Heterogenous specific binding assay employing a cycling reactant as label

• US 4,318,980 A
• Filed: 10/15/1979
• Issued: 03/09/1982
• 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): Wiseman, Thomas
• Application Number:
  US06/084,866
• Time in Patent Office: 876 Days
• Field of Search: 23/230 B, 435/7, 435/188, 435/810, 424/8, 424/12
• US Class Current: 435/7.5
• 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 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/966 involving an enzyme system with high turnover rate or complement magnified assay, e.g. multi-enzyme systems
• 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

Abstract

An improved heterogenous specific binding assay method which employs a substance having reactant activity, i.e., a reactant, 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 reactant. The reactant advantageously is an enzymatic reactant such as an enzyme substrate or coenzyme. The activity of the conjugated reactant as a constituent of a predetermined reaction system is utilized as means for monitoring the extent of binding of the labeled constituent in conventional heterogenous 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 predetermined monitoring reaction system in which the labeling substance is active and assessing reactant activity therein.

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

(1) a bound-phase of the labeled conjugate in which the specific binding substance therein is bound by a specific binding partner thereto and

(2) a free-phase of the labeled conjugate in which the specific binding substance therein is not bound by a specific binding partner thereto,

(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 the amount of said ligand in said liquid medium;

the improvement wherein said labeling substance is a participant in a cyclic chemical reaction of the following type;

##STR57## wherein said labeling substance is a non catalytic material selected from the group consisting of reactants A, reactants B, and said cycled reactant.

2. The method of claim 1 wherein said labeling substance is determined in said bound-phase or said free-phase by forming said cyclic chemical reaction therein and measuring the disappearance of one of reactants A or one of reactants B, or the appearance of one of products A or one of products B.

3. The method of claim 1 wherein said labeling substance is said cycled reactant.

4. The method of claim 3 wherein the reaction of reactants A to form products A and the reaction of reactants B to form products B are catalyzed by enzymes and said labeling substance is a coenzyme for said enzymes.

5. The method of claim 4 wherein the same enzyme catalyzes both the reaction of reactants A to form products A and the reaction of reactants B to form products B.

6. The method of claim 5 wherein said labeling substance is a coenzyme which cycles between oxidized and reduced forms while participating in said enzyme-catalyzed reactions.

7. The method of claim 6 wherein said labeling substance is flavin adenine dinucleotide.

8. The method of claim 3 wherein the reaction of reactants A to form products A and the reaction of reactants B to form products B are catalyzed by enzymes and said labeling substance is a substrate for said enzymes.

9. The method of claim 1 wherein said cyclic reaction is exponential in that one of products A or products B is also said cycled reactant.

10. The method of claim 9 wherein said labeling substance is said cycled reactant.

11. 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 pharmacological agents, and their receptors and binding substances.

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

13. The method of claim 1 wherein said labeling substance has a molecular weight of less than 9000.

14. In a reagent means for use in a heterogeneous specific binding assay method for determining a ligand in a liquid medium, which means includes (i) a labeled conjugate comprising a specific binding substance coupled to a labeling substance having a predetermined characteristic, which means and the ligand form a binding reaction system producing a bound-phase and a free-phase of the labeled conjugate, one of such phases being insoluble and the other being soluble, the amount of said predetermined characteristic of the labeling substance in said bound-phase or said free-phase being a function of the amount of the ligand present in the liquid medium under assay, and (ii) a specific binding partner of said ligand which binding partner is in a form which is insoluble in said liquid medium,

the improvement wherein said labeling substance is a participant in a cyclic chemical reaction of the following type:

##STR58## wherein said labeling substance is a non catalytic material selected from the group consisting of reactants A, reactants B, and said cycled reactant, wherein the conjugate and the immobilized specific binding partner are present in amounts sufficient to determine said ligand.

15. The means of claim 14 wherein said labeling substance is said cycled reactant.

16. The means of claim 15 wherein the reaction of reactants A to form products A and the reaction of reactants B to form products B are catalyzed by enzymes and said labeling substance is a substrate for said enzymes.

17. The means of claim 16 wherein said labeling substance is α

-aminoacid or α

-ketoacid, and said means additionally comprises L-aminoacid oxidase, glutamate, and transaminase.

18. The means of claim 16 wherein said labeling substance is oxidized or reduced glutathione, and said means additionally comprises nicotinamide adenine dinucleotide phosphate, glutathione reductase, dehydroascorbate, and dehydroascorbate reductase.

19. The means of claim 16 wherein said labeling substance is ascorbate or dehydroascorbate, and said means additionally comprises ascorbate oxidase, oxidized glutathione, and dehydroascorbate reductase.

20. The means of claim 16 wherein said labeling substance is oxidized or reduced cytochrome C and said means additionally comprises nicotinamide adenine dinucleotide phosphate, cytochrome C reductase, hydrogen peroxide, and cytochrome C peroxidase.

21. The means of claim 16 wherein said labeling substance is oxidized or reduced ferridoxin, and said means additionally comprises hydrogen, hydrogenase, nicotinamide adenine dinucleotide phosphate, and pyridine nucleotide reductase.

22. The means of claim 14 wherein the reaction of reactants A to form products A and the reaction of reactants B to form products B are catalyzed by enzymes and said labeling substance is a coenzyme for said enzyme.

