阅读说明：本技术 用于寨卡病毒免疫测定的方法和试剂 (Methods and reagents for Zika virus immunoassay ) 是由 S·帕蒂班德拉 K·辛格 于 2019-09-18 设计创作，主要内容包括：本文公开了用于检测来自受试者的生物样品中的抗寨卡IgM抗体和/或诊断受试者中的寨卡病毒感染的免疫测定方法和试剂。还公开了用于执行公开的方法的算法。公开的免疫测定方法、试剂和算法使得能够有效和可靠定性检测抗寨卡病毒抗体和快速确定人受试者中的寨卡病毒感染的假定阳性结果。(Disclosed herein are immunoassay methods and reagents for detecting anti-zika IgM antibodies in a biological sample from a subject and/or diagnosing zika virus infection in a subject. Algorithms for performing the disclosed methods are also disclosed. The disclosed immunoassay methods, reagents and algorithms enable efficient and reliable qualitative detection of anti-zika virus antibodies and rapid determination of putative positive results for zika virus infection in human subjects.)

1. A method of detecting anti-zika virus IgM antibodies in a biological sample from a human subject, the method 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 is provided,

labeled Zika virus antigens, and

a solid support comprising an anti-human IgM antibody,

wherein, in the presence of anti-Zika virus IgG antibodies, anti-Zika virus IgM antibodies, or anti-Zika virus IgG antibodies and anti-Zika virus IgM antibodies in the biological sample, a complex I is formed comprising (I) anti-Zika virus IgG antibodies, anti-Zika virus IgM antibodies, or anti-Zika virus IgG antibodies and anti-Zika virus IgM antibodies, (ii) labeled Zika virus antigens, and (iii) a solid support comprising anti-human IgM antibodies; and

b) detecting the complex I, the presence of the complex I indicating the presence of anti-zaka virus IgM antibody, anti-zaka virus IgG antibody, or anti-zaka virus IgG antibody and anti-zaka virus IgM antibody in the biological sample, and if the complex I is detected, performing the second immunoassay, the second immunoassay comprising:

c) incubating the biological sample with:

a solid support comprising anti-human IgM antibody, and

(ii) a labeled antigen of Zika virus,

wherein, in the presence of anti-Zika virus IgM antibody in the biological sample, a complex II is formed, the complex II comprising (i) a solid support comprising anti-human IgM antibody, (II) anti-Zika virus IgM antibody, and (iii) a labeled Zika virus antigen; and

b) detecting said complex II, the presence of said complex II indicating the presence of said anti-Zika virus IgM antibody in said biological sample.

2. The method of claim 1, wherein the label comprises an enzyme conjugate, a fluorescent probe, a radioisotope, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.

3. The method of claim 2, wherein the label comprises an Acridinium Ester (AE) or an analog thereof.

4. The method of claim 3, wherein the AE or analog thereof is DMAE, NSP-DMAE, HQYAE, ZAE, Iso-Di-ZAE, TSP-AE or HEG-GLU-AE.

5. The method of any preceding claim, wherein said Zika virus antigen is Zika virus NS1 antigen or an immunogenic fragment thereof.

6. The method of claim 5, wherein said Zika virus NS1 antigen or immunogenic fragment thereof is recombinant.

7. The method of any preceding claim, wherein the anti-human IgG Fc antibody is a monoclonal or polyclonal antibody.

8. The method of any preceding claim, wherein the anti-human IgG Fc antibody is a goat anti-human IgG Fc antibody.

9. The method of any preceding claim, wherein the anti-human IgM antibody is indirectly attached to the solid support.

10. The method of claim 9, wherein said anti-human IgM antibody is biotinylated and said solid support comprises streptavidin.

11. The method of claim 1, wherein the anti-human IgG Fc antibody is indirectly attached to the solid support.

12. The method of claim 11, wherein the anti-human IgG Fc antibody is biotinylated and the solid support comprises streptavidin.

13. The method of any preceding claim, wherein the biological sample is serum or plasma.

14. The method of claim 13, wherein the biological sample further comprises an anticoagulant.

15. The method of claim 14, wherein said anticoagulant comprises EDTA or heparin.

16. The method of any preceding claim, wherein the biological sample is obtained from the subject at least 8 days after the onset of symptoms of Zika virus infection or the risk of exposure to Zika virus.

17. The method of any preceding claim, wherein, in the first immunoassay, the anti-human IgM antibody and the solid support are present in a buffer comprising tricine, sodium chloride, Tween 20, disodium EDTA, a preservative, thiol-modified bovine serum albumin, and anti-human IgG Fc antibody, and wherein, in the second immunoassay, the IgM antibody and the solid support are present in a buffer comprising tricine, sodium chloride, Tween 20, disodium EDTA, a preservative, thiol-modified bovine serum albumin, but no anti-human IgG Fc antibody.

18. The method of any preceding claim, wherein the solid support comprises a column matrix material, a culture plate, a tube, a dish, a flask, a microtiter plate, a bead, or a combination thereof.

19. The method of any preceding claim, wherein the solid support comprises paramagnetic particles (PMP) or Latex Magnetic Particles (LMP).

20. The method of any one of the preceding claims, wherein said Zika virus antigen comprises an epitope tag, wherein said epitope tag is a 6-histidine tag, a hemagglutinin tag, glutathione-S-transferase, a maltose binding protein, or a chitin binding protein.

