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US 11,959,917 B2Granted
Methods and reagents for Zika virus immunoassays
AssigneeSiemens Healthcare Diagnostics Inc.
InventorsSai Patibandla, Krishna Singh
Life of this patent
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Abstract
Disclosed herein are immunoassay methods and reagents for detecting anti-Zika IgM antibody in a biological sample from a subject and/or diagnosing Zika virus infection in a subject. Also disclosed are algorithms for implementing the disclosed methods. The disclosed immunoassay methods, reagents, and algorithms enable efficient and reliable qualitative detection of anti-Zika virus antibodies and rapid determination of presumptive positive results for Zika virus infection in human subjects.
Claims33
A method of detecting an anti-Zika virus IgM antibody in a biological sample from a human subject, the method comprising a first immunoassay and a second immunoassay, wherein the first immunoassay comprises:
- a) incubating a first biological sample from the human subject with a labeled Zika virus antigen and
- a buffer comprising an anti-human IgG Fc antibody and a solid support comprising an anti-human IgM antibody,
- 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 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 presence of 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 wherein the second immunoassay comprises incubating a second biological sample from the human subject with a labeled Zika virus antigen and a solid support comprising an anti-human IgM antibody, wherein anti-human IgG Fc antibody is absent from the second immunoassay, and
- wherein, in the presence of an anti-Zika virus IgM antibody in the second 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 presence or absence of complex II, the presence of which indicates the presence of the anti-Zika virus IgM antibody in the second biological sample.
6 more elements
A method of detecting antibodies to Zika virus in a human subject, the method comprising performing a first immunoassay comprising:
- a) incubating a first biological sample from the subject with:
- a buffer comprising an anti-human IgG Fc antibody and a solid support comprising an anti-human IgM antibody,
- 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 presence of complex I, and
- performing a second immunoassay comprising:
- c) incubating a second biological sample from the subject with:
- a solid support comprising an anti-human IgM antibody, and
- a labeled Zika virus antigen,
- wherein anti-human IgG Fc antibody is absent from the second immunoassay,
- wherein, in the presence of an anti-Zika virus IgM antibody in the second 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 presence or absence of complex II, and
- di) if the complex II is not detected, determining that the human subject is negative for anti-Zika virus IgM antibodies, or
- dii) 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.
12 more elements
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 comprising an anti-human IgM antibody in a buffer comprising comprising tricine, sodium chloride, polyoxyethylene (20) sorbitan monolaurate, disodium ethylenediaminetetraacetic acid (EDTA), preservative, sulfhydryl-modified bovine serum albumin, and an anti-human IgG Fc antibody, and
- a labeled Zika virus antigen;
- and
- wherein the reagents for the second immunoassay comprise:
- a solid support comprising an anti-human IgM antibody, in a buffer comprising tricine, sodium chloride, polyoxyethylene (20) sorbitan monolaurate, disodium EDTA, preservative, sulfhydryl-modified bovine serum albumin, but without anti-human IgG Fc antibody, and
- a labeled Zika virus antigen.
4 more elements
Dependencies are inferred from the claim text.