Patent EP0185870A2 successfully refreshed from Google Patents.
EP0185870A2
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EP
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Immunoassays for denatured protein analytes, particularly HB Alc, and monoclonal antibodies thereto
Assignee: Bayer Corp
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Filing: 1985-10-17
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Granted: —
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Expires: 2005-10-17
Abstract
Abstract Binding of a particular protein by an antibody reagent involving denaturation of the protein and use of an antibody reagent specific for binding a linear peptide epitope therein. Denaturation by chemical or physical means effectively exposes or enhances the exposure of the linear peptide epitope for binding by the antibody reagent which is preferably raised against a synthetic peptide immunogen. The technique is particularly useful in performing immunoassays for protein analytes, such as a glycosylated protein, in aqueous test samples. Monoclonal antibodies are also provided which are specific for the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin and are therefore useful in the determination of Hb Alc.
Inventors
- 👤 William Dr. Knowles
- 👤 Vincent Dr. Marchesi
- 👤 Wallace Haigh
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-02 10:30
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/795; C07K14/805; C07K16/00; C07K16/18; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/72; G01N33/721; G01N33/723
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
65 words
An immunoassay method for determining a particular protein analyte in an aqueous test sample, characterized in that the test sample is treated to denature a significant amount of any such protein in the sample, the denatured sample is contacted with an antibody reagent specific for binding a linear peptide epitope in such particular protein, and binding of the antibody reagent to such protein is determined.
Claim 2
Dependent Claim
65 words
An immunoassay method for determining a particular protein analyte in an aqueous test sample, characterized in that the test sample is treated to denature a significant amount of any such protein in the sample, the denatured sample is contacted with an antibody reagent specific for binding a linear peptide epitope in such particular protein, and binding of the antibody reagent to such protein is determined.
Claim 3
Dependent Claim
54 words
The method of Claim 1 characterized in that the linear peptide epitope is substantially inaccessible to antibody binding in the native protein and the denaturation step is carried out to be sufficient to expose at least the region of the peptide epitope in the protein to make it sufficiently accessible for detectable antibody binding.
Claim 4
Independent Claim
120 words
The method of any of Claims 1 and 2 characterized in that the denaturation step is accomplished by combining the aqueous test sample with an aqueous solution of a chaotrope present at sufficient concentration and at a sufficient temperature to denature a significant amount of any of the particular protein analyte in the resulting mixture, and the resulting denatured mixture is maintained at a temperature and diluted sufficiently to reduce the concentration of the chaotrope to a level that has no significant denaturing effect on the antibody reagent subsequently contacted with the mixture, while maintaining the temperature and concentration of the chaotrope sufficiently high to substantially prevent renaturation of any denatured form of the protein analyte that had been produced.
Claim 5
Independent Claim
43 words
The method of any of Claims 1 to 3 characterized in that the antibody reagent is monoclonal and derived from immunization of an animal with a synthetic peptide immunogen comprising a residue of the linear peptide epitope linked to an immunogenic carrier material.
Claim 6
Independent Claim
60 words
The method of any of Claims 1 to 4 wherein the particular protein analyte to be determined is Hb Alc and the test sample is blood, characterized in that the antibody reagent is a monoclonal antibody, or a fragment thereof comprising an antibody combining site, which binds specifically to the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin.
Claim 7
Dependent Claim
55 words
A reagent system for the immunoassay determination of a particular protein analyte in an aqueous test sample, characterized in that it comprises: (1) an antibody reagent specific for binding a linear peptide epitope in such particular protein, and (2) a chemical agent capable of denaturing a significant amount of any such protein in the sample.
Claim 8
Dependent Claim
55 words
A reagent system for the immunoassay determination of a particular protein analyte in an aqueous test sample, characterized in that it comprises: (1) an antibody reagent specific for binding a linear peptide epitope in such particular protein, and (2) a chemical agent capable of denaturing a significant amount of any such protein in the sample.
Claim 9
Dependent Claim
15 words
The reagent system of Claim 6 characterized in that the chemical agent is a chaotrope.
Claim 10
Dependent Claim
38 words
The reagent system of Claim 6 characterized in that the antibody reagent is derived from immunization of an animal with a synthetic peptide immunogen comprising a residue of the linear peptide epitope linked to an immunogenic carrier material.
Claim 11
Dependent Claim
20 words
The reagent system of Claim 6 characterized in that it additionally comprises a labeled form of the linear peptide epitope.
Claim 12
Dependent Claim
61 words
The reagent system of any of Claims 6 to 9 wherein the particular protein analyte to be determined is Hb Alc and the test sample is blood, characterized in that the antibody reagent is a . monoclonal antibody, or a fragment thereof comprising an antibody combining site, which binds specifically to the glucosylatedN-terminal peptide sequence in the beta-subunit of human hemoglobin.
Claim 13
Dependent Claim
90 words
The reagent system of any of Claims 6 to 10 characterized in that the monoclonal antibody is obtained from a hybridoma derived from fusion of a myeloma cell and a lymphocyte from an animal immunized with a synthetic peptide immunogen of the formula: wherein Glyco-(NH)Val represents a nonenzymatic glucosylated valine residue, AA is a bond or one or more additional amino acid residues, R is a linking group, Carrier is an immunogenic carrier material, and n is on average from 1 to the number of available coupling sites on Carrier.
Claim 14
Dependent Claim
30 words
A monoclonal antibody, or a fragment thereof comprising an antibody combining site, characterized in that it binds specifically to the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin.
Claim 15
Dependent Claim
30 words
A monoclonal antibody, or a fragment thereof comprising an antibody combining site, characterized in that it binds specifically to the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin.
Claim 16
Dependent Claim
44 words
The monoclonal antibody or fragment thereof of Claim 12 characterized in that it binds specifically to a glucosylated peptide residue of the formula: wherein Glyco-(NH)Val represents a nonenzymatically glucosylated valine residue and AA is a bond or one or more additional amino acid residues.
Claim 17
Dependent Claim
33 words
The monoclonal antibody or fragment thereof of Claim 13 characterized in that AA is a sequence of from 1 to 12 amino acids corresponding to the N-terminus of the beta-subunit of human hemoglobin.
Claim 18
Dependent Claim
52 words
The monoclonal antibody or fragment of any of Claims 12 to 14 characterized in that it has been raised against an immunogen comprising a glucosylated peptide chemically linked to an immunogenic carrier material, the glucosylated peptide having at least 2 amino acid units corresponding to the N-terminus of the beta-subunit of hemoglobin.
Claim 19
Dependent Claim
23 words
A hybridoma cell line which secrets monoclonal antibody which binds specifically to the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin.
Claim 20
Dependent Claim
23 words
A hybridoma cell line which secrets monoclonal antibody which binds specifically to the glucosylated N-terminal peptide sequence in the beta-subunit of human hemoglobin.
References Cited (Backward Citations)
No citation records indexed.
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