21. The method of claim 1, further comprising determining the level of anti-Zika virus IgG antibodies, anti-Zika virus IgM antibodies, or anti-Zika virus IgG antibodies and anti-Zika virus IgM antibodies in the biological sample.

22. The method of claim 21, wherein the level of anti-Zika virus IgG antibodies, anti-Zika virus IgM antibodies, or anti-Zika virus IgG antibodies and anti-Zika virus IgM antibodies in the biological sample is directly proportional to the level of complex I or complex II detected.

23. The method of any preceding claim, wherein the detecting comprises measuring a signal from the marker and comparing the signal to a control signal from a biological sample known to be negative for anti-Zika virus antibodies.

24. The method of claim 1, wherein if said complex II is detected, performing a second immunoassay further comprises repeating steps c) and d) in at least duplicate.

25. The method of claim 24, wherein the method further comprises determining that the human subject is positive for anti-Zika virus IgM antibody if the complex II is detected in at least 2 equivalent repeats of the 3 repeats.

26. A method of detecting antibodies to zika virus in a human subject, the method comprising performing a first immunoassay comprising:

a) incubating a biological sample from the subject with:

a solid support comprising an anti-human IgM antibody,

anti-human IgG Fc antibodies, and

(ii) a labeled antigen of Zika virus,

wherein, in the presence of anti-Zika virus IgG antibodies, anti-Zika virus IgM antibodies, or anti-Zika virus IgG antibodies and anti-Zika virus IgM antibodies in the biological sample, a complex I is formed comprising (I) a solid support comprising anti-human IgM antibodies, (ii) anti-Zika virus IgG antibodies, anti-Zika virus IgM antibodies, or anti-Zika virus IgG antibodies and anti-Zika virus IgM antibodies, and (iii) labeled Zika virus antigens;

b) detecting said complex I, and

b_i) Determining that the subject is negative for antibodies to Zika virus if the complex I is not detected; or

b_ii) Determining that the subject is positive for anti-Zika virus antibodies if the complex I is detected.

27. The method of claim 26, further comprising performing a second immunoassay comprising:

c) incubating the biological sample with:

a solid support comprising anti-human IgM antibody, and

(ii) a labeled antigen of Zika virus,

wherein, in the presence of anti-Zika virus IgM antibody in the biological sample, a complex II is formed, the complex II comprising (i) a solid support comprising anti-human IgM antibody, (II) anti-Zika virus IgM antibody, and (iii) a labeled Zika virus antigen;

d) detecting said complex II, and

d_i) If said complex II is not detected, the human subject is determined to be negative for the anti-Zika virus IgM antibody.

28. The method of claim 27, further comprising:

d_ii) If said complex II is detected, repeating steps c) and d) at least in duplicate, and

e) determining that the human subject is positive for anti-Zika virus IgM antibody if the complex II is detected in at least 2 of the 3 replicates in an equivalent replicate.

29. The method of claim 26 or 27, wherein said detecting comprises measuring a signal from said marker and comparing said signal to a control signal from a biological sample known to be negative for anti-Zika virus antibodies.

30. A kit, comprising:

a solid support,

an anti-human IgM antibody which is capable of inhibiting the growth of an antigen,

anti-human IgG Fc antibodies, and

labeled Zika virus antigen.

31. The kit of claim 30, wherein the label comprises an enzyme conjugate, a fluorescent probe, a radioisotope, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.

32. The kit of claim 31, wherein said label is an Acridinium Ester (AE) or an analog thereof.

33. The kit of any one of claims 30 to 32, wherein the solid support comprises a column matrix material, a culture plate, a tube, a dish, a flask, a microtiter plate, a bead, a microparticle, or a combination thereof.

34. The kit of claim 33, wherein the solid support is paramagnetic particles (PMP) or Latex Magnetic Particles (LMP).

35. A kit comprising 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 which is capable of inhibiting the growth of an antigen,

anti-human IgG Fc antibodies, and

a labeled Zika virus antigen; and

wherein the reagents for the second immunoassay comprise:

a solid support,

anti-human IgM antibodies, and

labeled Zika virus antigen.

36. The kit of claim 35, further comprising instructions for performing a first and a second immunoassay, wherein the instructions direct the 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 the biological sample is determined to be positive for anti-zika virus antibodies in the first immunoassay.

Technical Field

Disclosed herein are methods of detecting anti-zika virus IgM antibodies 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 belonging to the Flaviviridae family (Musso D, Gubler DJ. Zika virus. Clin Microbiol Rev. 2016 Jul;29(3):487 524), which includes the closely related dengue, West Nile, Japanese encephalitis and yellow fever viruses (Rabe IB, Staples JE, Villanueva J, et al, inter prophylaxis for Interpretation of Zika virus antibody results, MMWR. 2016;65(21): 543) 546). In 1947, Zika virus was first isolated from rhesus monkeys in Wuganda Zika forest (Musso et al, 2016). In 1954, Zika virus infection in humans was first reported in Nigeria, and the first epidemic was reported in 2007 on the island of the Western Pacific ocean in Yapu (Yap), and later in 2013 and 2014 in the French Polish. Zika virus outbreaks first appeared in Brazil in America 3 months in 2015 and spread to several countries and regions by 2016 (Petersen LR, Jamieson DJ, Powers AM, Honein MA. Zika virus. N Engl J Med. 2016;374 (16): 1552) 1563 months.

