Claims for Patent: 4,810,631

Summary for Patent: 4,810,631

Title:	Signal enhancement in immunoassay by modulation of chemical catalysis
Abstract:	A method for immunoassay for a ligand suspected to be present in a fluid includes use of an enzyme, a metal ion catalyst for an indicator reaction and a blocked modulator for the catalyst. Ligand present in the fluid binds to an antiligand. The resulting bound fraction activates the enzyme to unblock the modulator. The free modulator activates or inhibits the catalyst thereby modulating the rate of an indicator reaction between a substrate and a redox reagent. The presence of absence of the ligand in the fluid is indicated by a signal, such as a color change or a rate of color change, consequent to the indicator reaction. The invention includes a kit of materials useful for performing the method of the invention.
Inventor(s):	Perlman; Michael E. (Durham, NC), Evans; Susan A. (Miami, FL)
Assignee:	Becton, Dickinson and Company (Franklin Lakes, NJ)
Application Number:	06/861,818
Patent Claims:	1. A method for detection of a ligand in a fluid comprising: (a) combining a fluid suspected of containing a ligand with an antiligand specific for said ligand, an enzyme, a metal ion catalyst, a substrate, a redox reagent and an inhibitor for the activity of said catalyst wherein said inhibitor is blocked by a group coupled thereto; (b) causing ligand present in said fluid to bind to said antiligand to provide a bound fraction whereby said enzyme removes said blocking group thereby providing free inhibitor, said free inhibitor inhibiting the catalytic activity of said catalyst for a reaction between said substrate and said redox reagent whereby a product is formed; and (c) detecting said ligand by a signal associated with said reaction. 2. The method in accordance with claim 1 wherein said antiligand is attached to a solid support. 3. The method in accordance with claim 2 further comprising combining said fluid with a second antiligand specific for said ligand, said second antiligand being coupled to said enzyme. 4. The method in accordance with claim 1 wherein said enzyme is the first component of complement. 5. The method in accordance with claim 1 wherein said enzyme is a hydrolase. 6. The method in accordance with claim 5 wherein said hydrolase is selected from the group of hydrolases consisting of a protease, an esterase, a glycosidase and a phosphatase. 7. The method in accordance with claim 1 wherein said ligand is selected from the group of ligands consisting of an antigen, an antibody and a hapten. 8. The method in accordance with claim 1 wherein said antiligand is selected from the group of antiligands consisting of an antigen, an antibody and and antibody complex. 9. The method in accordance with claim 1 wherein said redox reagent is selected from the group of reagents consisting of an oxidizing agent and a reducing agent. 10. The method in accordance with claim 9 wherein said oxidizing agent is selected from the group of agents consisting of hydrogen peroxide, periodate, bromate, chlorate, persulfate, m-chloroperbenzoic acid and oxygen. 11. The method in accordance with claim 1 wherein said free inhibitor is a metal binding agent. 12. The method in accordance with claim 11 wherein said metal binding agent is selected from the group of agents consisting of an amino acid, a thiol, a thiourea, a nitrogen heterocycle, a hydroxamate, a polyamine and a hydroxy acid. 13. The method in accordance with claim 12 wherein said metal binding agent is selected from the group of agents consisting of 8-hydroxyquinoline, benzylmercaptan, L-cysteine, ethylenediamine, salicylic acid and ethylenediamine tetraacetic acid. 14. The method in accordance with claim 1 wherein said blocking group is selected from the group of blocking groups consisting of a peptide, an amino acid, a carboxylic and, an alcohol, a carbohydrate and an orthophosphate. 15. The method in accordance with claim 1 wherein said catalyst is selected from the group of catalysts consisting of a metal ion and a metal ion complex. 16. The method in accordance with claim 15 wherein said metal ion is selected from the group of ions consisting of an ion of iron, cobalt, manganese, copper, vanadium, mercury, molybdenum and silver. 17. The method in accordance with claim 15 wherein said metal ion complex is selected from the group of complexes consisting of hemin, cobalamine, deuterohemin and iron (III) meso-tetraphenylporphine. 18. The method in accordance with claim 1 wherein said substrate is selected from the group of substrates consisting of aromatic amines, phenols and triarylmethanes. 19. The method in accordance with claim 1 wherein said signal is a color associated with said substrate. 20. The method in accordance with claim 1 wherein said signal is a color associated with said product. 