that is equivocal (i.e., “reactive”), then the algorithm can instruct the user or system to perform a second immunoassay comprising:
         c) incubating the biological sample with:
           a solid support comprising an anti-human IgM antibody, and   a labeled Zika virus antigen,   wherein, in the presence of an anti-Zika virus IgM antibody in the biological sample, a complex II is formed, the complex II comprising (i) the solid support comprising the anti-human IgM antibody, (ii) the anti-Zika virus IgM antibody, and (iii) the labeled Zika virus antigen;   
           d) detecting the complex II, and
           d i ) if the complex II is not detected, determining that the human subject is negative for anti-Zika virus IgM antibodies.   
               

     The algorithm can instruct the user or system to terminate the assays if the complex II is not detected in step d). Otherwise, the algorithm can proceed through the additional method steps comprising:
         d ii ) if the complex II is detected, repeating steps c) and d) at least in duplicate, and   e) if the complex II is detected in the equivalent of at least 2 of 3 replicates, determining that the human subject is positive for anti-Zika virus IgM antibodies.       

     In some embodiments of the methods and algorithms for implementing the methods disclosed herein, a biological sample known to have, or suspected of having, anti-Zika virus antibodies is incubated with a labeled Zika virus antigen and solid support having an anti-human IgM antibody bound thereto. In the absence of the anti-Zika virus antibodies, the labeled Zika virus antigen will not bind to or otherwise interact with the solid support. Thus, in the absence of the anti-Zika virus antibodies in the biological sample, the labeled Zika virus antigen remains in the solution and isolation of the solid support does not result in isolation of the labeled antigen. When the anti-Zika virus antibodies are present in the biological sample, on the other hand, the anti-Zika virus antibodies simultaneously bind to the anti-human IgM antibody bound to the solid support and the labeled Zika virus antigen, thereby linking the labeled antigen and the solid support and resulting in the formation of a solid support/labeled antigen complex. It is to be understood that the order in which the incubation takes place can be different from that described herein. Furthermore, the labeled and solid-support-bound reaction components can be rearranged in alternative embodiments of the presently described immunoassays. For example, the biological sample known to have, or suspected of having, anti-Zika virus antibodies can be incubated with a solid support having an unlabeled Zika virus antigen bound thereto followed by incubation with a labeled anti-human IgM antibody. In other embodiments, the biological sample known to have, or suspected of having, anti-Zika virus antibodies can be simultaneously incubated with a solid support having, on the one hand, an unlabeled anti-human IgM antibody bound thereto and a labeled Zika virus antigen, or, on the other hand, an unlabeled Zika virus antigen bound thereto and an anti-human IgM antibody. 
     The biological sample known to have, or suspected of having, anti-Zika virus antibody can be incubated in a reaction mixture for a period of time sufficient to achieve a partial reaction without allowing the reaction to achieve equilibrium, such as for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes about 18 minutes, about 19 minutes, or less than about 20 minutes. The labeled Zika virus antigen can be added and incubated with the biological sample for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes about 18 minutes, about 19 minutes, or less than about 20 minutes. The solid support having anti-human IgM antibody bound thereto can be added to the mixture of biological sample and labeled Zika virus antigen and incubated for a period of time sufficient to achieve a partial reaction without allowing the reaction to achieve equilibrium, such as for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes about 18 minutes, about 19 minutes, or less than about 20 minutes. In some embodiments, the incubating steps are performed in a total of about 10 minutes to about 20 minutes, about 20 minutes to about 30 minutes, about 30 minutes to about 40 minutes, about 40 minutes to about 50 minutes, or about 50 minutes to about an hour. The subsequent detecting can be performed in less than about 5 minutes, less than about 10 minutes, less than about 15 minutes, or less than about 20 minutes. It is to be understood that the amount of time needed for the assays or any step or steps thereof may vary based upon several factors including the level of the anti-Zika virus antibodies in the biological sample and the affinity of the anti-human IgM antibody for the anti-Zika virus antibodies in the biological sample. In some embodiments, incubating the biological sample with the reaction mixture can be performed for a period of time sufficient to enable the reaction to achieve equilibrium, such as on the order of 1 or more hours. Thus, the disclosed methods can be performed for any suitable amount of time. 
     The anti-human IgM antibody can be directly or indirectly linked to the solid support. Suitable techniques for directly linking the anti-human IgM antibody to the solid support include, for example, covalent attachment, adsorption, noncovalent interaction, or combinations thereof. In some embodiments, the anti-human IgM antibody can be directly linked to the solid support by N-hydroxysuccinimide (NHS) chemistry or by 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) NHS chemistry. Suitable techniques for indirectly linking the anti-human IgM antibody to the solid support include, for example, linking through a peptide, a protein, an antibody, a linker, or a combination thereof. In some embodiments, the anti-human IgM antibody can be indirectly linked to the solid support through streptavidin and biotin. For example, the anti-human IgM antibody can be biotinylated and the solid support can comprise streptavidin. It is to be understood that the same chemistry can be applied to any reaction component linked to the solid support in alternative embodiments of the disclosed immunoassays. 
     Exemplary solid supports include, but are not limited to, a column matrix material, a culture plate, a tube, a dish, a flask, a microtiter plate, a bead/particle, heat-killed formalin- (or other chemically)-fixed prokaryotic or eukaryotic cells, microscope slides, ACLAR® Film, or any other optically transparent polymer, or a combination thereof. The solid support can be fully or partially composed of plastic, cellulose, cellulose derivatives, nitrocellulose, glass, fiberglass, latex, or a combination thereof. In some embodiments, the solid support comprises a magnetic bead/particle. In some embodiments, the magnetic bead/particle is a paramagnetic particle (PMP). In some embodiments, the magnetic bead/particle is a latex magnetic particle (LMP). 
     The label can be any suitable label known to those skilled in the art to be useful for creating a detectable signal. Suitable detectable labels include, but are not limited to, enzyme conjugates (e.g., horseradish peroxidase (HRP), alkaline phosphatase, glucose oxidase, and β-galactosidase), fluorescent probes, radioactive isotopes, chemiluminescent compounds, bioluminescent compounds, or combination thereof. In some embodiments, the label is an acridinium ester (“AE”) or an analog thereof. Suitable AE analogs include: dimethyl acridinium ester (DMAE), N-sulfopropyl dimethyl acridinium ester (NSP-DMAE), high quantum yield acridinium ester (HQYAE, acridinium, 9-[[4-[[[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]carbonyl]-2,6-dimethylphenoxy]carbonyl]-2,7-bis(3,6,9,12,15,18-hexaoxanonadec-1-yloxy)-10-(3-sulfopropyl)-, inner salt), Zwitterionic acridinium ester (ZAE, Acridinium, 9-[[4-[[[3-[[3-[[5-[(2,5-dioxo-1-pyrrolidinyl)oxy]-1,5-dioxopentyl]amino]propyl]methyl(3-sulfopropyl)ammonio]propyl]amino]carbonyl]-2,6-dimethylphenoxy]carbonyl]-10-(3-sulfopropyl)-, bis(inner salt)), N-sulfopropyl-2-isopropoxy dimethyl acridinium ester (Iso-Di-ZAE), trisulfopropyl acridinium ester (TSP-AE), or N-sulfopropyl dimethyl acridinium ester with hexa(ethylene)glycol linker (HEG-GLU-AE). In some embodiments, the labeled Zika virus antigen comprises Zika NS1:NSP-DMAE-NHS. 
     The anti-human IgM antibody can be an IgA, IgD, IgG, IgE, or IgM isotype or a single domain format, such as a single-domain antibody from camelid. In some embodiments, the anti-human IgM antibody is an IgG isotype. In some embodiments, the anti-human IgM antibody is a commercially available anti-human IgM antibody. Aptamers that are specific for the human IgM antibodies can also be used. 
     In some embodiments, the immunoassays disclosed herein meet one or more of the following clinical requirements:
         a. ≥90% positive and negative agreement;   b. Repeatability 0.80-2.00 Index: ≤12.0% and &gt;2.00 Index: ≤8.0%;   c. Within-Run Precision 0.80-2.00 Index: ≤15.0% and &gt;2.00 Index: ≤10.0%;   d. Calibration Interval ≥7 Days;   e. On-Board Stability ≥14 Days;   f. Control System that is capable of maintaining consistent standardization &amp; performance.   g. Interference and Reproducibility       

