Method for detecting antibodies

• US 5,561,049 A
• Filed: 09/21/1994
• Issued: 10/01/1996
• Est. Priority Date: 09/21/1994
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Products GmbH (Siemens AG)
• Original Assignee: Behringwerke Aktiengesellschaft (Sanofi )
• Inventors: Vold, Barbara S., Ullman, Edwin F., Mehta, Harshvardhan B.
• Primary Examiner(s): SAUNDERS, DAVID A
• Application Number:
  US08/310,028
• Time in Patent Office: 741 Days
• Field of Search: 435/7.1, 435/7.4, 435/7.9, 435/7.91, 435/7.92, 435/7.93, 435/962, 435/971, 435/975, 436/506, 436/518, 436/537, 436/541
• US Class Current: 435/7.1
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 435/975 Kit

Abstract

This invention pertains to methods to detect antibodies in a sample. The methods use an amount of antigen that is up to 1000, preferably 10-100 times the minimum amount antigen that can be reliably detected and that is less than the maximum expected amount of antibodies in the sample. An antigen:antibody complex is formed and becomes bound to a binding agent that does not bind free antigen. The free antigen is then detected as a measure of antibodies present in the sample.

Claims

1. A method of determining the presence or amount of specific antibodies to an antigen, in a sample suspected of containing said antibodies, said method comprising the steps of:

(a) bringing together in an aqueous medium to form a mixture;

(i) said sample,

(ii) an antigen that binds said antibodies to form an antigen;

antibody complex, wherein the molar amount of said antigen added to said medium is Z, wherein Z is within the range of X to nX and Z is less than Y, where n is 5-1000, X is the minimum molar amount of said antigen that can be reliably detected when there are none of said antibodies present in a sample and Y is the maximum expected molar amount of said antibodies in said sample, and

(iii) a first binding agent that binds said complex and does not bind said antigen when said antigen is not part of said complex;

(b) adding to said mixture a second binding agent that selectively binds said antigen relative to binding said complex when said complex is bound to said first binding agent; and

(c) detecting said antigen bound to said second binding agent, the presence or amount thereof being related to the presence or amount of said antibodies in said sample.

2. The method of claim 1 wherein said first binding agent is selected from the group consisting of antibodies to immunoglobulins, complement factor C1q, rheumatoid factor, protein G and protein A.

3. The method of claim 2 wherein said first binding agent is bound to a suspendable solid phase or soluble polymer.

4. The method of claim 3 wherein said suspendable solid phase is a particle comprised of a material selected from the group consisting of polymers, ceramic and glass.

5. The method of claim 3 wherein said soluble polymer has a molecular weight of over 250,000.

6. The method of claim 1 wherein said detecting step involves the detection of enzyme activity, luminescence or light absorbance.

7. The method of claim 1 wherein said second binding agent is a receptor for said antigen and is bound to or capable of being bound to a support.

8. The method of claim 7 wherein said antigen is bound to a member of a signal producing system.

9. The method of claim 1 wherein said second binding agent is a first receptor that binds said antigen, which method further comprises contacting said mixture with a second receptor that binds said antigen, wherein at least one of said receptors is directly or indirectly bound to a label.

10. The method of claim 1 wherein said antigen is bound to a ligand and said second binding agent is a receptor for said ligand bound to a support.

11. The method of claim 10 which further comprises separating said support from said mixture and contacting said support with a receptor for said antigen.

12. The method of claim 1 wherein said antibodies are autoantibodies to glutamic acid decarboxylase or insulin.

13. A method of determining the presence or amount of antibodies against a certain antigen in a sample suspected of containing said antibodies, said method comprising the steps of:

(a) bringing together in an aqueous medium;

(i) said sample,

(ii) an antigen that binds said antibodies to form an antigen;

antibody complex, wherein the molar amount of said antigen added to said medium is Z, wherein Z is within the range of X to nX and Z is less than Y, where n is 5-1000, X is the minimum molar amount of said antigen that can be reliably detected when there are none of said antibodies present in a sample and Y is the maximum expected molar amount of said antibodies in said sample, and

(iii) a first binding agent that binds said complex and does not bind said antigen when said antigen is not part of said complex;

(b) contacting said medium with a second binding agent bound to a solid phase, wherein said second binding agent is a receptor that binds said antigen to form solid phase-bound antigen but does not bind said antigen;

antibody complex;

(c) detecting said antigen bound to said solid phase, the presence or amount thereof being related to the presence or amount of the antibodies in the sample.

14. The method of claim 13 wherein said first binding agent is selected from the group consisting of antibodies to immunoglobulins, complement factor C1q, rheumatoid factor, protein G and protein A.

15. The method of claim 14 wherein said first binding agent is bound to a suspendable solid phase or soluble polymer.

16. The method of claim 13 wherein said detection step comprises contacting said solid phase with one or more signal producing system members, and measuring the signal produced by said signal producing system members, the presence or amount thereof being related to the presence or amount of said antibodies in said sample.

17. The method of claim 16 wherein at least one of said signal producing system members is selected from the group consisting of fluorescers, enzymes, chemiluminescers, photosensitizers and suspendable particles.

18. A method of determining the presence or amount of antibodies in a sample suspected of containing said antibodies, said method comprising the steps of:

(a) bringing together in an aqueous medium;

(i) said sample,

(ii) an antigen that binds said antibodies to form an antigen;

antibody complex, wherein the molar amount of said antigen added to said medium is Z, wherein Z is within the range of X to nX and Z is less than Y, where n is 5-1000, X is the minimum molar amount of said antigen that can be reliably detected when there are none of said antibodies present in a sample and Y is the maximum expected molar amount of said antibodies in said sample, and

(iii) a first binding agent that binds said complex and does not bind said antigen when said antigen is not part of said complex;

(b) adding to said medium a second binding agent comprised of two receptors that bind said antigen, where at least one of said receptors is unable to bind effectively to said complex when said complex is bound to said first binding agent; and

(c) detecting the complex formed when said receptors bind said antigen, the presence or amount thereof being related to the presence or amount of said antibodies in said sample.

