Patent EP0458231A1 successfully refreshed from Google Patents.
EP0458231A1
Expired / Lapsed
EP
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Substrate composition, immunoassay and urea determination employing immobilized urease
Assignee: Becton Dickinson and Co
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Filing: 1991-05-18
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Granted: —
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Expires: 2011-05-18
Abstract
Abstract A method for enzyme immunoassay of a ligand includes urease as a label. A bound fraction of ligand and antiligand conjugated to urease is formed on a solid support. The urease component of the bound fraction is contacted with a substrate composition for urease which includes a compound converted to ammonia by the urease, a tetrazolium salt and a pH dependent reducing agent which reduces the tetrazolium salt when the pH of the assay medium has been raised by the ammonia. The tetrazolium salt is reduced to a colored insoluble formazan which precipitates as a detectable spot on the support. The invention includes the substrate composition and a kit of materials for performing the assay.
Inventors
- 👤 Thomas N. Stewart
- 👤 Glenn P. Vonk
- 👤 James P. Mapes
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-06 18:24
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/58; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/543; G01N33/54366; G01N33/54386; G01N33/54387; G01N33/58; G01N33/581; Y; Y10; Y10S; Y10S435/00; Y10S435/805; Y10S435/97; Y10S436/00; Y10S436/807; Y10S436/81; Y10S436/904
Claims Analysis
Total Claims: 10
Claim 1
Independent Claim
152 words
A method for detecting a ligand in a liquid comprising: a) passing a liquid suspected of containing a ligand through a porous membrane precoated with a capture antiligand whereby said ligand is captured on said membrane; b) passing a tracer solution comprising urease conjugated to one of the ligand and a detection antiligand through said membrane to give a bound fraction including said urease on said membrane; and c) passing a solution of a substrate composition through said membrane, said composition comprising a compound converted by urease to ammonia, a pH dependent reducing agent and a tetrazolium salt, said urease on the membrane converting said compound to ammonia, said ammonia raising the pH of the substrate solution thereby activating said reducing agent, the activated reducing agent reducing said tetrazolium salt to a formazan, said formazan precipitating as a colored spot on said membrane and indicating the presence of ligand in said liquid.
Claim 2
Dependent Claim
22 words
The method of Claim 1 wherein said tracer comprises urease conjugated to said detection antiligand and said tracer binds to said ligand.
Claim 3
Dependent Claim
22 words
The method of Claim 1 wherein said compound is selected from the group consisting of urea, N-methyl urea, semicarbazide, formamide and acetamide.
Claim 4
Dependent Claim
20 words
The method of Claim 1 wherein said reducing agent is selected from the group consisting of indoxyl and ascorbic acid.
Claim 5
Independent Claim
116 words
A method for detecting a ligand in a liquid comprising: a) contacting a liquid suspected of containing a ligand with a solid support whereby said ligand attaches to said support; b) contacting ligand attached to the support with a tracer comprising urease conjugated to an antiligand whereby said antiligand binds to the attached ligand and said urease becomes affixed to said support; and c) contacting urease affixed to said support with a substrate composition for said urease, said composition comprising a compound converted by said urease to ammonia, a tetrazolium salt and a pH-dependent reducing agent for said tetrazolium salt whereby a colored spot forms on said support if said ligand is present in said liquid.
Claim 6
Dependent Claim
12 words
The method of Claim 12 wherein said support is a porous membrane.
Claim 7
Independent Claim
69 words
A method for detecting urea in a liquid comprising: a) adsorbing urease onto a solid support; b) contacting said support with a solution suspected of containing urea; and c) contacting said support with a substrate composition for said urease, said composition comprising a tetrazolium salt and a pH-dependent reducing agent for said tetrazolium salt whereby a colored spot forms on said support if urea is present in said liquid.
Claim 8
Dependent Claim
45 words
A substrate composition for an enzyme immunoassay using urease as a label comprising a compound converted by said urease to ammonia, a tetrazolium salt and a reducing agent which reduces said tetrazolium salt to a formazan at a pH resulting from formation of said ammonia.
Claim 9
Dependent Claim
87 words
A kit of materials for performing an assay for a ligand comprising: a) an enclosure; b) a filter stack in said enclosure, said filter stack including a porous membrane and a pad of absorbent material in contact with said membrane; c) a tracer including urease conjugated to one of said ligand or a detection antiligand; and d) a substrate composition for said urease, said composition including a compound converted by said urease to ammonia, a tetrazolium salt and a pH dependent reducing agent for said tetrazolium salt.
Claim 10
Dependent Claim
76 words
The kit of Claim 23 further comprising a binder for said ligand on said support. a) an enclosure; b) a filter stack in said enclosure, said filter stack including a porous membrane having a capture antibody thereon and a pad of absorbent material in contact with said membrane; c) a tracer including urease conjugated to a detection antibody; and d) a substrate composition for said urease, said composition including urea, ascorbic acid and a tetrazolium salt.
References Cited (Backward Citations)
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