     In some embodiments, the immunoassays disclosed herein meet all of the above clinical requirements a.-f. 
     In some embodiments, the disclosed immunoassays are intended for in vitro diagnostic use in the presumptive qualitative detection of IgM antibodies to the Zika virus in serum and plasma (potassium EDTA or lithium heparin, each collected alongside a patient-matched serum specimen) specimens collected from individuals meeting Centers for Disease Control and Prevention (CDC) Zika virus clinical criteria (e.g. a history of clinical signs and symptoms associated with Zika virus infection) and/or CDC Zika virus epidemiological criteria (for instance, history of residence in or travel to a geographic region with active Zika transmission at the time of travel, or other epidemiological criteria for which Zika virus testing may be indicated). In some embodiments, specimens from symptomatic patients or returning travelers from endemic areas are not collected prior to 8 days after onset of symptoms or risk of exposure, respectively. 
     In some embodiments of the disclosed immunoassays, the first immunoassay, the “Zika Ab” assay, and the optional second immunoassay, the “Zika M” assay can be in 2-pass immunoassay format. The Zika Ab assay can be an antibody capture immunoassay using a 2-pass format. In the first pass, patient sample can be incubated in a cuvette, for example, with anti-human IgM antibody linked to a solid support. During incubation, solid-support-bound anti-human IgM antibody binds to antibodies from the patient sample. The captured antibodies can be washed and resuspended. In the second pass, the patient anti-Zika virus antibodies captured on the solid support can be incubated with Zika virus NS1 antigen labeled with any suitable chemiluminescent or other labeling reagent. The labeled NS1 antigen binds to patient Zika virus antibodies on the solid support during incubation. The NS1/solid support complex can be washed and subjected to, e.g., chemiluminescent detection. 
     The Zika M assay can be an IgM capture immunoassay using a 2-pass format. In the first pass the solid support, coated with anti-human IgM antibody and patient sample can be incubated in the cuvette for binding anti-Zika virus IgM antibodies from the patient sample to the solid support. The captured anti-Zika virus IgM antibodies can be washed and resuspended. In the second pass, the anti-Zika virus IgM antibodies captured on the solid support can be incubated with labeled NS1 for binding of the NS1 antigen to the captured anti-Zika virus IgM antibodies on immobilized on the solid support. The NS1/solid support complex can be washed and subjected to, e.g., chemiluminescent detection. 
     The disclosed methods can be performed manually or can be automated. For example, the disclosed methods can be performed using an ADVIA CENTAUR® Immunoassay System or an ATELLICA™ system. For example, a system can be automated to perform the following actions for both Zika Ab and Zika M assays:
         1. Dispense sample into a cuvette.   2. Dispense buffer containing solid support with anti-human IgM antibody bound thereto, and incubate for, e.g., 18.25 minutes at 37° C.   3. Separate/isolate solid support, aspirate, and wash the cuvette with wash reagent.   4. Dispense buffer containing chemiluminescent-labeled NS1 antigen and incubate, e.g., 18 minutes at 37° C.   5. Separate/isolate solid support, aspirate, and wash the cuvette with wash reagent.   6. Dispense chemiluminescence reagent to initiate the chemiluminescent reaction.   7. Report results according to a user-selected option.       

     In a particular embodiment, the disclosed immunoassays can be adapted for use on an ADVIA CENTAUR® Immunoassay System (Siemens Healthcare, AG), and/or an ADVIA CENTAUR® Immunoassay System can be automated to perform the above actions. 
     In some embodiments, a direct relationship exists between the level of anti-Zika virus antibodies (i.e., anti-Zika virus IgG antibodies or anti-Zika virus IgM antibodies) or anti-Zika virus IgM antibodies present in the patient sample and the amount of relative light units (RLUs) detected by the system. 
     In some embodiments, the immunoassays disclosed herein employ the following raw materials in the Zika Ab and/or Zika M assays: 
     Zika virus NS1 recombinant antigen: The disclosed immunoassays can use Zika virus NS1 recombinant antigen, for example, from Meridian Life Science. The Zika virus NS1 recombinant antigen can be expressed in insect cells and purified by affinity chromatography. 
     Anti-human IgM antibody: The anti-human IgM antibody can be a monoclonal antibody against human IgM. In some embodiments, it can be biotin-conjugated. In some embodiments, it can be produced by labelling an anti-human IgM monoclonal antibody with NHS-LC-LC-Biotin. 
     SERA-MAG Magnetic Streptavidin Microparticles (MG-SA): The SERA-MAG Magnetic Streptavidin Microparticles (MG-SA) microparticles (GE HealthCare Bio-Sciences Corp.) can be used as a solid support to prepare a wetcake comprising the solid support coated with the anti-human IgM antibody. 
     Goat Antiserum to Human IgG, Fc Specific: The Goat Antiserum to Human IgG, Fc Specific (“goat-anti-human IgG, Fc”), for example from Nittobo or Meridian Life Sciences, can be employed in the Zika Ab assay. 
     In some embodiments, the immunoassays disclosed herein comprise the following example reagent formulations. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Example Zika Ab Solid Support Buffer Formulation 
               