23. The means of claim 22 wherein said labeling substance is nicotinamide adenine dinucleotide or a reduced form thereof, and, in order to form said cyclic reaction, said means additionally comprises:

(1) one of the following groups of substances

(a) lactaldehyde and alcohol dehydrogenase;

(b) α

-ketoglutarate, a substance capable of releasing ammonia upon contact with said liquid medium, and glutamic dehydrogenase;

(c) acetaldehyde and alcohol dehydrogenase;

(d) α

-ketoglutarate, a substance capable of releasing carbon dioxide upon contact with said liquid medium, and isocitric dehydrogenase;

(e) dihydroxyacetone phosphate and α

-glycerol phosphate dehydrogenase;

(f) pyruvate and lactate dehydrogenase;

(g) 1,3-diphosphoglycerate and glyceraldehyde-3-phosphate dehydrogenase;

or

(h) oxaloacetate and malic dehydrogenase; and

(2) one of the following groups of substances other than the group having the same reference letter as the group selected from groups (1) above

(a) propanediol and alcohol dehydrogenase;

(b) glutamate and glutamic dehydrogenase;

(c) ethanol and alcohol dehydrogenase;

(d) isocitrate and isocitric dehydrogenase;

(e) L-α

-glycerol phosphate and α

-glycerol phosphate dehydrogenase;

(f) lactate and lactic dehydrogenase;

(g) glyceraldehyde-3-phosphate, phosphate, and glyceraldehyde-3-phosphate dehydrogenase;

or

(h) malate and malic dehydrogenase.

24. The means of claim 22 wherein said labeling substance is nicotinamide adenine dinucleotide phosphate or a reduced form thereof and, in order to form said cyclic reaction, said means additionally comprises:

(1) one of the following groups of substances

(a) oxidized glutathione and glutathione reductase;

(b) ρ

-benzoquinone and quinone reductase;

(c) nitrate and nitrate reductase;

(d) 6-phosphogluconate and glucose-6-phosphate dehydrogenase;

or

(e) α

-ketoglutarate, a substance capable of releasing ammonia upon contact with said liquid medium, and glutamic dehydrogenase; and

(2) one of the following groups of substances other than the group having the same reference letter as the group selected from groups (1) above

(a) reduced glutathione and glutathione reductase;

(b) hydroquinone and quinone reductase;

(c) nitrite and nitrate reductase;

(d) glucose-6-phosphate and glucose-6-phosphate dehydrogenase;

or

(e) glutamate and glutamic dehydrogenase.

25. The means of claim 22 wherein said labeling substance is flavin mononucleotide or a reduced form thereof, and said means additionally comprises nicotinamide adenine dinucleotide phosphate, oxidized cyctochrome C, and cyctochrome C reductase.

26. The means of claim 22 wherein said labeling substance is flavin adenine dinucleotide or a reduced form thereof, and said means additionally comprises D-aminoacid and D-aminoacid oxidase.

27. The means of claim 22 wherein said labeling substance is adenosine diphosphate or adenosine triphosphate, and said means additionally comprises phosphoenol pyruvate, pyruvate kinase, and adenosine triphosphatase.

28. The means of claim 22 wherein said labeling substance is coenzyme A or succinyl-coenzyme A, and said means additionally comprises α

-ketoglutarate, nicotinamide adenine dinucleotide, α

-ketoglutarate dehydrogenase, phosphate, guanosine diphosphate, and succinic thiokinase.

29. The means of claim 22 wherein said labeling substance is guanosine triphosphate or guanosine diphosphate, and said means additionally comprises oxaloacetate, phosphoenol pyruvate kinase, adenosine triphosphate, and nucleoside diphosphate kinase.

30. The means of claim 22 wherein said cyclic reaction is exponential in that one of products A or products B is also said cycled reactant.

31. The means of claim 30 wherein said labeling substance is adenosine triphosphate or adenosine diphosphate, and said means additionally comprises adenosine monophosphate, myokinase, phosphoenolpyruvate, and pyruvate kinase.

32. The means of claim 22 wherein the same enzyme catalyzes both the reaction of reactants A to form products A and the reaction of reactants B to form products B.

33. The means of claim 32 wherein said labeling substance is a coenzyme which cycles between oxidized and reduced forms while participating in said enzyme-catalyzed reactions.

34. The means of claim 33 wherein said labeling substance is flavin adenine dinucleotide.

35. The means of claim 14 wherein said cyclic reaction is exponential in that one of products A or products B is said cycled reactant.

36. The means of claim 35 wherein said labeling substance is said cycled reactant.

37. The means of claim 14 wherein said ligand is selected from the group consisting of antigens and antibodies thereto;

haptens and antibodies thereto; and

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

38. The means of claim 14 wherein at least one of the components of said means is incorporated with a carrier that is insoluble in said liquid medium.

39. The means of claim 38 wherein said carrier is a matrix that is absorbent relative to said liquid medium.

40. The means of claim 14 wherein said labeling substance has a molecular weight of less than 9000.

41. The means of claim 14 which comprises (i) a soluble labeled conjugate comprising said ligand, a specific binding analog thereof, or a specific binding partner thereof, coupled to said labeling substance, and (ii) an insoluble specific binding partner of said ligand.