Immunometric assays using monoclonal antibodies

• US 4,486,530 A
• Filed: 06/24/1981
• Issued: 12/04/1984
• Est. Priority Date: 08/04/1980
• Status: Expired due to Term

• Current Assignee: Hybritech, Inc. (Danaher Corporation)
• Original Assignee: Hybritech, Inc. (Danaher Corporation)
• Inventors: David, Gary S., Greene, Howard E.
• Primary Examiner(s): Marantz, Sidney
• Application Number:
  US06/323,498
• Time in Patent Office: 1,259 Days
• Field of Search: 23/230 B, 424/12, 435/7, 436/519, 436/523, 436/527, 436/529, 436/534, 436/537, 436/548, 436/800
• US Class Current: 435/7.91
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/576 for hepatitis
• CPC Class Codes: G01N 33/577 involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens; G01N33/53 - G01N33/576 take precedence
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

"Two-site" or "sandwich" immunometric assay techniques for determination of the presence and/or concentration of antigenic substances in fluids using monoclonal antibodies are described and compared to conventional assays using polyclonal antibodies. Also described are inhibition assays using complexes of antigens with a monoclonal antibody.

Claims

1. In an immunometric assay process to determine the presence or concentration of an antigenic substance in a fluid sample comprising forming a ternary complex of the antigenic substance, a first antibody and a second antibody bound to the antigen at a difference site than the first antibody by contacting the sample with said first and second antibodies, the improvement comprising employing a measured amount of a monoclonal antibody having an affinity for the antigenic substance of at least about 10⁸ liters/mole for each of said first and second antibodies.

2. A process according to claim 1 wherein a fluorescent chromophore is bonded to said first antibody and a chromophore capable of absorbing light at the wavelength emitted by the fluorescent chromophore is bonded to said second antibody, the monoclonal antibodies being capable of binding to the antigenic substance at sites which permit the light emitted by the fluorescing chromophore to be absorbed by the quenching chromophore.

3. A process according to claim 2 wherein the sample is contacted with a solution containing the first and second antibodies to form the ternary complex and the intensity of fluorescence determined and compared to the fluorescence of a standard sample free of said antigen or containing said antigen in a known amount.

4. A process according to claim 2 wherein the first antibody is bound to a solid carrier that is insoluble in the fluid sample and said second antibody is soluble in the fluid sample.

5. A process according to claim 4 wherein the fluid sample is simultaneously contacted with said first and second antibodies to form an insoluble ternary complex and the intensity of fluorescence of the ternary complex determined and compared to the fluorescence of a standard sample free of said antigen or containing said antigen in a known amount.

6. A process according to claim 2 wherein the second antibody is bound to a solid carrier that is insoluble in the fluid sample and said first antibody is soluble in the fluid sample.

7. A process according to claim 6 wherein the fluid sample is simultaneously contacted with said first and second antibodies to form an insoluble complex and the intensity of fluorescence of the fluid determined and compared to the fluorescence of a standard sample free of said antigen or containing said antigen in a known amount.

8. A process according to claims 4 or 6 wherein the sample is first contacted with the first antibody to form an antibody:

antigen binary complex and then contacted with the second antibody to form the ternary complex and the intensity of the fluorescence of the complex or the fluid determined and compared to a standard sample free of said antigen or containing the antigen in a known amount.

9. A process according to claims 2, 3, 4, 5, 6, or 7 wherein the fluorescent chromophore is fluorescein and the chromophore capable of absorbing emitted light is rhodamine.

10. A process according to claim 8 wherein the fluorescent chromophore is fluorescein and the chromophore capable ot absorbing emitted light is rhodamine.

11. A process according to claim 1 wherein said first antibody is bound to particles insoluble in the fluid sample and said second antibody is bound to particles insoluble in the fluid sample and wherein the sample is contacted with a suspension of the particles for a time sufficient to cause formation of the ternary complex whereby agglomeration of the particles bound to said first and second antibodies occurs.

12. A process according to claim 11 wherein the size of the particles is in the range of from about 0.2μ

to about 10μ

.

13. A process according to claim 12 wherein the particles are selected from the group consisting of particles of latex, silica, glass, cells, polyacrylamide, polymethyl methacrylate and agarose.

14. A process according to claim 13 wherein the particles are latex particles.

15. A process according to claims 11, 12, 13, or 14 wherein the particles binding the first antibody are of different color than the particles binding the second antibody.

16. A process according to claims 12, 13, or 14 wherein the size of the particles is in the range of from about 1.0 to 10μ

.

17. A process according to claim 15 wherein the size of the particles is in the range of from about 1.0 to 10μ

.

18. A process according to claims 11, 12, 13, or 14 wherein the turbidity of the sample after formation of the ternary complex is determined and related to the turbidity of a control sample known to be free of the antigen or to contain a known amount of the antigen.

19. A process according to claims 11, 12, 13, or 14 wherein a fluorescent chromophore is bound to said first antibody and a chromophore capable of absorbing light at the wavelength emitted by the fluorescent chromopore is bound to the second antibody and wherein, after said contacting, the intensity of fluorescence is determined and compared to the fluorescence of a standard sample free of said antigen or containing said antigen in a known amount.

20. A process according to claim 19 wherein the fluorescent chromophore is flurescein and the chromophore capable of absorbing emitted light is rhodamine.

21. A process according to claim 1 wherein an enzyme is bound to the first antibody and a substance is bound to the second antibody whereby the enzyme interacts with the substance to permit detection of the antibody:

antigen;

antibody complex.

22. A process according to claim 21 wherein the substance bound to the second antibody is an enzyme.

23. A process according to claim 22 whereby the first antibody bound enzyme catalyzes a reaction which produces a product required by a reaction catalyzed by the second antibody bound enzyme.

24. A process according to claim 23 whereby the product of the reaction catalyzed by the first bound enzyme is consumed in the reaction catalyzed by the second bound enzyme.

25. A process according to claim 23 whereby the product of the reaction catalyzed by the first bound enzyme allosterically interacts with the second bound enzyme.

26. A process according to claim 25 whereby the product of the reaction catalyzed by the first bound enzyme allosterically activates the second bound enzyme.

27. A process according to claim 25 whereby the product of the reaction catalyzed by the first bound enzyme allosterically inhibits the second bound enzyme.

28. A process according to claims 23, 24, 25, 26 or 27 wherein the reaction catalyzed by the second antibody bound enzyme produces a detectable product.

29. A process according to claim 28 wherein the formation of the detectable product is detected by colorimetry, fluorimetry, luminescence or spectrophotometry.

30. A process according to claims 23, 24, 25, 26 or 27 wherein the reaction catalyzed by the second antibody bound enzyme consumes a detectable substance.

31. A process according to claim 30 wherein the consumption of the detectable substance is detected by colorimetry, fluorimetry, luminescence or spectrophotometry.

32. A process according to claim 21 wherein the substance on the second antibody undergoes a reaction catalyzed by the first antibody bound enzyme.

33. A process according to claim 32 wherein the reaction produces a substance detectable by colorimetry, fluorimetry, luminescence or spectrophotometry.