            
           
           
               
               
               
            
               
                 Component 
                 Conc. 
                 Function 
               
               
                   
               
               
                 Tricine 
                 18.0 g/L  
                 Buffer Salt 
               
               
                 Sodium Chloride 
                 15.0 g/L  
                 Ionic Strength 
               
               
                 Disodium EDTA 
                 0.7 g/L  
                 Chelating Agent 
               
               
                 MICROPROTECT ™  
                 2.50 mL/L 
                 Preservative/Anti-microbial 
               
               
                 preservative 
                   
                   
               
               
                 TWEEN ®20 detergent 
                 2.20 g/L  
                 Detergent (Blocking Agent) 
               
               
                 Sodium Azide 
                 0.9 g/L  
                 Preservative/Anti-microbial 
               
               
                 Heat Inactivated Goat Serum 
                 50.0 mL/L 
                 Protein (Blocking Agent) 
               
               
                 BSA, Sulfhydryl Modified 
                 10.0 g/L  
                 Protein (Blocking Agent) 
               
               
                 Goat Anti-Human IgG FC 
                 1.35 g/L  
                 Protein (Blocking Agent) 
               
               
                 Zika M/Zika Ab Wetcake 
                 0.1 g/L  
                 Capture Human IgM 
               
               
                   
                   
                 Antibodies 
               
               
                   
               
               
                 pH = 8.0 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Example Zika Ab Ancillary Well Reagent Formulation 
               
            
           
           
               
               
               
            
               
                 Component 
                 Conc. 
                 Function 
               
               
                   
               
               
                 EPPS 
                 25.3 g/L  
                 Buffer Salt 
               
               
                 Sodium Chloride 
                 15.0 g/L  
                 Ionic Strength 
               
               
                 TWEEN ®20 detergent 
                 2.20 g/L  
                 Detergent (Blocking Agent) 
               
               
                 Disodium EDTA 
                 0.70 g/L  
                 Chelating Agent 
               
               
                 MICROPROTECT ™  
                  1.0 mL/L 
                 Preservative/Anti-microbial 
               
               
                 preservative 
                   
                   
               
               
                 Sodium Azide 
                 0.9 g/L  
                 Preservative/Anti-microbial 
               
               
                 BSA, Sulfhydryl Modified 
                 10.0 g/L  
                 Protein (Blocking Agent) 
               
               
                 Heat Inactivated Goat Serum 
                  400 mL/L 
                 Protein (Blocking Agent) 
               
               
                   
               
               
                 pH = 8.0 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Example Zika Ab Visualization Buffer Formulation 
               
            
           
           
               
               
               
            
               
                 Component 
                 Conc. 
                 Function 
               
               
                   
               
               
                 EPPS 
                 25.3 g/L  
                 Buffer Salt 
               
               
                 Sodium Chloride 
                 15.0 g/L  
                 Ionic Strength 
               
               
                 TWEEN ®20 detergent 
                 2.20 g/L  
                 Detergent (Blocking Agent) 
               
               
                 Disodium EDTA 
                 0.70 g/L  
                 Chelating Agent 
               
               
                 MICROPROTECT ™  
                  1.0 mL/L 
                 Preservative/Anti-microbial 
               
               
                 preservative 
                   
                   
               
               
                 Sodium Azide 
                 0.9 g/L  
                 Preservative/Anti-microbial 
               
               
                 BSA, Sulfhydryl Modified 
                 10.0 g/L  
                   
               
               
                 Zika M/ZikaAb Visualization  
                 0.40 mg/L 
                 Zika Antibodies detection 
               
               
                 Buffer (Zika NS1:  
                   
                   
               
               
                 NSP-DMAE-NHS) 
               
               
                   
               
               
                 pH = 8.0 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Example Zika M Solid Support Buffer Formulation 
               
            
           
           
               
               
               
            
               
                 Component 
                 Conc. 
                 Function 
               
               
                   
               
               
                 Tricine 
                 18.0 g/L  
                 Buffer Salt 
               
               
                 Sodium Chloride 
                 15.0 g/L  
                 Ionic Strength 
               
               
                 Disodium EDTA 
                 0.7 g/L  
                 Chelating Agent 
               
               
                 MICROPROTECT ™  
                 2.50 mL/L 
                 Preservative/Anti-microbial 
               
               
                 preservative 
                   
                   
               
               
                 TWEEN ®20 detergent 
                 2.20 g/L  
                 Detergent (Blocking Agent) 
               
               
                 Pluronic P-105 
                 5.0 g/L  
                 Detergent (Blocking Agent) 
               
               
                 Sodium Azide 
                 0.9 g/L  
                 Preservative/Anti-microbial 
               
               
                 Heat Inactivated Goat Serum 
                 50.0 mL/L 
                 Protein (Blocking Agent) 
               
               
                 BSA, Sulfhydryl Modified 
                 10.0 g/L  
                 Protein (Blocking Agent) 
               
               
                 Zika M/Zika Ab Wetcake 
                 0.1 g/L  
                 Capture Human IgM 
               
               
                   
                   
                 Antibodies 
               
               
                   
               
               
                 pH = 8.0 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Example Zika M Ancillary Well Reagent Formulation 
               
            
           
           
               
               
               
            
               
                 Component 
                 Conc. 
                 Function 
               
               
                   
               
               
                 EPPS 
                 25.3 g/L  
                 Buffer Salt 
               
               
                 Sodium Chloride 
                 15.0 g/L  
                 Ionic Strength 
               
               
                 TWEEN ®20 detergent 
                 2.20 g/L  
                 Detergent (Blocking Agent) 
               