Zika virus infection is mainly caused by infected mosquitoes (Aedes aegypti: (A. aegypti)Aedes aegypti) ) the transmission of bites. However, transmission from mother to fetus during pregnancy and transmission by sexual contact with infected partners has been reported (supra). Potential transmission by transfusion has been recorded (Musso et al, 2016). Most of those infected with Zika virus showed mild symptoms or no symptoms (no symptoms developed) (Zika: The basics of The virus and how to protect against agains it. CDC's response to Zika. targets for diseases Control and preservation view. https:// www _ CDC _ gov/Zika/pdfs/fs-Zika-basics _ pdf. 2017, day 27. accession: 2016, 8, 12). Common symptoms include fever, rash, arthralgia, and redness, and these symptoms can be observed for up to one week (Petersen et al, 2016)). Zika virus infection in pregnant women may cause a small head deformity in the fetus, which is a major public health problem. An association between Zika virus infection and Guillain-Barre syndrome, a neurological disease that causes temporary paralysis, has also been reported (Musso et al, 2016; Petersen et al, 2016).

During zika virus infection, viremia was expected to persist for one week after symptom onset, and zika virus-specific IgM antibodies were reported to develop during the first week after symptom onset, and were expected to persist for up to 12 weeks (Rabe et al, 2016). Detection of Zika virus-specific IgM antibodies was used for diagnosis and appropriate clinical management of suspected Zika virus-infected patients (supra).

The emergence of Zika virus infection outbreaks in America in 2015-2016 required urgent development of diagnostic tests to detect Zika virus-specific IgM antibodies in individuals recently infected with Zika virus for appropriate clinical management of them.

Immunoassays for the detection of anti-zika IgM antibodies in biological samples from human subjects have been developed, but available immunoassays have various drawbacks, including: 1) non-specific reactions with serum/plasma from normal donors and pregnant women, resulting in poor specificity in the non-endemic population of zika; 2) cross-reactivity with zika IgG antibodies in samples from human subjects, resulting in poor specificity in the zika circulating population; 3) the need to test a single sample with up to 3 different antigens in the same assay increases the time and cost to produce a result; and/or 4) the need to test each sample with at least two different assays also increases the time and cost to produce results. Accordingly, there remains a need for immunoassay methods and reagents for reliably and efficiently detecting anti-Zika virus IgM antibodies in a biological sample from a subject.

SUMMARY

Disclosed herein are methods of detecting anti-zika virus IgM antibodies in a biological sample from a human subject, the method 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 anti-human IgM antibodies, wherein, in the presence of the anti-zika virus IgG antibodies, the anti-zika virus IgM antibodies, or the anti-zika virus IgG antibodies and the anti-zika virus IgM antibodies in the biological sample, a complex I is formed comprising (I) the anti-zika virus IgG antibodies, the anti-zika virus IgM antibodies, or the anti-zika virus IgG antibodies and the anti-zika virus IgM antibodies, (ii) the labeled zika virus antigen, and (iii) the solid support comprising the anti-human IgM antibodies; and b) detecting complex I, the presence of complex I indicating the presence of anti-zaka virus IgM antibody, anti-zaka virus IgG antibody, or anti-zaka virus IgG antibody and anti-zaka virus IgM antibody in the biological sample, and if the complex I is detected, performing the second immunoassay, the second immunoassay comprising: c) incubating the biological sample with a solid support comprising anti-human IgM antibodies and labeled zika virus antigens, wherein, in the presence of anti-zika virus IgM antibodies in the biological sample, a complex II is formed comprising (i) a solid support comprising anti-human IgM antibodies, (II) anti-zika virus IgM antibodies, and (iii) labeled zika virus antigens; and b) detecting said complex II, the presence of said complex II indicating the presence of said anti-Zika virus IgM antibody in said biological sample.

Also disclosed is a method of detecting antibodies to zika virus in a subject, the method comprising performing a first immunoassay comprising:

a) incubating a biological sample from the subject with:

a solid support comprising an anti-human IgM antibody,

anti-human IgG Fc antibodies, and

(ii) a labeled antigen of Zika virus,

wherein, in the presence of anti-Zika virus IgG antibodies, anti-Zika virus IgM antibodies, or anti-Zika virus IgG antibodies and anti-Zika virus IgM antibodies in the biological sample, a complex I is formed comprising (I) a solid support comprising anti-human IgM antibodies, (ii) anti-Zika virus IgG antibodies, anti-Zika virus IgM antibodies, or anti-Zika virus IgG antibodies and anti-Zika virus IgM antibodies, and (iii) labeled Zika virus antigens;

b) detecting said complex I, and

b_i) Determining that the subject is negative for antibodies to Zika virus if the complex I is not detected; or

b_ii) Determining that the subject is positive for anti-Zika virus antibodies if the complex I is detected.

The disclosed methods may further comprise performing a second immunoassay comprising:

c) incubating the biological sample with:

a solid support comprising anti-human IgM antibody, and

(ii) a labeled antigen of Zika virus,

wherein, in the presence of anti-Zika virus IgM antibody in the biological sample, a complex II is formed, the complex II comprising (i) a solid support comprising anti-human IgM antibody, (II) anti-Zika virus IgM antibody, and (iii) a labeled Zika virus antigen;

d) detecting said complex II, and

d_i) If said complex II is not detected, the human subject is determined to be negative for the anti-Zika virus IgM antibody.