19. The method of claim 18 wherein said first binding agent is selected from the group consisting of antibodies to immunoglobulins, complement factor C1q, rheumatoid factor, protein G and protein A.

20. The method of claim 19 wherein said first binding agent is bound to a suspendable solid phase or soluble polymer.

21. The method of claim 18 wherein said detecting step involves the detection of luminescence or light absorbance.

22. The method of claim 21 wherein at least one of said receptors is bound to a detectable label selected from the group consisting of fluorescers, chemiluminescers and photosensitizers.

23. A method of determining the presence or amount of glutamic acid decarboxylase autoantibodies in a sample suspected of containing said autoantibodies, comprising the steps of:

(a) bringing together in an aqueous medium;

(i) said sample,

(ii) glutamic acid decarboxylase antigen that binds said autoantibodies to form an antigen;

autoantibody complex, wherein the molar amount of said antigen added to said medium is Z, wherein Z is within the range of X to nX and Z is less than Y, where n is 5-1000, X is the minimum molar amount of said antigen that can be reliably detected when there are none of said antibodies present in a sample and Y is the maximum expected molar amount of said antibodies in said sample, and

(iii) a first binding agent that binds said complex, wherein said first binding agent is a receptor for said autoantibodies and is bound to a material selected from the group consisting of a suspendable solid phase and a soluble polymer;

(b) adding to said medium a second binding agent that selectively binds said antigen relative to binding said complex when said complex is not separated from said medium; and

(c) detecting the binding of said second binding agent to said antigen, the binding thereof being related to the presence or amount of said autoantibodies in the sample.

24. The method of claim 23 wherein said first binding agent is selected from the group consisting of antibodies to immunoglobulins, complement factor C1q, rheumatoid factor, protein G and protein A.

25. The method of claim 24 wherein said first binding agent is protein A and said soluble polymer is dextran.

26. The method of claim 23 wherein said detecting step involves the detection of enzyme activity, luminescence or light absorbance.

27. The method of claim 23 wherein said second binding agent is a receptor for said antigen and is bound to or capable of being bound to a support.

28. The method of claim 27 wherein said antigen is bound to a member of a signal producing system.

29. The method of claim 23 wherein said second binding agent is a first receptor for said antigen and which method further comprises contacting said medium with a second receptor that binds said antigen, wherein at least one of said receptors is bound to a member of a signal producing system.

30. The method of claim 23 wherein said antigen is bound to a ligand and said second binding agent is a receptor for said ligand bound to a support.

31. The method of claim 30 which further comprises separating said support from said medium and contacting said support with a second receptor for said antigen bound to a member of a signal producing system.

32. The method of claim 23 wherein the amount of said antigen added to said medium is 0.01-12.0 fmol.

33. The method of claim 32 wherein the amount of said antigen added to said medium is 0.025-7.5 fmole.

34. The method of claim 33 wherein the amount of said antigen added to said medium is 0.1-2.0 fmole.

35. In an assay for determining the presence or amount of antibodies in a sample suspected of containing said antibodies for use in the detection or monitoring of a human disease, comprising the steps of combining in an aqueous medium said sample and an antigen that binds said antibodies to form a mixture comprised of an antigen:

antibody complex and free antigen; and

detecting said free antigen, the presence or amount thereof being related to the presence or amount of said antibodies in the sample;

wherein the improvement comprises using a molar amount of antigen that is Z, wherein Z is within the range of X to nX and Z is less than Y, where n is 10-100, X is the minimum molar amount of said antigen that can be reliably detected when there are no antibodies present in a sample and Y is the maximum expected molar amount of said antibodies in said sample.

36. In an assay for determining the presence or amount of antibodies in a sample suspected of containing said antibodies for use in the detection or monitoring of a human disease, comprising the steps of combining in an aqueous medium said sample and an antigen that binds said antibodies to form a mixture comprised of an antigen:

antibody complex and free antigen; and

detecting said free antigen, the presence or amount thereof being related to the presence or amount of said antibodies in the sample;

wherein the improvement comprises adding a first binding agent that binds said complex but does not bind said antigen, followed by the addition of a second binding agent that binds said free antigen but does not bind antigen when it is part of said complex and detecting said free antigen but not said antigen;

antibody complex.

37. A kit for use in a method for detecting antibodies, comprising in a packaged combination:

(a) an antigen that binds said antibodies to form an antigen;

antibody complex, wherein the molar amount of said antigen is Z, wherein Z is within the range of X to nX and Z is less than Y, where n is 5-1000, X is the minimum molar amount of said antigen that can be reliably detected when there are no antibodies present in a sample and Y is the maximum expected molar amount of said antibodies in said sample,

(b) a first binding agent that binds said complex without binding said antigen when said antigen is not part of said complex, said first binding agent being bound to a suspendable solid phase or a soluble polymer, and

(c) a second binding agent that selectively binds said antigen relative to binding said complex when said complex is bound to said first binding agent, said second binding agent being bound to a label or a solid phase or a soluble polymer.

38. The kit of claim 37 wherein said antibodies are glutamic acid decarboxylase autoantibodies, said antigen is glutamic acid decarboxylase, and said first binding agent is a receptor for said complex and is bound to a soluble polymer.

39. The kit of claim 37 wherein said antibodies are insulin autoantibodies, said antigen is insulin, and said first binding agent is a receptor for said complex and is bound to a soluble polymer.