Claims for Patent: 5,231,004

Summary for Patent: 5,231,004

Title:	Use of heme-containing proteins as stabilizers for enzyme-labeled immunoreactants
Abstract:	Heme-containing proteins, such as cytochrome c, are useful in admixture with enzyme-labeled immunoreactants, such as peroxidase-labeled antibodies or fragments thereof. The heme-containing proteins and enzyme-labeled immunoreactants can be supplied in a buffered composition as part of a test kit. The buffered composition comprising the heme-containing protein and peroxidase-labeled immunoreactant excludes 4\'-hydroxyacetanilide, which is a phenolic electron transfer agent. The composition can be used in immunoassays for detecting various immunologically reactive species, such as hCG, and chlamydial or gonococcal antigens.
Inventor(s):	Warren, III; Harold C. (Rush, NY), Boyer; Bradley P. (Rochester, NY)
Assignee:	Eastman Kodak Company (Rochester, NY)
Application Number:	07/522,441
Patent Claims:	1. A test kit useful for the determination of an immunologically reactive ligand, said kit comprising, in individual packaging: a. a buffered composition comprising a water-soluble, non-particulate enzyme-labeled immunoreactant in admixture with a heme-containing protein which reduces background in enzyme label immunoassays, but said composition excluding 4'-hydroxyacetanilide, said immunoreactant being a receptor for said ligand, said heme-containing protein and said immunoreactant being present in said composition at a weight ratio of from about 500:1 to about 10:1, and b. a dye-providing composition which provides a dye in the presence of said enzyme label. 2. The test kit of claim 1 wherein said immunoreactant is an antibody or F(ab').sub.2 fragment thereof. 3. The test kit of claim 2 wherein said antibody or F(ab').sub.2 fragment thereof specifically binds to either a chlamydial antigen or to human chorionic gonadotropin. 4. The test kit of claim 1 wherein said immunoreactant is labeled with peroxidase, alkaline phosphatase, acid phosphatase, .beta.-galactosidase, glucoamylase, glucose oxidase, acetylcholineesterase, catalase, lysozyme, malate dehydrogenase, glucose-6-phosphate dehydrogenase or .beta.-amylase, and said heme-containing protein is cytochrome, myoglobin, hemoglobin, hematin, heme, hemin or a mixture of any of these. 5. The test kit of claim 4 wherein said immunoreactant is labeled with peroxidase, said dye-providing composition comprises a triarylimidazole leuco dye which provides a dye in the presence of peroxidase and hydrogen peroxide, and said heme-containing protein is cytochrome c. 6. The test kit of claim 1 further comprising a disposable test device including a microporous membrane fitted therein. 7. A method for the determination of an immunologically reactive ligand comprising: A. contacting an immunologically reactive ligand of interest with a buffered composition comprising a water-soluble, non-particulate enzyme-labeled receptor for said ligand, said enzyme-labeled receptor being in admixture with a heme-containing protein which reduces background in immunoassays, but said composition excluding 4'-hydroxyacetanilide, said heme-containing protein and said enzyme-labeled receptor being present in said composition at a weight ratio of from about 500:1 to about 10:1, so as to form an enzyme-labeled immunological complex of said ligand with said enzyme-labeled receptor, and B. after separation of uncomplexed enzyme-labeled receptor from said complex, detecting the presence of either said uncomplexed enzyme-labeled receptor or said enzyme-labeled complex as an indication of the presence of said ligand. 8. The method of claim 7 wherein said ligand is either a chlamydial or gonococcal antigen or human chorionic gonadotropin and said enzyme-labeled receptor is an antibody of F(ab').sub.2 fragment thereof that specifically bind to said chlamydial or gonococcal antigen or human chorionic gonadotropin, respectively. 9. The method of claim 8 wherein said enzyme-labeled receptor is a peroxidase-labeled F(ab').sub.2 antibody fragment, and said heme-containing protein is cytochrome, myoglobin, hemoglobin, hematin, heme, hemin or a mixture of any of these. 10. A method for the determination of a chlamydial or gonococcal antigen in a biological specimen comprising: A. contacting antigen extracted from chlamydial or gonococcal organisms suspected of being present in a biological specimen with a microporous membrane to bind said antigen thereto, B. contacting said antigen bound with a buffered composition comprising a water-soluble non-particulate enzyme-labeled antibody or F(ab').sub.2 fragment thereof which specifically binds to said antigen, said enzyme-labeled antibody or F(ab').sub.2 fragment thereof being in admixture with a heme-containing protein, but said composition excluding 4'-hydroxyacetanilide, so as to form an enzyme-labeled immunological complex between said antigen and said enzyme-labeled antibody or F(ab').sub.2 fragment thereof bound to said membrane, C. contacting the bound complex with a composition which provides a dye in the presence of said enzyme, and D. detecting the presence of said dye on said membrane as an indication of the presence or amount of said chlamydial or gonococcal organisms in said specimen. 