Reducing interference in ligand-receptor binding assays

• US 4,737,456 A
• Filed: 05/09/1985
• Issued: 04/12/1988
• Est. Priority Date: 05/09/1985
• Status: Expired due to Term

• Current Assignee: Dade Behring Marburg GmbH (Siemens AG)
• Original Assignee: Syntec Incorporated (Atos SE)
• Inventors: Gibbons, Iam, Weng, Litai
• Primary Examiner(s): Warden, Robert J.
• Assistant Examiner(s): NOT, DEFINED
• Application Number:
  US06/732,471
• Time in Patent Office: 1,069 Days
• Field of Search: 435/4, 435/7, 435/810, 436/518, 436/537, 436/547, 436/548, 436/808, 436/817, 436/818, 436/819, 436/825, 935/108, 935/110
• US Class Current: 435/7.92
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• 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 435/975 Kit
• CPC Class Codes: Y10S 436/808 Automated or kit
• CPC Class Codes: Y10S 436/817 Steroids or hormones
• CPC Class Codes: Y10S 436/818 Human chorionic gonadotropin
• CPC Class Codes: Y10S 436/819 Multifunctional antigen or antibody
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample

Abstract

A method for assaying for a ligand analyte which is a member of a specific binding pair ("sbp member") consisting of ligand and its complementary receptor is disclosed. The ligand analyte has a binding site in common with an interfering substance present in a sample suspected of containing the analyte. The interfering substance has at least two binding sites. The method comprises combining in an assay medium without intervening separation (1) the sample, (2) a blocking receptor which does not bind to the ligand and does bind to the interfering substance, thereby blocking the binding of a common receptor to the interfering substance, and (3) a common receptor which binds to the common binding site. Any additional members of a signal producing system capable of producing a detectable signal in relation to the amount of analyte in the sample are added to the assay medium. Next, the assay medium is examined for the presence of a detectable signal. The method has application to both heterogeneous and homogeneous assays.

Claims

1. A method for assaying for a ligand which is a member of a specific binding pair ("sbp member ") consisting of said ligand and its complementary receptor wherein said ligand has a binding site in common with an interfering substance present in a sample suspected of containing said ligand, said interfering substance having at least two binding sites, which method comprises

combining without intervening separation (1) said sample with (2) a blocking receptor, which does not bind to said ligand and does bind to said interfering substance and thereby blocks binding of a common receptor to said interfering substance, and (3) a common receptor which binds to said common binding site, to provide an assay medium, and

determining the extent of binding of said common receptor, the extent of binding being related to the amount of said analyte in said sample.

2. The method of claim 1 wherein said common receptor is an antibody.

3. The method of claim 1 wherein said blocking receptor is a monoclonal antibody.

4. The method of claim 1 wherein said ligand is a protein.

5. The method of claim 1 wherein said blocking and common receptors are antibodies.

6. The method of claim 1 wherein said ligand is human thyroid stimulating hormone (hTSH) and said interfering substance is human chorionic gonadotropin (hCG).

7. The method of claim 6 wherein said common receptor recognizes the alpha sub-unit of hTSH and hCG and said blocking receptor is specific for the beta sub-unit of hCG.

8. The method of claim 1 which is an enzyme linked immunosorbent assay.

9. The method of claim 1 wherein said common receptor is immobilized.

10. The method of claim 1 wherein said common receptor is conjugated to a label.

11. The method of claim 10 wherein said label is an enzyme.

12. The method of claim 10 wherein said label is a radioisotope.

13. The method of claim 10 wherein said label is part of a signal producing system and said determining is carried out by examining said assay medium for the presence of a detectible signal.

27. The method of claim 6 wherein said protein analyte is human thyroid stimulating hormone (hTSH) and said interfering substance is human chorionic gonadotropin.

14. In an assay method for a ligand which is a member of a specific binding pair ("sbp member") consisting of said ligand and its complementary receptor wherein said ligand has a binding site in common with a binding site of an interfering substance having at least two binding sites, said method employing a receptor recognizing the common binding site, the improvement which comprises adding to an assay medium containing said analyte and said interfering substance, without intervening separation, a specific binding compound which blocks the binding between said receptor and said interfering substance and does not block the binding between said receptor and ligand.

15. The method of claim 14 wherein said receptor is an antibody.

16. The method of claim 14 wherein said receptor is a monoclonal antibody.

17. The method of claim 14 wherein said ligand is a protein.

18. The method of claim 14 wherein said compound is an antibody that binds to said interfering substance.

19. In an assay method for an antigen which is a member of a specific binding pair ("sbp member") consisting of said antigen and its complementary antibody wherein said antigen has at least two epitopic sites, one of said epitopic sites being common to an interfering substance having at least two epitopic sites, said method employing a common antibody, said common antibody recognizing the common epitopic site,

the improvement which comprises combining with an assay medium containing said antigen and said interfering substance an amount of a blocking antibody sufficient to block the binding between said common antibody and said intefering substance wherein said blocking antibody does not block the binding between said common antibody and said antigen.

20. The method of claim 19 wherein said common antibody is a monoclonal antibody.

21. The method of claim 19 wherein said antigen is a protein.

22. The method of claim 19 wherein said antigen is human thyroid stimulating hormone (hTSH) and said interfering substance is human chorionic gonadotropin (hCG).

23. The method of claim 22 wherein said common antibody recognizes the alph sub-unit of TSH and HCG and said blocking antibody binds only to intact HCG comprised of both the alpha and beta sub-units.

24. The method of claim 19 which is an enzyme linked immunosorbent assay.

25. The method of claim 19 wherein said common antibody is immobilized.

26. In an assay method for a protein analyte having alpha and beta sub-units, wherein one of said sub-units is common to a sub-unit of an interfering protein having alpha and beta sub-units, said method employing a common antibody which recognizes the sub-unit common to said protein analyte and said interfering substance and an antibody specific for the non-common sub-unit of said analyte, the improvement which comprises combinine with said assay medium containing said interfering substance an amount of a blocking antibody that binds specifically to said interfering subtance but not to its separate alpha and beta sub-units, said blocking antibody blocking the binding of said interfering substance to said common antibody.

28. A composition comprising one or both of (1) a common receptor that specifically binds to a binding site common to an endogenous interfering substance and a ligand suspected of being present in a sample and (2) members of a signal producing system capable of producing a detectible signal in the presence of said ligand in combination with (3) an amount of a blocking receptor which does not bind to said ligand and does bind to said endogenous interfering substance and thereby blocks the binding between said common receptor and said endogenous interfering substance.

29. The composition of claim 28 wherein said blocking receptor is an antibody.

30. The composition of claim 28 wherein said common receptor is a monoclonal antibody.

31. The composition of claim 28 wherein said ligand is a protein.

32. The composition of claim 28 wherein said blocking and common receptors are antibodies.

33. The composition of claim 28 wherein said ligand is human thyroid stimulating hormone (hTSH) and said interfering substance is human chorionic gonadotropin (hCG).

34. The composition of claim 33 wherein said common receptor recognizes the alpha sub-unit of hTSH and hCG and said blocking receptor binds only to intact hCG comprised of both alpha and beta sub-units of hCG.

35. The composition of claim 28 which additionally comprises a third receptor specific for non-common site on said ligand.

36. A kit for use in an assay method, comprising in packaged combination

the composition of claim 28, and

ancillary reagents as required.