The disclosed method may further comprise:

d_ii) If said complex II is detected, repeating steps c) and d) at least in duplicate, and

e) determining that the human subject is positive for anti-Zika virus IgM antibody if the complex II is detected in at least 2 of the 3 replicates in an equivalent replicate.

Also disclosed is a kit comprising: a solid support, an anti-human IgM antibody, an anti-human IgG Fc antibody and a labeled Zika virus antigen. The kit may 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, anti-human IgM antibody and labeled zika virus antigen. The kit may further comprise instructions for performing the first and second immunoassays, wherein the instructions direct the 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 the biological sample is determined to be positive for anti-zika virus antibodies in the first immunoassay.

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 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 thereof; however, the methods and kits are not limited to the specific embodiments disclosed. In the drawings:

figure 1 is a schematic diagram of one embodiment of a "zika test" algorithm whereby the results of determining antibody negativity or putative zika positivity against zika virus 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 drawings, 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 of possible mechanisms or modes of action or reasons for improvement is intended to be illustrative only, and the disclosed methods and kits should not be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

Throughout this document, the present specification relates to methods of detecting antibodies and methods of diagnosing Zika virus infection. Where the disclosure describes or claims features or embodiments relating to methods of detecting antibodies, such features or embodiments are equally applicable to methods of diagnosing Zika virus infection. Likewise, where the disclosure describes or claims features or embodiments related to a method of diagnosing Zika virus infection, such features or embodiments are equally applicable to a method of detecting an antibody.

Where a range of numerical values is stated 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 possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within said range to the same extent as if each of those narrower ranges were explicitly stated. Where a range of values is specified herein as being greater than a specified value, the range is still limited and is bounded at its upper end by values operable in the context of the invention as described herein. Where a range of values is specified herein as being less than a specified value, the range is still bounded at 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 understood 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 are used throughout the description and claims that refer to various aspects of the specification. Such terms are 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 the examples encompassed by the term "consisting essentially of and" consisting of; similarly, the term "consisting essentially of is intended to include the examples encompassed by the term" consisting of.

Disclosed herein are immunoassays and methods for detecting anti-zika virus IgM antibodies 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 anti-zika virus IgG antibodies, anti-zika virus IgM antibodies, or anti-zika virus IgG antibodies and anti-zika virus IgM antibodies in the biological sample, a complex I is formed comprising (I) anti-zika virus IgG antibodies, anti-zika virus IgM antibodies, or anti-zika virus IgG antibodies and anti-zika virus IgM antibodies, (ii) labeled zika virus antigens, and (iii) a solid support comprising anti-human IgM antibodies. The method can further comprise detecting the complex I, the presence of the complex I indicating the presence of anti-zika virus IgM antibody, anti-zika virus IgG antibody, or anti-zika virus IgG antibody and anti-zika virus IgM antibody in the biological sample. If the complex I is detected, the method can further comprise performing a second immunoassay comprising incubating the biological sample with a solid support comprising anti-human IgM antibodies and labeled zika virus antigen. In the presence of anti-Zika virus IgM antibody in the biological sample, a complex II is formed, the complex II comprising (i) a solid support comprising anti-human IgM antibody, (II) anti-Zika virus IgM antibody, and (iii) a labeled Zika virus antigen. The method may further comprise detecting the complex II, the presence of the complex II indicating the presence of the anti-zika virus IgM antibody in the biological sample.

In some embodiments, the zika virus antigen is a 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 the Fc region of a human IgG antibody. For example, the anti-human IgG Fc antibody can be a monoclonal or 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 may be a goat antibody against human IgG Fc.

Without wishing to be bound by theory, it is contemplated that the anti-human IgG Fc antibody may be bound to or otherwise coupled to a solid support during the incubation reaction. For example, the anti-human IgG Fc can be non-covalently bound to a solid support. Thus, in some embodiments, the anti-human IgG Fc antibody is indirectly attached 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 attached 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 attached to the solid support via a biotin-streptavidin interaction. In some embodiments, the anti-human IgG Fc antibody is indirectly attached to the solid support. In addition, the anti-human IgG Fc antibody may be biotinylated and thus also indirectly attached to a streptavidin-containing solid support.

The biological sample may be serum or plasma, and it may further comprise an anticoagulant, including, for example, EDTA or heparin. In some embodiments, the biological sample is from a human. A biological sample is obtained from a human subject at least 8 days after the onset of symptoms of a zika virus infection or the risk of exposure to a zika virus. In some embodiments, the biological sample must be obtained from the human subject at least 8 days after the onset of symptoms of Zika virus infection or the risk of exposure to Zika virus to ensure accurate detection of anti-Zika virus IgM antibodies in the biological sample.

In a first immunoassay ("zika Ab" assay), the anti-human IgM antibody and solid support may be present in a buffer comprising tricine, sodium chloride, Tween 20, disodium EDTA, a preservative, thiol-modified bovine serum albumin, and anti-human IgG Fc antibody. In a second immunoassay ("zika M" assay), IgM antibodies and solid support may be present in a buffer comprising tricine, sodium chloride, Tween 20, disodium EDTA, preservative, thiol-modified bovine serum albumin, but no anti-human IgG Fc antibodies.