11. The method of claim 10 wherein said chlamydial antigen is contacted with a peroxidase-labeled antibody or F(ab').sub.2 fragment thereof which binds thereto in admixture with a heme-containing protein which is cytochrome, myoglobin, hemoglobin, hematin, heme, hemin or a mixture of any of these, and said dye-providing composition comprises a triarylimidazole leuco dye which provides a dye in the presence of peroxidase and hydrogen peroxide. 12. A method for the determination of an immunologically reactive ligand comprising: detecting the presence or amount of either an uncomplexed water-soluble, non-particulate enzyme-labeled receptor for an immunologically reactive ligand of interest or an enzyme-labeled complex of said enzyme-labeled receptor bound to said ligand, after said ligand is contacted with a buffered composition comprising said enzyme-labeled receptor in admixture with a heme-containing protein, but said composition excluding 4'-hydroxyacetanilide, and after uncomplexed receptor is separated from any complex formed between said ligand and enzyme-labeled receptor. 13. A method for the substantial elimination of background in an assay for the determination of an immunologically reactive ligand, said method comprising: detecting the presence or amount of either an uncomplexed, water-soluble, non-particulate enzyme-labeled receptor for an immunologically reactive ligand of interest or an enzyme-labeled complex of said enzyme-labeled receptor bound to said ligand, after said ligand is contacted with a buffered composition comprising said enzyme-labeled receptor in admixture with a heme-containing protein which reduces background in said assay, but said composition excluding 4'-hydroxyacetanilide, said heme-containing protein and said enzyme-labeled receptor being present in said composition at a weight ratio of from about 500:1 to about 10:1, and after uncomplexed receptor is separated from any complex formed between said ligand and enzyme-labeled receptor, said heme-containing protein selected from the group consisting of cytochrome c, myoglobin, hemoglobin, hematin, heme, hemin and a mixture of any of these. 14. In an enzyme immunoassay employing antibodies or antigens conjugated with an enzyme to form enzyme conjugates for detecting the presence or amount of a ligand in a specimen, wherein the improvement comprises adding a buffered composition to a specimen, said composition comprising: a water-soluble, non-particulate conjugate of an enzyme and an immunoreactant which is specifically reactive with said ligand or with a receptor therefor, and a heme-containing protein which reduces background in said enzyme immunoassay, but said composition excluding 4'-hydroxyacetanilide, said heme-containing protein and said conjugate being present in said composition at a weight ratio of from about 500:1 to about 10:1. 15. A method for the determination of a chlamydial or gonococcal antigen in a biological specimen comprising: A. contacting antigen extracted from chlamydial or gonococcal organisms suspected of being present in a biological specimen with a microporous membrane to bind said antigen thereto, B. contacting antigen bound to said membrane with an unlabeled antibody of F(ab').sub.2 fragment thereof directed to said antigen, so as to form an unlabeled immunological complex between said antigen and said unlabeled antibody or F(ab').sub.2 fragment thereof bound to said membrane, C. contacting said complex bound to said membrane with a buffered composition comprising a water-soluble, non-particulate enzyme-labeled anti-antibody or F(ab').sub.2 fragment thereof directed to said unlabeled antibody, said enzyme-labeled anti-antibody of F(ab').sub.2 fragment thereof being in admixture with a heme-containing protein which reduces background in enzyme label immunoassays, but said composition excluding 4'-hydroxyacetanilide, said heme-containing protein and said labeled anti-antibody or F(ab').sub.2 fragment thereof being present in said composition at a weight ratio of from about 500:1 to about 10:1. so as to form an enzyme-labeled immunological complex among said antigen, unlabeled antibody and enzyme-labeled anti-antibody bound to said membrane, D. contacting said complex bound to said membrane composition which provides a dye in the presence of said enzyme-labeled anti-antibody, and E. detecting the presence of said dye on said membrane as an indication of the presence or absence of said organisms in said specimen.

Details for Patent 5,231,004

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	01/15/1974		2040-01-28
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	12/27/1984		2040-01-28
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	02/15/1985		2040-01-28
Ferring Pharmaceuticals Inc.	NOVAREL	chorionic gonadotropin	For Injection	017016	02/16/1990		2040-01-28
Bel-mar Laboratories, Inc.	CHORIONIC GONADOTROPIN	chorionic gonadotropin	Injection	017054	03/26/1974		2040-01-28
Fresenius Kabi Usa, Llc	CHORIONIC GONADOTROPIN	chorionic gonadotropin	For Injection	017067	03/05/1973		2040-01-28