               
                 Pluronic P-105 
                 5.0 g/L  
                 Detergent (Blocking Agent) 
               
               
                 Disodium EDTA 
                 0.70 g/L  
                 Chelating Agent 
               
               
                 MICROPROTECT ™  
                  1.0 mL/L 
                 Preservative/Anti-microbial 
               
               
                 preservative 
                   
                   
               
               
                 Sodium Azide 
                 0.9 g/L  
                 Preservative/Anti-microbial 
               
               
                 BSA, Sulfhydryl Modified 
                 10.0 g/L  
                 Protein (Blocking Agent) 
               
               
                 Heat Inactivated Goat Serum 
                  400 mL/L 
                 Protein (Blocking Agent) 
               
               
                   
               
               
                 pH = 8.0 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Example Zika M Visualization Buffer Formulation 
               
            
           
           
               
               
               
            
               
                 Component 
                 Conc. 
                 Function 
               
               
                   
               
               
                 EPPS 
                 25.3 g/L  
                 Buffer Salt 
               
               
                 Sodium Chloride 
                 15.0 g/L  
                 Ionic Strength 
               
               
                 TWEEN ®20 detergent 
                 2.20 g/L  
                 Detergent (Blocking Agent) 
               
               
                 Pluronic P-105 
                 5.0 g/L  
                 Detergent (Blocking Agent) 
               
               
                 Disodium EDTA 
                 0.70 g/L  
                 Chelating Agent 
               
               
                 MICROPROTECT ™  
                  1.0 mL/L 
                 Preservative/Anti-microbial 
               
               
                 preservative 
                   
                   
               
               
                 Sodium Azide 
                 0.9 g/L  
                 Preservative/Anti-microbial 
               
               
                 BSA, Sulfhydryl Modified 
                 10.0 g/L  
                 Protein (Blocking Agent) 
               
               
                 Zika M/ZikaAb Visualization  
                 0.40 mg/L 
                 Zika Antibodies detection 
               
               
                 Buffer (Zika NS1:  
                   
                   
               
               
                 NSP-DMAE-NHS) 
               
               
                   
               
               
                 pH = 8.0 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Example Zika M/Zika Ab Wetcake Formulation 
               
            
           
           
               
               
               
            
               
                 Component 
                 Conc. 
                 Function 
               
               
                   
               
               
                 Tricine 
                 18.0 g/L  
                 Buffer Salt 
               
               
                 Sodium Chloride 
                 15.0 g/L  
                 Ionic Strength 
               
               
                 Disodium EDTA 
                 0.7 g/L  
                 Chelating Agent 
               
               
                 MICROPROTECT ™  
                 2.50 mL/L 
                 Preservative/Anti-microbial 
               
               
                 preservative 
                   
                   
               
               
                 TWEEN ®20 detergent 
                 2.20 g/L  
                 Detergent (Blocking Agent) 
               
               
                 Sodium Azide 
                 0.9 g/L  
                 Preservative/Anti-microbial 
               
               
                 BSA, Sulfhydryl Modified 
                 10.0 g/L  
                 Protein (Blocking Agent) 
               
               
                 SERA-MAG Magnetic  
                  5 g/L 
                 Solid support for analyte 
               
               
                 Streptavidin 
                   
                   
               
               
                 Microparticles (MG-SA) 
                   
                 capture conjugate 
               
               
                 Conj Biotin Ab huIgM 
                  0.04 mg/mg 
                 Coat Biotin conjugate on 
               
               
                   
                   
                 MG-SA 
               
               
                   
               
               
                 pH = 8.0 
               
            
           
         
       
     
     Suitable biological samples for detecting the anti-Zika virus antibody include any biological sample from a subject that contains, or is suspected of containing, anti-Zika virus antibody including, but not limited to serum or plasma. 
     The immunoassays disclosed herein employ a Zika virus antigen. In some embodiments, the Zika virus antigen is Zika virus NS1 antigen. In some embodiments, the Zika virus antigen is recombinant Zika virus NS1 antigen. The Zika virus antigen can be expressed in heterologous cells, for example, insect cells. In some embodiments, the Zika antigen further comprises an epitope tag. The epitope tag can be at the N-terminus or at the C-terminus of the Zika virus antigen. The epitope tag can be any suitable tag known to persons skilled in the art including, but not limited to, a 6-histidine tag, a hemagglutinin tag, a glutathione-S-transferase, a maltose binding protein, or a chitin binding protein. In some embodiments, the Zika virus antigen comprises a C-terminal 6-histidine tag. 
     Further disclosed herein are kits. The kits can comprise a solid support, an anti-human IgM antibody, an anti-human IgG Fc antibody, and a labeled Zika virus antigen. In some embodiments, the kits can comprise reagents for a first immunoassay and reagents for a second immunoassay, wherein the reagents for the first immunoassay comprise: a solid support, an anti-human IgM antibody, an anti-human IgG Fc antibody, and a labeled Zika virus antigen; and wherein the reagents for the second immunoassay comprise: a solid support, an anti-human IgM antibody, and a labeled Zika virus antigen. The kits can further comprise instructions for performing the first and the second immunoassays. The instructions can direct a user or system to perform the first immunoassay to determine the presence or absence of anti-Zika virus antibodies in a biological sample from a human subject. The instructions can further direct the user or system to perform the second immunoassay only if it is determined in the first immunoassay that the biological sample is positive for anti-Zika virus antibodies. 
     Suitable solid supports and labels for any of the kits disclosed herein include those solid supports and labels disclosed for the methods above. 
     EXAMPLES 
     The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments. 
     Development of a Highly Specific and Efficient Anti-Zika IgM Antibody Immunoassay 
     A “Zika Ab” assay with anti-human IgG Fc antibodies present in the reaction buffer was prepared alongside another “Zika M” assay without anti-human IgG Fc antibodies. Using both Zika Ab and Zika M assays, a Zika Test algorithm was developed ( FIG.  1   ). Per the Zika Test algorithm, each biological sample is tested first by the Zika Ab assay (including the anti-human IgG Fc antibodies) and only reactive samples in which either anti-Zika IgM or anti-Zika IgG or anti-Zika IgM and anti-Zika IgG antibodies are detected are to be subsequently tested with the Zika M assay. 
     Because the Zika Ab assay provides high specificity in the non-endemic population and can detect both Zika-specific IgG and IgM antibodies, this assay identifies biological samples (i.e., individuals) containing Zika-specific antibodies. Subsequent testing of these samples with the Zika M assay without anti-human IgG Fc antibodies, which detects only Zika-specific IgM antibodies, identifies those samples (i.e., individuals) with anti-Zika IgM antibodies. These individuals are presumed recently infected with Zika virus. 
     Because the Zika Test algorithm requires testing by the Zika Ab assay one time only, and a majority of the tested population are expected to be negative for Zika infection, a majority of samples will be tested with the Zika Ab assay only. Therefore, the Zika Test algorithm provides a simple solution to achieve high specificity and sensitivity in assaying for recent Zika infection in an individual. 
     Materials 
     A reagent formulation was finalized for the Zika Ab immunoassay (Table 8) and for the Zika M assay (Table 9). 
     
