Systems and methods for detection of analytes in biological fluids
First Claim
Patent Images
1. A method for detecting an antimicrobial antibody in a sample, comprising:
- providing a first component comprising biotin linked, through polyethylene glycol, to albumin;
providing a second component comprising a biotin-binding moiety conjugated to a microbial antigen or hapten that binds the antibody to be detected in the sample;
providing a third component comprising the microbial antigen or hapten conjugated to a label;
contacting the sample suspected of comprising the antibody with the second and third components such that a first mixture is formed comprising a triple immune complex in which the antibody, if present in the sample, binds to the antigen or hapten moiety of each of the second and third components;
contacting the first mixture with the first component, whereby the biotin of the first component binds to the biotin-binding moiety of the second component, such that a second mixture is formed comprising a triple immune complex bound to the first component;
contacting the second mixture with a solid phase so as to immobilize the triple immune complex;
separating the mixture comprising uncomplexed components from the solid phase; and
detecting whether the label is associated with the solid phase, wherein the detection of the label indicates the presence of the triple immune complex in the mixture and therefore of the antimicrobial antibody in the sample.
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Abstract
The invention provides a heterogeneous immunoassay for detection of antibodies and antigens based on specific antigen-antibody immune complex formation with multiple antigen-bearing conjugate components. The invention further provides means for optimizing the assay format for the detection of both low and high-affinity antibodies, and provides means for quantitative detection of both antibody and the corresponding antigen present in a sample.
33 Citations
45 Claims
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1. A method for detecting an antimicrobial antibody in a sample, comprising:
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providing a first component comprising biotin linked, through polyethylene glycol, to albumin; providing a second component comprising a biotin-binding moiety conjugated to a microbial antigen or hapten that binds the antibody to be detected in the sample; providing a third component comprising the microbial antigen or hapten conjugated to a label; contacting the sample suspected of comprising the antibody with the second and third components such that a first mixture is formed comprising a triple immune complex in which the antibody, if present in the sample, binds to the antigen or hapten moiety of each of the second and third components; contacting the first mixture with the first component, whereby the biotin of the first component binds to the biotin-binding moiety of the second component, such that a second mixture is formed comprising a triple immune complex bound to the first component; contacting the second mixture with a solid phase so as to immobilize the triple immune complex; separating the mixture comprising uncomplexed components from the solid phase; and detecting whether the label is associated with the solid phase, wherein the detection of the label indicates the presence of the triple immune complex in the mixture and therefore of the antimicrobial antibody in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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24. A method for detecting an antimicrobial antibody in a sample, comprising:
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providing a first component comprising biotin linked, through polyethylene glycol, to albumin; providing a second component comprising a biotin-binding moiety conjugated to a microbial antigen or hapten that binds the antibody; providing a third component comprising the microbial antigen or hapten conjugated to a label; contacting the first component with a solid phase so as to immobilize the first component; contacting the sample suspected of comprising the antibody with the second and third components such that a mixture is formed comprising a triple immune complex in which the antibody, if present in the sample, binds to the antigen or hapten moiety of each of the second and third components; contacting the mixture with the immobilized first component so as to immobilize the triple immune complex wherein the triple immune complex is immobilized by binding between the biotin of the first component to the biotin-binding moiety of the second component; separating the solid phase from the uncomplexed components; and detecting whether the label is associated with the solid phase, wherein the detection of the label indicates the presence of the triple immune complex in the mixture and therefore of the antimicrobial antibody in the sample. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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Specification