The methods disclosed herein can further comprise determining the level of anti-zika virus IgG antibodies, anti-zika virus IgM antibodies, or anti-zika virus IgG antibodies and anti-zika virus IgM antibodies in the biological sample. In some embodiments, the level of anti-zika virus IgG antibodies, anti-zika virus IgM antibodies, or anti-zika virus IgG antibodies and anti-zika virus IgM antibodies in the biological sample is directly proportional to the level of complex I or complex II detected. Detecting a signal as used herein can include measuring a signal from a 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 case where complex II is detected in the zika M assay, the method may further comprise the step of repeating the zika M assay in at least duplicate, and determining that the human subject is positive for anti-zika virus IgM antibody if complex II is detected in at least 2 of the 3 repeats.

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 is an algorithm 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 step of detecting antibodies to zika virus in a subject in a manner that increases the efficiency and reliability of the methods disclosed herein. For example, the algorithm can be used to reduce the number of assays required to determine that a particular sample is negative, e.g., for anti-Zika virus IgM antibodies. In some embodiments, the algorithm comprises a set of instructions to guide a user or an automated system by a method of detecting antibodies to zika virus in a biological sample. An example algorithm may instruct a user or system to perform a method of detecting antibodies to zika virus in a subject, the method comprising performing a first immunoassay comprising:

a) incubating a biological sample from the subject with:

a solid support comprising an anti-human IgM antibody,

anti-human IgG Fc antibodies, and

(ii) a labeled antigen of Zika virus,

wherein, in the presence of anti-Zika virus IgG antibodies, anti-Zika virus IgM antibodies, or anti-Zika virus IgG antibodies and anti-Zika virus IgM antibodies in the biological sample, a complex I is formed comprising (I) a solid support comprising anti-human IgM antibodies, (ii) anti-Zika virus IgG antibodies, anti-Zika virus IgM antibodies, or anti-Zika virus IgG antibodies and anti-Zika virus IgM antibodies, and (iii) labeled Zika virus antigens;

b) detecting said complex I, and

b_i) Determining that the subject is negative for antibodies to Zika virus if the complex I is not detected; or

b_ii) Determining that the subject is positive for anti-Zika virus antibodies if the complex I is detected.

If in step b_iWherein complex I is not detected, the algorithm may further instruct the user or system to terminate the assay, thereby reducing the number of assays, the amount of reagent, and detecting the presence of complex I in the biological sampleThe time required for the anti-Zika virus antibody of (1). However, if the result obtained in step b) is ambiguous (i.e., "reactive"), the algorithm may instruct the user or system to perform a second immunoassay comprising:

c) incubating the biological sample with:

a solid support comprising anti-human IgM antibody, and

(ii) a labeled antigen of Zika virus,

wherein, in the presence of anti-Zika virus IgM antibody in the biological sample, a complex II is formed, the complex II comprising (i) a solid support comprising anti-human IgM antibody, (II) anti-Zika virus IgM antibody, and (iii) a labeled Zika virus antigen;

d) detecting said complex II, and

d_i) If said complex II is not detected, the human subject is determined to be negative for the anti-Zika virus IgM antibody.

If the complex II is not detected in step d), the algorithm may instruct the user or the system to terminate the assay. Otherwise, the algorithm may proceed with additional method steps, including:

d_ii) If said complex II is detected, repeating steps c) and d) at least in duplicate, and

e) determining that the human subject is positive for anti-Zika virus IgM antibody if the complex II is detected in at least 2 of the 3 replicates in an equivalent replicate.

In some embodiments of the methods and algorithms for performing the methods disclosed herein, a biological sample known to have or suspected of having anti-zika virus antibodies is incubated with labeled zika virus antigen and a solid support having human IgM antibodies bound thereto. In the absence of the anti-Zika virus antibodies, the labeled Zika virus antigens will not bind to or otherwise interact with a solid support. Thus, in the absence of anti-zika virus antibodies in the biological sample, the labeled zika virus antigen remains in solution and separation of the solid support does not result in separation of the labeled antigen. In another aspect, when the anti-Zika virus antibody is present in the biological sample, the anti-Zika virus antibody simultaneously binds 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 understood that the order in which incubation occurs may differ from that described herein. Furthermore, in an alternative embodiment of the presently described immunoassay, labeled and solid support bound reaction components may be rearranged. For example, a biological sample known to have or suspected of having anti-Zika virus antibodies can be incubated with a solid support having unlabeled Zika virus antigens bound thereto, followed by incubation with labeled anti-human IgM antibodies. In other embodiments, a biological sample known to have or suspected of having anti-zika virus antibodies may be incubated simultaneously with: on the one hand, a solid support having unlabeled anti-human IgM antibody bound thereto and labeled Zika virus antigen, or on the other hand, a solid support having unlabeled Zika virus antigen bound thereto and anti-human IgM antibody.

A biological sample known to have or suspected of having anti-zika virus antibodies can be incubated in the reaction mixture for a period of time sufficient to achieve a partial reaction without allowing the reaction to equilibrate, such as 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 may 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 a mixture of the biological sample and labeled zika virus antigen and incubated for a period of time sufficient to effect a partial reaction without allowing the reaction to equilibrate, such as 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 step is performed for 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 one hour. Subsequent testing may 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 will be appreciated that the amount of time required for the assay or any one or more steps thereof may vary based on several factors, including the level of anti-zika virus antibodies in the biological sample and the affinity of anti-human IgM antibodies for anti-zika virus antibodies in the biological sample. In some embodiments, the incubation of the biological sample with the reaction mixture may be performed for a period of time sufficient to enable the reaction to reach equilibrium, such as about 1 or more hours. Thus, the disclosed methods may be performed for any suitable amount of time.