       
         
           
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Zika Ab assay formulation with goat-anti-human IgG Fc 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Ancillary Reagent  
                 Visualization Reagent  
               
               
                 Buffer (pH 8.0) 
                 (pH 8.0) 
                 (pH 8.0) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Component 
                 Conc. 
                 Component 
                 Conc. 
                 Component 
                 Conc. 
               
               
                   
               
               
                 Tricine 
                 18.0 g/L  
                 EPPS 
                 25.3 g/L  
                 EPPS 
                 25.3 g/L  
               
               
                 Sodium  
                 15.0 g/L  
                 Sodium  
                 15.0 g/L  
                 Sodium  
                 15.0 g/L  
               
               
                 Chloride 
                   
                 Chloride 
                   
                 Chloride 
                   
               
               
                 TWEEN ®20 
                 2.20 g/L  
                 TWEEN ®20 
                 2.20 g/L  
                 TWEEN ®20 
                 2.20 g/L  
               
               
                 detergent 
                   
                 detergent 
                   
                 detergent 
                   
               
               
                 Disodium  
                 0.7 g/L  
                 Disodium  
                 0.70 g/L  
                 Disodium  
                 0.70 g/L  
               
               
                 EDTA 
                   
                 EDTA 
                   
                 EDTA 
                   
               
               
                 MICRO- 
                 2.50 mL/L 
                 MICRO- 
                  1.0 mL/L 
                 MICRO- 
                  1.0 mL/L 
               
               
                 PROTECT ™  
                   
                 PROTECT ™ 
                   
                 PROTECT ™  
                   
               
               
                 preservative 
                   
                 preservative 
                   
                 preservative 
                   
               
               
                 Sodium Azide 
                 0.9 g/L  
                 Sodium Azide 
                 0.9 g/L  
                 Sodium Azide 
                 0.9 g/L  
               
               
                 BSA,  
                 10.0 g/L  
                 BSA,  
                 10.0 g/L  
                 BSA,  
                 10.0 g/L  
               
               
                 Sulfhydryl 
                   
                 Sulfhydryl 
                   
                 Sulfhydryl 
                   
               
               
                 Modified 
                   
                 Modified 
                   
                 Modified 
                   
               
               
                 Heat  
                 50.0 mL/L  
                 Heat  
                  400 mL/L 
                   
                   
               
               
                 Inactivated 
                   
                 Inactivated 
                   
                   
                   
               
               
                 Goat Serum 
                   
                 Goat Serum 
                   
                   
                   
               
               
                 Goat  
                 1.35 g/L  
                   
                   
                   
                   
               
               
                 Anti-Human 
                   
                   
                   
                   
                   
               
               
                 IgGFC 
               
               
                   
               
            
           
           
               
            
               
                 Raw Materials 
               
            
           
           
               
               
            
               
                 Component 
                 Conc. 
               
               
                   
               
               
                 Zika M Visualization Buffer  
                 0.40 mg/L  
               
               
                 (Zika NS1: NSP-DMAE-NHS) 
                   
               
               
                 SERA-MAG ® Magnetic Streptavidin 
                  0.1 mg/mL 
               
               
                 Microparticles 
                   
               
               
                 Biotinylated anti-human IgM 
                 0.04 mg/mg 
               
               
                 monoclonal antibody 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 Zika M assay formulation without goat-anti-human IgG Fc 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Buffer  
                 Ancillary Reagent  
                 Visualization Reagent  
               
               
                 (pH 8.0) 
                 (pH 8.0) 
                 (pH 8.0) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Component 
                 Conc. 
                 Component 
                 Conc. 
                 Component 
                 Conc. 
               
               
                   
               
               
                 Tricine 
                 18.0 g/L  
                 EPPS 
                 25.3 g/L  
                 EPPS 
                 25.3 g/L  
               
               
                 Sodium  
                 15.0 g/L  
                 Sodium  
                 15.0 g/L  
                 Sodium  
                 15.0 g/L  
               
               
                 Chloride 
                   
                 Chloride 
                   
                 Chloride 
                   
               
               
                 TWEEN ®20 
                 2.20 g/L  
                 TWEEN ®20 
                 2.20 g/L  
                 TWEEN ®20 
                 2.20 g/L  
               
               
                 detergent 
                   
                 detergent 
                   
                 detergent 
                   
               
               
                 Disodium  
                 0.7 g/L  
                 Disodium  
                 0.70 g/L  
                 Disodium  
                 0.70 g/L  
               
               
                 EDTA 
                   
                 EDTA 
                   
                 EDTA 
                   
               
               
                 MICRO- 
                 2.50 mL/L 
                 MICRO- 
                  1.0 mL/L 
                 MICRO- 
                 1.0 mL/L 
               
               
                 PROTECT ™  
                   
                 PROTECT ™  
                   
                 PROTECT ™  
                   
               
               
                 preservative 
                   
                 preservative 
                   
                 preservative 
                   
               
               
                 Sodium Azide 
                 0.9 g/L  
                 Sodium Azide 
                 0.9 g/L  
                 Sodium Azide 
                 0.9 g/L  
               
               
                 BSA,  
                 10.0 g/L  
                 BSA,  
                 10.0 g/L  
                 BSA,  
                 10.0 g/L  
               
               
                 Sulfhydryl 
                   
                 Sulfhydryl 
                   
                 Sulfhydryl 
                   
               
               
                 Modified 
                   
                 Modified 
                   
                 Modified 
                   
               
               
                 Heat  
                 50.0 mL/L 
                 Heat  
                  400 mL/L 
                   
                   
               
               
                 Inactivated 
                   
                 Inactivated 
                   
                   
                   
               
               
                 Goat Serum 
                   
                 Goat Serum 
               
               
                   
               
            
           
           
               
            
               
                 Raw Materials 
               
            
           
           
               
               
            
               
                 Component 
                 Conc. 
               
