Detection of multiple antigens or antibodies

• US 5,763,158 A
• Filed: 02/06/1997
• Issued: 06/09/1998
• Est. Priority Date: 02/06/1997
• Status: Expired due to Fees

• Current Assignee: United States Secretary of the Army
• Original Assignee: The United States Of America As Represented By The Secretary Of The Army
• Inventors: Bohannon, Robert C.
• Primary Examiner(s): LEARY, LOUISE N
• Application Number:
  US08/796,772
• Time in Patent Office: 488 Days
• Field of Search: 435/4, 435/7.1, 435/12, 435/21, 435/28, 435/14, 435/29, 435/34, 435/235.1, 435/968, 422/50, 422/52, 422/55, 422/68.1
• US Class Current: 435/4
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Abstract

A method for simultaneously testing a sample for the presence of multiple rget antigens or antibodies in the sample, which comprises presenting the sample to a plurality of different binding sites, wherein at least two of the sites are binding sites for different known target antigens or antibodies, each known binding site is composed of at least one molecule of a ligand-enzyme complex attached to a support, and the ligand-enzyme complex is a ligand attached to an enzyme in proximity to the enzyme'"'"'s active site such that the enzymatic activity of the ligand-enzyme complex is changed when the target antibody or antigen is present in the sample. The ligand-enzyme complex embraces nonenzymatic reporter molecules such as electrochemiluminescent compounds. The method also includes assaying each binding site for a change in enzymatic or other reporter activity compared to a control value. A device for performing simultaneous detection of multiple target antigens or antibodies is also disclosed.

Claims

1. A method for simultaneously testing a sample for the presence of multiple target antigens or antibodies in the sample, comprising:

(a) presenting the sample to a plurality of different binding sites arranged in a known order with respect to each other, wherein at least two of the sites are binding sites for different known target antigens or antibodies, each known binding site comprising at least one molecule of a ligand-enzyme complex attached to a support, the ligand-enzyme complex comprising a ligand attached to an enzyme in proximity to the enzyme'"'"'s active site such that the enzymatic activity of the ligand-enzyme complex is changed when the target antigen or antibody is present in the sample; and

(b) assaying each binding site for a change in enzymatic activity compared to a control value.

2. The method of claim 1, further comprising, before the presenting step, generating the control value, comprising the steps of:

providing the binding sites with a first aliquot of a substrate, said substrate producing a detectable signal upon reaction with the enzyme, and

measuring the signal produced by each binding site to provide the control value.

3. The method of claim 2, further comprising

washing the binding sites to remove the substrate between the generating and presenting steps;

and

providing the binding sites with a second aliquot of the substrate before the assaying step.

4. The method of claim 1, wherein at least one of the known binding sites further comprises a mAb bound to the ligand and at least partially blocking the active site of the enzyme, wherein said mAb has a greater binding affinity for the target antigen or antibody than for the ligand.

5. The method of claim 4, wherein at least one of the binding sites is a negative control site and the assaying step comprises:

providing the binding sites with a substrate for the enzyme, said substrate producing a detectable signal upon reaction with the enzyme;

generating the control value by measuring the signal produced by the negative control site; and

measuring the signal produced by the known binding sites.

6. The method of claim 4, wherein the enzyme is a molecule labeled with a reporter compound and the mAb is a molecule labeled with a quencher compound.

7. The method of claim 6, wherein the reporter compound is ruthenium (II) tris(bipyridyl) chelate and the quencher compound adsorbs light at 620 nm.

8. The method of claim 1, wherein at least one of the known binding sites comprises a plurality of molecules of the ligand-enzyme complex.

9. The method of claim 1, wherein the enzyme is selected from the group consisting of alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta-glucuronidase, luciferase, and urease.

10. The method of claim 9, wherein the enzyme is alkaline phosphatase.

11. The method of claim 4, wherein the ligand comprises a peptide derived from an antigenic site of the target antigen or antibody.

12. The method of claim 4, wherein the ligand comprises a nucleic acid or oligonucleotide.

13. The method of claim 11, wherein the known binding sites are specific for target molecules of biologic origin.

14. The method of claim 13, wherein the target molecules are selected from the group consisting of ebola virus, hanta virus, and lassa virus.

15. A device for simultaneously testing a sample for the presence of multiple target antigens or antibodies in the sample, comprising a plurality of different binding sites arranged in a known order with respect to each other, wherein at least two of the sites are binding sites for different known target antigens or antibodies, each known binding site comprising at least one molecule of a ligand-enzyme complex attached to a support, the ligand-enzyme complex comprising a ligand attached to an enzyme in proximity to the enzyme'"'"'s active site such that the enzymatic activity of the ligand-enzyme complex is changed when the target antigen or antibody is present in the sample.

16. The device of claim 15, wherein the ligand comprises a binding partner of the target antigen or antibody.

17. The device of claim 15, wherein the ligand comprises a nucleic acid or oligonucleotide.

18. The device of claim 15, wherein at least one of said known binding sites further comprises a mAb bound to the ligand and at least partially blocking the active site of the enzyme, wherein said mAb has a greater binding affinity for the target antigen or antibody than for the ligand.

19. The device of claim 15, wherein at least one of said known binding sites comprises a plurality of molecules of the ligand-enzyme complex.

20. The device of claim 15, further comprising a positive control binding site and a negative control binding site.

21. The device of claim 20, wherein the support is selected from the group consisting of glass beads, plastic beads, magnetic particles, nitrocellulose particles and latex particles, and the binding sites are arranged in a known order in a tube.

22. The device of claim 21, wherein the tube is provided with a plurality of luminometers, said luminometers being disposed in the vicinity of the binding sites and operably linked to a microprocessor having means for analyzing information received from the binding sites and means for reporting detection of target antigens or antibodies in the sample.

23. The device of claim 15, wherein the enzyme is selected from the group consisting of alkaline phosphatase, horseradish peroxidase, beta-galactosidase, beta-glucuronidase, luciferase, and urease.

24. The device of claim 23, wherein the enzyme is alkaline phosphatase.

25. The device of claim 18, wherein the enzyme is a molecule labeled with a reporter compound and the mAb is a molecule labeled with a quencher compound.

26. The device of claim 25, wherein the reporter compound is ruthenium (II) tris(bipyridyl) chelate and the quencher compound adsorbs light at 620 nm.

27. The device of claim 18, wherein the ligand comprises a peptide derived from an antigenic site of the target antigen or antibody.

28. The device of claim 27, wherein the known binding sites are specific for target molecules of biologic origen.

29. The device of claim 28, wherein the target molecules are selected from the group consisting of ebola virus, hanta virus, and lassa virus.

30. The device of claim 15, wherein the support is selected from a membrane and a plastic strip.

31. The device of claim 30, wherein the support is provided with a substrate for the enzyme and means for retaining the enzyme until the device is exposed to the sample.