The anti-human IgM antibody can be directly or indirectly attached to the solid support. Suitable techniques for directly linking the anti-human IgM antibody to the solid support include, for example, covalent attachment, adsorption, non-covalent interactions, or combinations thereof. In some embodiments, the anti-human IgM antibody can be directly attached to the solid support by N-hydroxysuccinimide (NHS) chemistry or by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) NHS chemistry. Suitable techniques for indirectly attaching the anti-human IgM antibody to the solid support include, for example, attachment via a peptide, protein, antibody, linker, or combination thereof. In some embodiments, the anti-human IgM antibody can be indirectly attached 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 understood that in alternative embodiments of the disclosed immunoassays, the same chemicals may be applied to any of the reaction components attached to the solid support.

Exemplary solid supports include, but are not limited to, column matrix material, culture plates, tubes, dishes, flasks, microtiter plates, beads/particles, heat-killed formalin- (or other chemical) -fixed prokaryotic or eukaryotic cells, microscope slides, ACLAR @ thin films, or any other optically clear polymer, or combinations thereof. The solid support may be composed in whole or in part of plastic, cellulose derivatives, nitrocellulose, glass fiber, latex, or a combination thereof. In some embodiments, the solid support comprises magnetic beads/particles. In some embodiments, the magnetic beads/particles are paramagnetic particles (PMPs). In some embodiments, the magnetic beads/particles are Latex Magnetic Particles (LMPs).

The label may be any suitable label known to those skilled in the art to be useful for generating a detectable signal. Suitable detectable labels include, but are not limited to, enzyme conjugates (e.g., horseradish peroxidase (HRP), alkaline phosphatase, glucose oxidase, and beta-galactosidase), fluorescent probes, radioisotopes, chemiluminescent compounds, bioluminescent compounds, or combinations thereof. In some embodiments, the label is an acridinium ester ("AE") or analog thereof. Suitable AE analogs include dimethylacridine ester (DMAE), N-sulfopropyl dimethylacridine 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) ammonium ] propyl ] amino ] carbonyl ] -2, 6-dimethylphenoxy ] carbonyl ] -10- (3-sulfopropyl) -, bis (inner salt)), N-sulfopropyl-2-isopropoxydimethylacridinate (Iso-Di-ZAE), trissulfopropylacridinate (TSP-AE) or N-sulfopropyldimethylacridinate with a 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 may be in the form of an IgA, IgD, IgG, IgE or IgM isotype or single domain, such as a single domain antibody from a 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 specific for human IgM antibodies may also be used.

In some embodiments, the immunoassays disclosed herein meet one or more of the following clinical requirements:

a. more than or equal to 90 percent of positive and negative are consistent;

b. repeatability 0.80-2.00 index: 12.0% and > 2.00 index: less than or equal to 8.0 percent;

c. the intra-operation accuracy is 0.80-2.00 index: ≦ 15.0% and > 2.00 index: less than or equal to 10.0 percent;

d. the calibration interval is more than or equal to 7 days;

e. the stability on the plate is more than or equal to 14 days;

f. a control system capable of maintaining consistent standardization and performance.

g. Interference and reproducibility.

In some embodiments, the immunoassays disclosed herein meet all of the above clinical requirements a.

In some embodiments, the disclosed immunoassays are intended for in vitro diagnostic use in the putative qualitative detection of IgM antibodies to zika virus in serum and plasma (potassium EDTA or lithium heparin, each collected with patient-matched serum samples) samples collected from individuals that meet the clinical criteria for zika virus (e.g., history of clinical signs and symptoms associated with zika virus infection) and/or CDC zika virus epidemiological criteria (e.g., history of residency or traveling to geographical regions with active zika transmission when traveling, or other epidemiological criteria that may indicate zicar virus testing) of the disease control and prevention center (CDC). In some embodiments, samples from symptomatic patients or travelers returning from endemic areas are not collected until 8 days after symptom onset or exposure, respectively.

In some embodiments of the disclosed immunoassays, the first immunoassay ("zika Ab" assay) and optional second immunoassay ("zika M" assay) can be a 2-pass immunoassay format. The zika Ab assay may be an antibody capture immunoassay using a 2-pass format. In the first pass, the patient sample can be incubated in a cuvette, for example, with anti-human IgM antibody attached to a solid support. During incubation, the solid support bound anti-human IgM antibody binds to the antibody from the patient sample. The captured antibody 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. During incubation, the labeled NS1 antigen bound to the patient zika virus antibody on the solid support. The NS 1/solid support complex can be washed and subjected to, for example, chemiluminescent detection.

The zika M assay may be an IgM capture immunoassay using a 2-pass format. In the first pass, the solid support coated with anti-human IgM antibodies and the patient sample can be incubated in a 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 captured anti-Zika virus IgM antibodies on the solid support may be incubated with labeled NS1 for binding NS1 antigen to the captured anti-Zika virus IgM antibodies immobilized on the solid support. The NS 1/solid support complex can be washed and subjected to, for example, chemiluminescent detection.

The disclosed methods may be performed manually or may be automated. For example, the disclosed methods may use ADVIA CENTAUR ® immunoassay system or ATELLICA @^TMAnd (4) carrying out the system. For example, for both the Zika Ab and Zika M assays, the system may be automated to perform the following actions:

1. the samples were dispensed into cuvettes.

