System and methods for detection of Bacillus anthracis related analytes in biological fluids

• US 7,262,019 B2
• Filed: 05/24/2004
• Issued: 08/28/2007
• Est. Priority Date: 05/03/2001
• Status: Expired due to Fees

• Current Assignee: Immunetics, Inc (Oxford Immunotec Ltd.)
• Original Assignee: Immunetics, Inc (Oxford Immunotec Ltd.)
• Inventors: Kovalenko, Victor
• Primary Examiner(s): McKelvey; Terry
• Assistant Examiner(s): Davis; Deborah A.
• Application Number:
  US10/852,644
• Publication Number:
  US 20070178542A1
• Time in Patent Office: 1,191 Days
• Field of Search: 436/518, 436/538, 436/543, 436/8, 436/808, 435/7.1, 435/7.9, 435/7.92, 435/32, 424/184.1
• US Class Current: 435/7.5
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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. In preferred embodiments, the invention can be practiced to detect presence in biological fluid samples of antibodies to Bacillus anthracis related antigens.

Claims

1. A method for detecting an antibody that binds to a Bacillus anthracis antigen in a sample, comprising:

providing a first component comprising biotin linked through a polyethylene glycol, to albumin;

providing a second component comprising a biotin-binding moiety conjugated to said antigen;

providing a third component comprising said antigen conjugated to a label;

contacting the first component with a solid phase so as to immobilize the first component;

contacting a sample suspected of comprising said 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 of each of the second and third components; and

, uncomplexed third component;

contacting the mixture with the immobilized first component so as to immobilize the triple immune complex but not uncomplexed third component, wherein triple immune complex is immobilized by the binding between the biotin of the first component to the biotin-binding moiety of the second component;

separating the mixture comprising uncomplexed third component 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 antibody in the sample.

2. 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 had been exposed to an immunogenic amount of Bacillus anthracis.

3. 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.

4. The method of claim 3, wherein the microbial infection is Anthrax.

5. The method of claim 1, wherein the antigen is selected from the group consisting of natural peptides, synthetic peptides, natural polypeptides, and synthetic polypeptides.

6. The method of claim 1, wherein the antigen is Protective Antigen protein of B. anthracis.

7. The method of claim 6, wherein the antigen is recombinant Protective Antigen protein of B. anthracis.

8. The method of claim 1, wherein said antigen is a recombinant fusion protein comprising at least one epitope of a Bacillus anthracis antigen.

9. The method of claim 8, wherein the Bacillus anthracis antigen is Protective Antigen.

10. The method of claim 1, wherein the sample is selected from serum, saliva, intranasal swab, throat swab, and bronchoalveolar lavage fluid.

11. The method of claim 1, wherein the biotin-binding moiety is avidin or streptavidin.

12. 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.

13. The method of claim 12, wherein the label is horse radish peroxidase.

14. The method of claim 1, wherein the third component comprises said antigen conjugated directly to a label.

15. The method of claim 1, wherein the third component comprises said antigen conjugated indirectly to a label through a natural polymer or a chemical spacer.

16. The method of claim 1, wherein the solid phase comprises a plastic surface, a porous membrane or a non-porous membrane.

17. The method of claim 16, wherein the plastic surface is polystyrene.

18. The method of claim 1, wherein the second component and the third component are contacted concurrently with the sample.

19. The method of claim 1, wherein the second component and the third component are contacted concurrently with the sample before contacting the sample with the first component.

20. The method of claim 1, wherein the second component and the third component are contacted concurrently with the sample and the solid phase having the first component immobilized thereon.

21. The method of claim 1, further comprising varying the amount of antigen conjugated to the second or the third component to determine a relative affinity of the antibody suspected of being present in the sample.

27. The kit of claim 22, further comprising instructions for practicing the method of claim 1.

22. A kit comprising a first component comprising biotin linked through a polyethylene glycol spacer, to albumin, a second component comprising a biotin-binding moiety conjugated to a Bacillus anthracis antigen and a third component comprising said antigen conjugated to a label.

23. The kit of claim 22, wherein the second and third components are supplied as dry or liquid samples, wherein the dry or liquid samples may be separate or mixtures of the second and third components.

24. The kit of claim 22, wherein the second component is avidin or streptavidin conjugated to said antigen.

25. The kit of claim 22, wherein said antigen is Protective Antigen protein of B. anthracis.

26. The kit of claim 25, wherein said antigen is recombinant Protective Antigen protein derived from B. anthracis.