Systems and methods for detection of analytes in biological fluids
View Patent ↗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.
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.
2. The method of claim 1 , wherein the biotin-binding moiety is avidin or streptavidin.
3. The method of claim 1 , wherein the label is selected from the group consisting of: an enzyme, a fluorescent probe, a chemiluminescent probe, a metal, a non-metal colloidal particle, a polymeric dye particle and a pigment molecule.
4. The method of claim 3 , wherein the label is horse radish peroxidase.
5. The method of claim 1 , wherein the label is colloidal gold.
6. The method of claim 1 , wherein the solid phase comprises a plastic surface, a porous membrane or a non-porous membrane.
7. The method of claim 6 , wherein the plastic surface is polystyrene.
8. The method of claim 1 , wherein the third component comprises an antigen or hapten conjugated directly to a label.
9. The method of claim 1 , wherein the third component comprises an antigen or hapten conjugated indirectly to a label through a natural polymer or a chemical spacer.
10. The method of claim 1 , wherein the second and third component conjugates each comprise two or more different antigens or haptens.
11. The method of claim 1 , wherein the second and third components each comprise two or more different antigens or haptens chemically linked into one type of conjugate containing ligand-binding moiety or detector label.
12. The method of claim 1 , wherein the quantity of antigen or hapten in the third component is equal or greater than the quantity of antigen or hapten in the second component.
13. The method of claim 12 , wherein the ratio of antigen or hapten in the third component to antigen or hapten in the second component is greater than about 2:1.
14. The method of claim 12 , wherein the ratio of antigen or hapten in the third component to antigen or hapten in the second component is greater than about 5:1.
15. The method of claim 1 , wherein the quantity of antigen or hapten in the third component is less than the quantity of antigen or hapten in the second component.
16. The method of claim 1 , wherein the second component and the third component are contacted concurrently with the sample.
17. The method of claim 1 , wherein the antigen or hapten moiety of the second component, the third component or both are mixtures comprising two or more different antigens or haptens to which antibodies present in the sample will bind.
18. The method of claim 1 , further comprising varying the amount of antigens or haptens conjugated to the second or the third component to determine a relative affinity of the antibody suspected of being present in the sample.
19. The method of claim 1 , wherein the antibody, if present in the sample, indicates that a human or animal from whom the sample was obtained has a microbial infection.
20. The method of claim 19 , wherein the microbial infection is Lyme disease.
21. The method of claim 1 , wherein the antigen is C6 peptide derived from VLSe protein of B. burgdorferi.
22. The method of claim 1 , wherein the antigen is C10 peptide derived from OspC protein of B. burgdorferi.
23. The method of claim 1 , wherein the antigens are selected from the group consisting of natural peptides, synthetic peptides, recombinant peptides, natural polypeptides, synthetic polypeptides, and recombinant polypeptides.
24. 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;
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.
25. The method of claim 24 , wherein the biotin-binding moiety is avidin or streptavidin.
26. The method of claim 24 , wherein the label is selected from the group consisting of: an enzyme, a fluorescent probe, a chemiluminescent probe, a metal, a non-metal colloidal particle, a polymeric dye particle and a pigment molecule.
27. The method of claim 26 , wherein the label is horse radish peroxidase.
28. The method of claim 24 , wherein the label is colloidal gold.
29. The method of claim 24 , wherein the solid phase comprises a plastic surface, a porous membrane or a non-porous membrane.
30. The method of claim 29 , wherein the plastic surface is polystyrene.
31. The method of claim 24 , wherein the third component comprises an antigen or hapten conjugated directly to a label.
32. The method of claim 24 , wherein the third component comprises an antigen or hapten conjugated indirectly to a label through a natural polymer or a chemical spacer.
33. The method of claim 24 , wherein the second and third component conjugates each comprise two or more different antigens or haptens.
34. The method of claim 24 , wherein the second and third components each comprise two or more different antigens or haptens chemically linked into one type of conjugate containing ligand-binding moiety or detector label.
35. The method of claim 24 , wherein the quantity of antigen or hapten in the third component is equal or greater than the quantity of antigen or hapten in the second component.
36. The method of claim 35 , wherein the ratio of antigen or hapten in the third component to antigen or hapten in the second component is greater than about 2:1.
37. The method of claim 35 , wherein the ratio of antigen or hapten in the third component to antigen or hapten in the second component is greater than about 5:1.
38. The method of claim 24 , wherein the quantity of antigen or hapten in the third component is less than the quantity of antigen or hapten in the second component.
39. The method of claim 24 , wherein the antigen or hapten moiety of the second component, the third component or both are mixtures comprising two or more different antigens or haptens to which antibodies present in the sample will bind.
40. The method of claim 24 , further comprising varying the amount of antigens or haptens conjugated to the second or the third component to determine a relative affinity of the antibody suspected of being present in the sample.
41. The method of claim 24 , wherein the antibody, if present in the sample, indicates that a human or animal from whom the sample was obtained has a microbial infection.
42. The method of claim 41 , wherein the microbial infection is Lyme disease.
43. The method of claim 24 , wherein the antigen is C6 peptide derived from VLSe protein of B. burgdorferi.
44. The method of claim 24 , wherein the antigen is C10 peptide derived from OspC protein of B. burgdorferi.
45. The method of claim 24 , wherein the antigens are selected from the group consisting of natural peptides, synthetic peptides, recombinant peptides, natural polypeptides, synthetic polypeptides, and recombinant polypeptides.