2. The buffer containing the solid support with anti-human IgM antibody bound thereto is dispensed and incubated, for example, at 37 ℃ for 18.25 minutes.

3. The solid support is detached/separated, the cuvette is aspirated and washed with washing reagent.

4. The buffer containing the chemiluminescent-tagged NS1 antigen is dispensed and incubated, for example, at 37 ℃ for 18 minutes.

5. The solid support is detached/separated, the cuvette is aspirated and washed with washing reagent.

6. Dispensing a chemiluminescent reagent to initiate a chemiluminescent reaction.

7. Reporting the result according to the option selected by the user.

In a particular embodiment, the disclosed immunoassays may be suitable for use on the ADVIA CENTAUR immunoassay system (Siemens Healthcare, AG), and/or the ADVIA CENTAUR immunoassay system may be automated to perform the actions described above.

In some embodiments, there is a direct relationship 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 a 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 immunoassay may use Zika virus NS1 recombinant antigen, e.g., 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 to human IgM. In some embodiments, it may be biotin-conjugated. In some embodiments, it may be produced by labeling an anti-human IgM monoclonal antibody with NHS-LC-biotin.

SERA-MAG magnetic streptavidin microparticles (MG-SA): SERA-MAG magnetic streptavidin microparticles (MG-SA) microparticles (GE HealthCare Bio-Sciences Corp.) can be used as a solid support to prepare a wet cake comprising a solid support coated with anti-human IgM antibody.

Goat antiserum specific for human IgG, Fc: for example, goat antiserum specific for human IgG, Fc from Nittobo or Meridian Life Sciences ("goat anti-human IgG, Fc") can be used in the zika Ab assay.

In some embodiments, the immunoassays disclosed herein include the following example reagent formulations.

TABLE 1 example Zika Ab solid support buffer formulations

TABLE 2 example Zika Ab auxiliary well reagent formulations

TABLE 3 example Zika Ab visualization buffer formulation

TABLE 4 example Zika M solid support buffer formulation

TABLE 5 example Zika M auxiliary well reagent formulation

TABLE 6 example Zika M visualization buffer formulation

TABLE 7 example Zika M/Zika Ab Wet cake formulation

Suitable biological samples for detecting anti-Zika virus antibodies include any biological sample from a subject that contains or is suspected of containing anti-Zika virus antibodies, including, but not limited to, serum or plasma.

The immunoassays disclosed herein employ zika virus antigens. In some embodiments, the zika virus antigen is a zika virus NS1 antigen. In some embodiments, the zika virus antigen is a recombinant zika virus NS1 antigen. The Zika virus antigen may be expressed in a heterologous cell, such as an insect cell. In some embodiments, the zika antigen further comprises an epitope tag. The epitope tag may be at the N-terminus or C-terminus of the Zika virus antigen. The epitope tag may be any suitable tag known to those skilled in the art, including but not limited to a 6-histidine tag, a hemagglutinin tag, 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 kit 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 kit may 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, anti-human IgM antibody and labeled zika virus antigen. The kit may further comprise instructions for performing the first and second immunoassays. The instructions can direct a user or system to perform a first immunoassay to determine the presence or absence of anti-zika virus antibodies in a biological sample from a human subject. The instructions may further instruct the user or system to perform the second immunoassay only if the biological sample is determined to be positive for anti-zika virus antibodies in the first immunoassay.

Suitable solid supports and labels for use in any of the kits disclosed herein include those disclosed for the above methods.

Examples

The following examples are provided to further describe some embodiments disclosed herein. The examples are intended to illustrate, but not to limit, the disclosed embodiments.

Development of highly specific and effective immunoassay for anti-Zika IgM antibody

A "zika Ab" assay with anti-human IgG Fc antibodies present in the reaction buffer was prepared, along with another "zika M" assay without anti-human IgG Fc antibodies. Using both the zika Ab and the zika M assays, a zika test algorithm was developed (fig. 1). According to the zika test algorithm, each biological sample is first tested by the zika Ab assay (including anti-human IgG Fc antibodies), and then only reactive samples in which anti-zika IgM or anti-zika IgG or anti-zika IgM and anti-zika IgG antibodies are detected are tested with the zika M assay.

Because the zika Ab assay provides high specificity in non-circulating populations and can detect both zika-specific IgG and IgM antibodies, the assay identifies biological samples (i.e., individuals) that contain zika-specific antibodies. These samples were then tested with a zika M assay without anti-human IgG Fc antibody (which only detects zika-specific IgM antibodies), identifying those samples (i.e., individuals) that had anti-zika IgM antibodies. These individuals were presumed to have recently been infected with Zika virus.

Because the zika test algorithm only needs to test through the zika Ab assay once, and most test populations are expected to be negative for zika infection, most samples will only be tested with the zika Ab assay. Thus, the Zika test algorithm provides a simple solution to achieve high specificity and sensitivity in determining recent Zika infections in individuals.

Material

Finally, reagent preparations for the Zika Ab immunoassay (Table 8) and the Zika M assay (Table 9) were determined.

TABLE 8 Zika Ab assay formulation with goat anti-human IgG Fc

TABLE 9 Zika M assay formulation without goat anti-human IgG Fc

Results

Omission of goat anti-human IgG Fc from the solid support buffer solves the IgG detection problem, which has been confirmed by IgM blocking experiments (table 10) and dilution studies of the zika IgG humanized monoclonal antibody (table 11).