               
                   
               
               
                 Zika M Visualization Buffer  
                 0.40 mg/L  
               
               
                 (Zika NS1: NSP-DMAE-NHS) 
                   
               
               
                 SERA-MAG ® Magnetic Streptavidin 
                  0.1 mg/mL 
               
               
                 Microparticles 
                   
               
               
                 Biotinylated anti-human IgM  
                 0.04 mg/mg 
               
               
                 monoclonal antibody 
               
               
                   
               
            
           
         
       
     
     Results 
     Omitting goat-anti-human IgG Fc from the solid support buffer resolved the IgG detection issue, which was demonstrated through IgM blocking experiments (Table 10) and Zika IgG humanized monoclonal antibody dilution study (Table 11). 
     IgM Blocking Study 
     The blocking experiments (Table 10) were performed by pre-treating wetcake (SERA-MAG Magnetic Streptavidin Microparticles (MG-SA) (GE HealthCare Bio-Sciences Corp.) coated with anti-human IgM antibody) with non-specific human IgM (I8260, Sigma). Samples from Zika-positive several bleed panels were tested with the two formulations, one formulation with goat-anti-human IgG Fc (see Table 8) and another formulation without goat-anti-human IgG Fc (see Table 9) in which the wetcake had or had not been incubated with IgM blocker. Reactivity was analyzed by measuring relative light units (RLUs) and, the percent inhibition was calculated in the following manner: 
       % Inhibition=[(No IgM Blocking−Background)−(With IgM Blocking−Background)]/(No IgM−locking Background)
 
     Using the formulation with goat-anti-human IgG Fc, incubation with non-specific human IgM led to an average inhibition of only 39%. Using the formulation without goat-anti-human IgG Fc, incubation with non-specific human IgM resulted in 103% inhibition. This demonstrated that, in samples suspected to have Zika IgG, reactivity could be completely eliminated in the no goat-anti-human IgG Fc formulation (Table 10) by blocking with human IgM. Thus, the assay formulation without goat-anti-human IgG Fc does not bind human anti-Zika IgG. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 IgM Blocking: Compare formulation having goat-anti-human 
               
               
                 IgG Fc to formulation having no goat-anti-human IgG Fc 
               
            
           
           
               
               
               
            
               
                   
                 Formulation with Goat Fc 
                 Formulation No Goat Fc 
               
               
                   
                 (Zika Ab) 
                 (Zika M) 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 No IgM 
                 With IgM 
                   
                 No IgM 
                 With IgM 
                   
               
               
                 Sample 
                 Sample ID 
                 Blocking 
                 Blocking 
                 Inhibition 
                 Blocking 
                 Blocking 
                 Inhibition 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Normal 
                 2437115 
                 13,767 
                 14,360 
                 N/A 
                 12,843 
                 19,639 
                 N/A 
               
               
                 Donor 
                 2437117 
                 13,357 
                 14,023 
                 N/A 
                 15,222 
                 21,813 
                 N/A 
               
               
                   
                 2439148 
                 13,142 
                 14,471 
                 N/A 
                 10,806 
                 19,493 
                 N/A 
               
               
                   
                 2439153 
                 11,625 
                 14,950 
                 N/A 
                 13,146 
                 23,991 
                 N/A 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Average Background 
                 12,973 
                 14,451 
                 N/A 
                 13,004 
                 21,234 
                 N/A 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Zika 
                 SD TDS0116V2 
                 202,052 
                 135,059 
                 36% 
                 N/A 
                 N/A 
                 N/A 
               
               
                 Positive 
                 SD TDS0116V8 
                 512,102 
                 319,638 
                 39% 
                 N/A 
                 N/A 
                 N/A 
               
               
                 Serial 
                 S-TDS0067V1 
                 102,777 
                 76,821 
                 31% 
                 N/A 
                 N/A 
                 N/A 
               
               
                 Draws 
                 S-TDS0067V8 
                 579,790 
                 384,744 
                 35% 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
                 S-TDS0123V1 
                 110,617 
                 79,051 
                 34% 
                 23,639 
                 19,361 
                 118%  
               
               
                   
                 S-TDS0123V8 
                 1,926,160 
                 1,257,580 
                 35% 
                 68,364 
                 22,422 
                 98% 
               
               
                   
                 SD TDS0143V2 
                 1,306,188 
                 833,212 
                 37% 
                 126,956 
                 22,986 
                 98% 
               
               
                   
                 SD TDS0143V8 
                 923,575 
                 618,585 
                 34% 
                 30,083 
                 24,555 
                 81% 
               
               
                   
                 S-TDS0150V2 
                 52,752 
                 37,819 
                 41% 
                 66,876 
                 18,124 
                 106%  
               
               
                   
                 S-TDS0150V8 
                 791,043 
                 494,667 
                 38% 
                 97,975 
                 20,642 
                 101%  
               
               
                   
                 S-TDS0156V1 
                 79,066 
                 57,542 
                 35% 
                 29,499 
                 19,103 
                 113%  
               
               
                   
                 S-TDS0156V8 
                 548,211 
                 339,358 
                 39% 
                 39,531 
                 21,001 
                 101%  
               
               
                   
                 S-TDS0171V1 
                 N/A 
                 N/A 
                 N/A 
                 37,815 
                 20,504 
                 103%  
               
               
                   
                 S-TDS0171V2 
                 1,862,393 
                 1,147,510 
                 39% 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
                 SD TDS0171V8 
                 725,385 
                 389,415 
                 47% 
                 118,221 
                 23,580 
                 98% 
               
               
                   
                 S-TDS0220V1 
                 332,984 
                 212,381 
                 38% 
                 39,967 
                 20,799 
                 102%  
               
               
                   
                 S-TDS0220V8 
                 550,814 
                 368,590 
                 34% 
                 32,397 
                 22,852 
                 92% 
               
               
                   
                 S-TDS0246V1 
                 104,627 
                 74,876 
                 34% 
                 74,422 
                 23,043 
                 97% 
               
               
                   
                 S-TDS0246V8 
                 568,712 
                 351,616 
                 39% 
                 123,372 
                 25,502 
                 96% 
               
