Heterogenous chemiluminescent specific binding assay

• US 4,380,580 A
• Filed: 06/21/1979
• Issued: 04/19/1983
• Est. Priority Date: 04/10/1978
• Status: Expired due to Term

• Current Assignee: Miles Laboratories, Inc. (Bayer AG)
• Original Assignee: Miles Laboratories, Inc. (Bayer AG)
• Inventors: Christner, James E., Carrico, Robert J., Boguslaski, Robert C.
• Primary Examiner(s): Marantz, Sidney
• Application Number:
  US06/050,681
• Time in Patent Office: 1,398 Days
• Field of Search: 23/230 B, 424/8, 424/12, 252/408, 435/7, 436/518
• US Class Current: 435/5
• CPC Class Codes: C07D 209/48 with oxygen atoms in positions 1 and 3, e.g. phthalimide
• CPC Class Codes: C07D 237/32 with oxygen atoms directly attached to carbon atoms of the nitrogen-containing ring
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/97 Test strip or test slide
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/817 Steroids or hormones
• CPC Class Codes: Y10S 436/818 Human chorionic gonadotropin

Abstract

A heterogeneous specific binding assay which employs a substance having reactant activity, i.e., a reactant, in a chemiluminescent reaction as a labeling substance in the detection of a ligand in a liquid medium. The method is carried out using reagent means which comprises, as its labeled constituent, a conjugate formed of a specific binding substance coupled to the chemiluminescent reactant. The activity of the conjugated reactant in the chemiluminescent reaction, i.e., the production of light, is utilized as means for monitoring the extent of binding of the labeled constituent in conventional heterogeneous specific binding assay schemes. The presence of a ligand in a liquid medium may be determined following conventional competitive binding manipulative techniques. After the necessary separation of the bound- and free-phases resulting in the specific binding reaction system, the extent of binding of the labeled constituent is determined by contacting either phase with the necessary materials to form the chemiluminescent monitoring reaction and measuring the light produced.

Claims

1. In a heterogeneous specific binding assay method for determining a ligand in a liquid medium, which method comprises the steps of:

(a) contacting said liquid medium with reagent means including a labeled conjugate comprising a specific binding substance coupled to a labeling substance, said reagent means and the ligand forming a binding reaction system producing a bound-phase and a free-phase of the labeled conjugate;

(b) separating said bound-phase and said free-phase; and

(c) determining said labeling substance in said bound-phase or said free-phase as a function of said ligand in said liquid medium,

the improvement wherein said labeling substance is a chemiluminescent reactant and wherein said chemiluminescent label is determined in said bound-phase or said free-phase by forming the chemiluminescent reaction therein and measuring the light produced.

2. The method of claim 1 wherein the rate of said chemiluminescent reaction is measured as a function of said ligand in said medium.

3. The method of claim 1 wherein the light produced by said chemiluminescent reaction is measured as the total amount of light produced or a portion thereof.

4. The method of claim 1 wherein the light produced by said chemiluminescent reaction is measured as the peak intensity of light produced.

5. The method of claim 1 wherein said chemiluminescent label is luciferin, luminol, pyrogallol, or isoluminol.

6. The method of claim 1 wherein said chemiluminescent label is luminol or isoluminol, or a derivative thereof.

7. The method of claim 1 wherein said chemiluminescent reaction is enzymatic.

8. The method of claim 7 wherein said chemiluminescent label is a substrate for an enzyme involved in the enzymatic chemiluminescent reaction.

9. The method of claim 1 wherein said chemiluminescent reaction is non-enzymatic.

10. The method of claim 1 wherein said ligand is selected from the group consisting of antigens and antibodies thereto;

haptens and antibodies thereto; and

hormones, vitamins, metabolites, and pharmocological agents, and their receptors and binding substances.

11. The method of claim 1 wherein said liquid medium is a biological fluid.

12. A heterogeneous competitive binding assay method for determining a ligand in a liquid medium, comprising the steps of:

(a) contacting said liquid medium with (1) an insoluble form of one of said ligand or a specific binding partner thereof, and (2) a conjugate comprising the other of said ligand and binding partner labeled with a substance which is a reactant in a chemiluminescent reaction, to form a reaction mixture comprising a bound-phase and a free-phase of said labeled conjugate,

(b) separating said bound-phase and said free-phase,

(c) forming said chemiluminescent reaction in one of said bound-phase and said free-phase, and

(d) measuring the light produced as a function of said ligand in said liquid medium.

13. The method of claim 12 wherein said chemiluminescent label is luciferin, luminol, pyrogallol, or isoluminol.

14. The method of claim 12 wherein said chemiluminescent label is luminol or isoluminol, or a derivative thereof.

15. The method of claim 12 wherein said chemiluminescent reaction is enzymatic.

16. The method of claim 15 wherein said chemiluminescent label is a substrate for an enzyme involved in the enzymatic chemiluminescent reaction.

17. The method of claim 12 wherein said chemiluminescent reaction is non-enzymatic.

18. The method of claim 12 wherein said ligand is an antigen or hapten and said liquid medium is contacted with an insoluble form of an antibody thereto and with a chemiluminescent labeled form of said ligand.

19. A sandwich-type heterogeneous specific binding assay method for determining a ligand in a liquid medium, which method comprises the steps of:

(a) contacting said liquid medium with (1) an insoluble form of a specific binding partner to said ligand, and (2) a labeled conjugate comprising a specific binding partner to said ligand labeled with a substance which is a reactant in a chemiluminescent reaction, to form a bound-phase comprising said labeled conjugate bound to ligand bound to said insoluble binding partner and a free-phase comprising said labeled conjugate not so bound,

(b) separating said bound-phase and said free-phase,

(c) forming said chemiluminescent reaction is one of said separated phases, and

(d) measuring the light produced as a function of the ligand in the medium under assay.