IgM blocking study

Blocking experiments (Table 10) were performed by pre-treating wet cake (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 several Zika positive blood collection groups were tested with two formulations, one with goat anti-human IgG Fc (see Table 8) and the other without goat anti-human IgG Fc (see Table 9), where the wet cake had been incubated with or without IgM blocking agent. Reactivity was analyzed by measuring Relative Light Units (RLU) and percent inhibition was calculated in the following manner:

。

incubation with a preparation with goat anti-human IgG Fc with non-specific human IgM resulted in an average inhibition of only 39%. Incubation with non-specific human IgM using a preparation without goat anti-human IgG Fc resulted in 103% inhibition. This indicates that reactivity in the no goat anti-human IgG Fc preparation (table 10) can be completely eliminated by blocking with human IgM in samples suspected of having zika IgG. Thus, the assay formulation without goat anti-human IgG Fc did not bind human anti-zika IgG.

Table 10 IgM blocking: the preparation with goat anti-human IgG Fc was compared with the preparation without goat anti-human IgG Fc

Dilution study of humanized monoclonal antibody to Zika IgG

The cross-reactivity of the 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 monoclonal antibody IgG specific to zika NS 1. Monoclonal antibody (ZKA35) was obtained from HUMABS BioMed. Serial dilutions of ZKA35 (0.001 ug/ml to 1.0 ug/ml) were tested with the zika Ab assay and the zika M assay as well as the prototype zika total assay (which was designed to detect both anti-zika virus IgM and IgG antibodies).

The reactivity measured for each ZKA35 IgG dilution with each zika virus was calculated as signal (RLU)/cutoff (S/Co). Based on the reactivity of samples from the normal population and zika PCR positive individuals, cutoff values of 42500, 70000, and 35000 were used for the zika M assay, zika Ab assay, and zika total assay, respectively. S/Co ≧ 1.0 is considered reactive with ZKA35 IgG.

Total Zika assay to detect both Zika IgG and IgM antibodies was reacted with dilutions containing 0.063 to 1.0 μ g/mL ZKA35 IgG (Table 11). The Zika Ab assay reacted with dilutions containing 0.125 to 1.0 μ g/mL ZKA35 IgG and showed approximately 10-fold lower reactivity than the Zika total assay at the highest ZKA35 IgG concentration. For the zika M assay, none of the ZKA35 IgG dilutions tested reacted (table 11). These results indicate that although some zika IgG cross-reactivity was seen with the zika Ab assay, the zika M assay did not cross-react with zika-specific IgG at concentrations up to 1 μ g/mL.

TABLE 11 results from a serial dilution study of Zika IgG (ZKA35)

Standardization

The cutoff indices for the Zika Ab and Zika M assays were set based on testing putative Zika virus negative normal donor samples (including pregnant women), Zika virus PCR positive serial extracts, and dengue/West Nile virus positive cross-reactive samples (SeraCare panel) from continental USA. Initial values of the Zika Ab and Zika M standards were determined as follows: the above mentioned populations were tested on small lots of standards based on cut-off values during early development. A second, larger batch of standards is then established and values are assigned from the previous small batch. These larger batches (zika Ab standard batch #16KL240 and zika M standard batch #17CL059) served as anchor standards for standardization. Gold standards can be traced back to the anchoring standard. Both the zika Ab and the zika M were assigned master curves, and the calibration values were traceable to internal gold standards. Zika Ab and zika M gold standards were used to evaluate and validate reagent performance and in-process tests as well as value assignment for new batches of standards, calibrators, controls, and medical decision pools.

Zika Ab assay used 6-level standards formulated with defibrinated and dialyzed human plasma. The Zika M assay also used 6-level standards formulated with defibrinated and dialyzed human plasma. The zika Ab and zika M standards were constructed by spiking the zika IgM antibody positive pools into the zika Ab and zika M negative plasma pools, respectively. Zika Ab and Zika M standards levels S02-S06 were spiked with Zika M positive pools. The lowest Zika Ab and Zika M standard level S01 was the non-spiked Zika Ab and Zika M negative human plasma pool.

Zika Ab and Zika M assay used 2-level calibrators formulated with defibrinated and dialyzed human plasma. High calibrators (above cut-off) of zika Ab and zika M were prepared by spiking zika IgM antibody positive pools into zika Ab and zika M negative plasma pools, respectively. The zika Ab and zika M low calibrators (below the cut-off value) were non-incorporated zika Ab and zika M negative human plasma pools.

Zika Ab and Zika M assays of the Zika test A2-point calibration curve fit based on a 4 PL-weighted curve fitting algorithm was used.

Both Zika Ab and Zika M assays used a 2-level control formulated with defibrinated, dialyzed human plasma. Zika Ab and Zika M positive controls were prepared by spiking Zika IgM antibody positive pools into Zika Ab and Zika gM negative plasma pools, respectively. Zika Ab and Zika IgM negative controls were both non-incorporated Zika Ab and Zika M negative human plasma pools.

Zika Ab and zika M assays each had one level of Medical Decision Pool (MDP) formulated with defibrinated and dialyzed human plasma. Zika Ab and zika M medical decision pools were prepared by spiking zika M antibody positive pools into zika Ab and zika M negative plasma pools, respectively.

It will be understood by those skilled in the art that many changes and modifications may be made to the preferred and exemplary embodiments of the present invention, and that such changes and modifications may 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 this present invention.