               
                   
                 S-TDS0257V2 
                 227,322 
                 135,499 
                 44% 
                 387,771 
                 26,883 
                 98% 
               
               
                   
                 S-TDS0257V8 
                 298,172 
                 201,619 
                 34% 
                 51,558 
                 15,038 
                 116%  
               
               
                   
                 S-TDS0270V1 
                 29,598 
                 21,317 
                 59% 
                 133,410 
                 21,266 
                 100%  
               
               
                   
                 S-TDS0270V2 
                 509,633 
                 296,302 
                 43% 
                 216,768 
                 25,458 
                 98% 
               
               
                   
                 S-TDS0270V8 
                 246,886 
                 160,573 
                 38% 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
                 S-TDS0271V1 
                 41,728 
                 32,103 
                 39% 
                 19,625 
                 18,204 
                 146%  
               
               
                   
                 S-TDS0271V8 
                 562,101 
                 368,303 
                 36% 
                 25,690 
                 18,868 
                 119%  
               
               
                   
                 S-TDS0282V2 
                 290,474 
                 134,788 
                 57% 
                 523,732 
                 35,679 
                 97% 
               
               
                   
                 S-TDS0282V8 
                 150,834 
                 101,864 
                 37% 
                 49,105 
                 18,758 
                 107%  
               
               
                   
                 S-TDS0284V2 
                 324,150 
                 49,351 
                 89% 
                 1,850,327 
                 75,090 
                 97% 
               
               
                   
                 S-TDS0284V8 
                 82,944 
                 55,043 
                 42% 
                 159,110 
                 23,832 
                 98% 
               
               
                   
                 S-TDS0288V2 
                 343,600 
                 233,380 
                 34% 
                 71,657 
                 24,542 
                 94% 
               
               
                   
                 S-TDS0288V8 
                 188,297 
                 141,843 
                 27% 
                 24,637 
                 19,485 
                 115%  
               
               
                   
                 S-TDS0289V1 
                 195,875 
                 127,818 
                 38% 
                 23,389 
                 19,912 
                 113%  
               
               
                   
                 S-TDS0289V8 
                 860,416 
                 535,146 
                 39% 
                 27,448 
                 21,552 
                 98% 
               
               
                   
                 S-TDS0310V2 
                 2,952,971 
                 1,954,619 
                 34% 
                 155,367 
                 28,164 
                 95% 
               
               
                   
                 S-TDS0310V8 
                 1,039,104 
                 650,438 
                 38% 
                 50,448 
                 21,629 
                 99% 
               
               
                   
                 S-TDS0314V1 
                 509,816 
                 323,250 
                 38% 
                 60,409 
                 19,429 
                 104%  
               
               
                   
                 S-TDS0314V8 
                 896,907 
                 563,005 
                 38% 
                 65,282 
                 21,897 
                 99% 
               
               
                   
                 S-TDS0315V2 
                 733,792 
                 445,873 
                 40% 
                 33,626 
                 21,826 
                 97% 
               
               
                   
                 S-TDS0315V8 
                 332,319 
                 209,223 
                 39% 
                 22,858 
                 18,108 
                 132%  
               
               
                   
                 S-TDS0322V2 
                 1,111,591 
                 727,669 
                 35% 
                 67,715 
                 20,621 
                 101%  
               
               
                   
                 S-TDS0322V8 
                 630,740 
                 407,999 
                 36% 
                 58,654 
                 25,406 
                 91% 
               
               
                   
                 S-TDS0345V2 
                 99,900 
                 57,264 
                 51% 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
                 S-TDS0345V8 
                 141,480 
                 94,846 
                 37% 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
                 S-TDS0362V2 
                 195,550 
                 114,925 
                 45% 
                 202,942 
                 28,319 
                 96% 
               
               
                   
                 S-TDS0362V8 
                 251,220 
                 157,971 
                 40% 
                 33,200 
                 20,973 
                 101%  
               
               
                   
                 S-TDS0396V1 
                 114,005 
                 78,597 
                 37% 
                 N/A 
                 N/A 
                 N/A 
               
               
                   
                 S-TDS0396V2 
                 1,451,870 
                 975,558 
                 33% 
                 95,521 
                 21,929 
                 99% 
               
               
                   
                 S-TDS0396V8 
                 634,572 
                 386,259 
                 40% 
                 38,965 
                 18,728 
                 110%  
               
               
                   
                 S-TDS0401V2 
                 906,881 
                 569,003 
                 38% 
                 27,095 
                 19,024 
                 116%  
               
               
                   
                 S-TDS0401V8 
                 297,200 
                 187,950 
                 39% 
                 27,929 
                 18,227 
                 120%  
               
               
                   
                 S-TDS0422V1 
                 369,715 
                 226,977 
                 40% 
                 136,705 
                 24,481 
                 97% 
               
               
                   
                 S-TDS0422V8 
                 222,534 
                 141,486 
                 39% 
                 62,710 
                 20,654 
                 101%  
               
               
                   
                 S-TDS0468V1 
                 158,078 
                 113,324 
                 32% 
                 69,662 
                 22,190 
                 98% 
               
               
                   
                 S-TDS0468V2 
                 554,736 
                 359,027 
                 36% 
                 225,839 
                 23,850 
                 99% 
               
               
                   
                 S-TDS0468V8 
                 729,933 
                 473,088 
                 36% 
                 98,505 
                 21,813 
                 99% 
               
               
                   
                 S-TDS0478V2 
                 475,450 
                 293,242 
                 40% 
                 103,273 
                 21,893 
                 99% 
               
               
                   
                 S-TDS0478V8 
                 435,118 
                 278,252 
                 38% 
                 21,930 
                 20,127 
                 112%  
               
               
                   
                 S-TDS0499V2 
                 1,254,676 
                 821,953 
                 35% 
                 64,277 
                 21,236 
                 100%  
               
               
                   
                 S-TDS0499V8 
                 912,477 
                 582,515 
                 37% 
                 30,958 
                 20,927 
                 102%  
               
               
                   
               
            
           
         
       
     