21. The method in accordance with claim 1 wherein said signal is light associated with said product. 22. The method in accordance with claim 21 wherein said light is chemiluminescence. 23. The method in accordance with claim 21 wherein said light is fluorescence. 24. The method in accordance with claim 1 wherein an incubation causes step (b). 25. The method in accordance with claim 1 wherein said blocked inhibitor is attached to a solid support. 26. The method in accordance with claim 25 further comprising separating the fluid phase of said mixture from said solid support. 27. The method in accordance with claim 1 further comprising combining said fluid with a promotor for said catalytic activity. 28. The method in accordance with claim 27 wherein said promotor is selected from the group consisting of 2,2'-dipyridyl and 1,10-phenanthroline. 29. A method for detection of a ligand in a fluid comprising: (a) combining a fluid suspected of containing a ligand with the first component of complement, an antiligand specific for said ligand, a metal ion catalyst, an oxidizing agent, a substrate for said oxidizing agent, and a metal ion binding agent said binding agent being coupled to a peptide which blocks its binding properties; (b) causing ligand present in said fluid to bind to said antiligand, said binding activating said first component of complement, said activated first component of complement removing said peptide thereby providing free binding agent which binds said metal ion catalyst whereby the catalytic activity of said catalyst for a reaction between said substrate and said oxidizing agent is reduced; and (c) detecting said ligand by inhibition of a color change associated with said reaction. 30. The method in accordance with claim 29 wherein said metal binding agent coupled to said peptide is benzyl N.sup.2 -carbobenzyloxy-L-arginine thioester hydrochloride. 31. A method for detection of a ligand in a fluid comprising: (a) combining a fluid suspected of containing a ligand with a first antiligand specific for said ligand attached to a solid support and a second antiligand having coupled thereto a hydrolase; (b) causing ligand present in said fluid to bind to said first and second antiligands to give a bound phase and a free phase; (c) separating said bound phase from said free phase; (d) contacting said bound phase with an oxidizing agent, a substrate for said oxidizing agent, a metal ion catalyst and a metal ion binding agent, said metal ion binding agent being coupled to a blocking group which blocks its binding properties, wherein said hydrolase removes said blocking group thereby providing free binding agent which binds said metal ion catalyst, thereby inhibiting the catalytic activity of said catalyst for a reaction between said substrate and said oxidizing agent; and (e) detecting said ligand by inhibition of a color change associated with said reaction. 32. A method for determination of the concentration of a ligand in a fluid comprising: (a) combining the first component of complement, a substrate, a redox reagent, a metal ion catalyst, an inert inhibitor for said catalyst, a fluid containing an unknown quantity of a ligand and an antiligand for said ligand; (b) causing said antiligand to bind to said ligand, said binding enabling said first component of complement to actuate said inert inhibitor, said actuated inhibitor inhibiting the catalytic activity of said catalyst for a reaction between said substrate and said redox reagent; (c) measuring a signal associated with said reaction; and (d) comparing the magnitude of said signal with the magnitude of a signal associated with said reaction when steps (a) to (c) are repeated with fluid samples containing known quantities of said ligand. 33. A kit of materials for performing an assay for a ligand in a fluid comprising an antiligand for a ligand, an enzyme and a blocked inhibitor for a metal catalyst. 34. The kit in accordance with claim 33 wherein said enzyme is conjugated to said antiligand. 35. The kit in accordance with claim 33 wherein at least one of said blocked inhibitor and said antiligand is attached to a solid support. 36. The kit in accordance with claim 33 further comprising at least one fluid sample containing ligand of known concentration. 37. The kit in accordance with claim 33 further comprising a fluid sample substantially free of ligand. 38. The kit in accordance with claim 33 further comprising at least one other reagent selected from the group of reagents consisting of substrates, metal ion catalysts, redox reagents, antigens, antibodies and complexes thereof, buffers and saline. 39. The kit in accordance with claim 33 further comprising one or more containers.

Details for Patent 4,810,631

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Recordati Rare Diseases, Inc.	PANHEMATIN	hemin for injection	For Injection	101246	07/20/1983		2040-01-28