20. The method of claim 19 wherein steps (a) and (b) are accomplished by:

(i) first contacting said liquid medium with said insoluble binding partner, to form a reaction mixture comprising, as a first bound-phase, ligand from said medium bound to said insoluble binding partner,

(ii) separating said first-bound phase from the reaction mixture,

(iii) then contacting said separated first bound-phase with said chemiluminescent labeled conjugate to form a second bound-phase comprising said labeled conjugate bound to said first bound-phase and a free-phase comprising said labeled conjugate not so bound, and

(iv) separating said second bound-phase and said free-phase.

21. The method of claim 19 wherein said specific binding partner in said labeled conjugate is the same as that in said insoluble form.

22. The method of claim 19 wherein said chemiluminescent label is luciferin, luminol, pyrogallol, or isoluminol.

23. The method of claim 19 wherein said chemiluminescent label is luminol or isoluminol, or a derivative thereof.

24. The method of claim 19 wherein said chemiluminescent reacton is enzymatic.

25. The method of claim 24 wherein said chemiluminescent label is a substrate for an enzyme involved in the enzymatic chemiluminescent reaction.

26. The method of claim 19 wherein said chemiluminescent reaction is non-enzymatic.

27. The method of claim 19 wherein said ligand is an antigen and wherein said insoluble form of a specific binding partner thereof is an insoluble form of an antibody thereto and said labeled conjugate is a chemiluminescent labeled form of an antibody thereto.

28. A heterogeneous method of determining the presence of a ligand in a medium suspected of containing said ligand which comprises:

bringing together in an aqueous liquid zone;

(1) said medium;

(2) a chemiluminescent labeled ligand; and

(3) a binding partner having sites capable of bonding to said ligand and said chemiluminescent labeled ligand;

the concentration of said binding partner being such that there are insufficient binding sites to combine with all of said labeled ligand present in the three component mixture; and

analyzing in said zone for the effect of said medium on the amount of chemiluminescent labeled ligand bound to said binding partner by physically separating free chemiluminescent labeled ligand from chemiluminescent labeled ligand bound to said binding partner and measuring chemiluminescence in either of the separated free and bound phases.

29. A method according to claim 28 wherein said ligand is insulin.

30. A method according to claim 28 wherein said chemiluminescent labeled ligand is an insulin-luminol conjugate.

31. A method according to claim 28 wherein said ligand is digoxin.

32. A method according to claim 31 wherein said chemiluminescent labeled ligand is a digoxin-luminol conjugate.

33. A method according to claim 28 wherein said binding partner is an antibody.

34. A method according to claim 28 wherein said ligand is a drug, its analog or metabolite.

35. A method according to claim 34 wherein said drug is selected from the group consisting of morphine, barbiturates, diphenylhydantoin, and metabolites and analogs thereof.

36. A method according to claim 28 wherein said ligand is a protein.

37. A method according to claim 36 wherein said protein is selected from the group consisting of polypeptides, antibodies, hormones, insulin, chorionic gondotropin, bradykinin, and thyroid binding globulin.

38. A method according to claim 28 wherein said ligand is a steroid.

39. A method according to claim 28 wherein said ligand is a vitamin.

40. The method of claim 28 wherein said binding partner is isolated and the amount of chemiluminescent ligand bound thereto is determined by adding an activator and measuring the light emitted.

41. The method of claim 28 wherein said chemiluminescent ligand is a conjugate of a ligand with a chemiluminescent substance selected from the group consisting of luciferin and luminol.

42. Reagent means for use in a heterogeneous competitive binding assay for determining a ligand in a liquid medium, comprising (a) an insoluble form of one of said ligand or a specific binding partner thereof, and (b) a conjugate comprising the other of said ligand and binding partner labeled with a substance which is a reactant in a chemiluminescent reaction.

43. The reagent means of claim 42 wherein said chemiluminescent label is luciferin, luminol, pyrogallol, or isoluminol.

44. The reagent means of claim 42 wherein said chemiluminescent label is luminol or isoluminol, or a derivative thereof.

45. The reagent means of claim 42 wherein said chemiluminescent reaction is enzymatic.

46. The reagent means of claim 45 wherein said chemiluminescent label is a substrate for an enzyme involved in the enzymatic chemiluminescent reaction.

47. The reagent means of claim 42 wherein said chemiluminescent reaction is non-enzymatic.

48. The reagent means of claim 42 wherein said ligand is an antigen or hapten and wherein said means comprises an insoluble form of an antibody thereto and a chemiluminescent labeled form of said ligand.

49. Reagent means for use in a sandwich-type heterogeneous binding assay for determining a ligand in a liquid medium, comprising (a) an insoluble form of a specific binding partner of said ligand, and (b) a conjugate comprising a specific binding partner of said ligand labeled with a substance which is a reactant in a chemiluminescent reaction.

50. The reagent means of claim 49 wherein said specific binding partner in said labeled conjugate is the same as that in said insoluble form.

51. The reagent means of claim 49 wherein said chemiluminescent label is luciferin, luminol, pyrogallol, or isoluminol.

52. The reagent means of claim 49 wherein said chemiluminescent label is luminol or isoluminol, or a derivative thereof.

53. The reagent means of claim 49 wherein said chemiluminescent reaction is enzymatic.

54. The reagent means of claim 53 wherein said chemiluminescent label is a substrate for an enzyme involved in the enzymatic chemiluminescent reaction.

55. The reagent means of claim 49 wherein said chemiluminescent reaction is non-enzymatic.

56. The reagent means of claim 49 wherein said ligand is an antigen and wherein said insoluble form of a specific binding partner thereof is an insoluble form of an antibody thereto and said labeled conjugate is a chemiluminescent labeled form of an antibody thereto.