     Zika IgG Humanized Monoclonal Antibody Dilution Study 
     The cross-reactivity of Zika Ab assay (with goat anti-human Fc) and the Zika M assay (without goat anti-human Fc) to Zika-specific IgG was further evaluated using a monoclonal antibody IgG specific to Zika NS1. The monoclonal antibody (ZKA35) was obtained from HUMABS BioMed. Serial dilutions (0.001 ug/ml to 1.0 ug/ml) of ZKA35 were tested with the Zika Ab assay and the Zika M assay, as well as a prototype Zika Total assay (which was designed to detect both anti-Zika virus IgM and IgG antibodies). 
     The reactivity of each ZKA35 IgG dilutions with the respective Zika virus assays was calculated as signal (RLU)/cut-off (S/Co). Based on reactivity of samples from normal population and Zika PCR positive individuals, cut-offs of 42500, 70000 and 35000 were used for the Zika M assay, the Zika Ab assay, and the Zika Total assay, respectively. The S/Co of ≥1.0 was considered reactive with ZKA35 IgG. 
     The Zika Total assay, which detects both Zika IgG and IgM antibodies, was reactive with dilutions containing 0.063 μg/mL to 1.0 μg/mL of ZKA35 IgG (Table 11). The Zika Ab assay was reactive with dilutions containing 0.125 μg/mL to 1.0 μg/mL of ZKA35 IgG and showed approximately 10-fold lower reactivity compared to Zika Total assay at the highest ZKA35 IgG concentration. For the Zika M assay, all tested ZKA35 IgG dilutions were nonreactive (Table 11). These results demonstrate that while some Zika IgG cross-reactivity is seen with the Zika Ab assay, the Zika M assay does not cross-react with Zika specific IgG up to concentration of 1 μg/mL. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Results from Zika IgG (ZKA35) Serial Dilution Study 
               
            
           
           
               
               
               
               
               
            
               
                 Zika IgG 
                   
                   
                   
                   
               
               
                 (ZKA35) 
               
               
                 Concentration 
                   
                 Zika M 
                 Zika Ab 
                 Zika Total 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 (μg/mL) 
                 Sample ID 
                 RLU 
                 S/Co 
                 RLU 
                 S/Co 
                 RLU 
                 S/Co 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1.000 
                 MS012017A 
                 15,600 
                 0.37 
                 508,892 
                 7.27 
                 2,752,938 
                 78.66 
               
               
                 0.500 
                 MS012017B 
                 13,976 
                 0.33 
                 265,539 
                 3.79 
                 1,034,048 
                 29.54 
               
               
                 0.250 
                 MS012017C 
                 14,142 
                 0.33 
                 144,352 
                 2.06 
                 351,344 
                 10.04 
               
               
                 0.125 
                 MS012017D 
                 13,683 
                 0.32 
                 81,223 
                 1.16 
                 134,427 
                 3.84 
               
               
                 0.063 
                 MS012017E 
                 14,590 
                 0.34 
                 48,646 
                 0.69 
                 59,543 
                 1.70 
               
               
                 0.031 
                 MS012017F 
                 14,258 
                 0.34 
                 31,549 
                 0.45 
                 31,522 
                 0.90 
               
               
                 0.016 
                 MS012017G 
                 14,163 
                 0.33 
                 22,745 
                 0.32 
                 19,300 
                 0.55 
               
               
                 0.008 
                 MS012017H 
                 14,224 
                 0.33 
                 18,546 
                 0.26 
                 13,457 
                 0.38 
               
               
                 0.004 
                 MS012017I 
                 15,134 
                 0.36 
                 17,399 
                 0.25 
                 10,455 
                 0.30 
               
               
                 0.002 
                 MS012017J 
                 14,775 
                 0.35 
                 16,247 
                 0.23 
                 9,070 
                 0.26 
               
               
                 0.001 
                 MS012017K 
                 13,716 
                 0.32 
                 15,543 
                 0.22 
                 8,675 
                 0.25 
               
               
                   
               
            
           
         
       
     
     Standardization 
     The cutoff indices of the Zika Ab and Zika M assays were set based on testing presumed Zika virus negative normal donor samples (including pregnant women) from the US mainland, Zika virus PCR positive serial draws, and Dengue/West Nile virus positive cross-reactive samples (SeraCare Panel). The initial value of Zika Ab and Zika M standards was determined by testing the population mentioned above on a small lot of standards based on the cutoff during early development. Then a second larger lot of standards was built and values assigned from the previous small lots. These larger lots (Zika Ab Standards lot #16KL240 and Zika M standards lot #17CL059) served as the Anchor Standards for the standardization. Gold Standards are traceable to the Anchor Standards. Both Zika Ab and Zika M were assigned master curves and calibrator values were traceable to the internal gold standards. The Zika Ab and Zika M Gold Standards were used for evaluation and confirmation of reagent performance and in-process testing and value assignment of new lots of standards, calibrators, controls and medical decision pools. 
     The Zika Ab assay uses 6-level standards formulated with defibrinated and dialyzed human plasma. The Zika M assay also uses 6-level standards formulated with defibrinated and dialyzed human plasma. The Zika Ab and Zika M standards were built by spiking Zika IgM antibody positive pools into the Zika Ab and Zika M negative plasma pools respectively. The Zika Ab and Zika M standard levels S02-S06 are spiked with Zika M positive pools. The lowest Zika Ab and Zika M standard level S01 is unspiked Zika Ab and Zika M negative human plasma pools. 
     The Zika Ab and Zika M assays use 2 level calibrators formulated with defibrinated and dialyzed human plasma. The Zika Ab and Zika M high calibrator (above cutoff) was prepared by spiking Zika IgM antibody positive pools into the Zika Ab and Zika M negative plasma pools respectively. The Zika Ab and Zika M low calibrators (below cutoff) are unspiked Zika Ab and Zika M negative human plasma pools. 
     The Zika Ab and Zika M assays of the Zika Test use a 2-point calibration curve fit based on a 4PL-weighted curve fitting algorithm. 
     The Zika Ab and Zika M assays both use 2-level controls formulated with defibrinated, dialyzed human plasma. The Zika Ab and Zika M positive controls were prepared by spiking Zika IgM antibody positive pools into the Zika Ab and Zika gM negative plasma pools respectively. Both the Zika Ab and Zika IgM negative controls were unspiked Zika Ab and Zika M negative human plasma pools. 
     The Zika Ab and Zika M assays each have one level of Medical Decision Pools (MDPs) formulated with defibrinated and dialyzed human plasma. The Zika Ab and Zika M medical decision pools were prepared by spiking Zika M antibody positive pools into the Zika Ab and Zika M negative plasma pools respectively. 
     Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred and exemplified embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.