US4658022A
Expired / Lapsed
US
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Binding of antibody reagents to denatured protein analytes
Assignee: MILES INC. A CORP. OF INDIANA
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Filing: 1985-09-27
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Granted: 1987-04-14
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Expires: 2005-09-27
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.
Inventors
- 👤 William J. Knowles
- 👤 Vincent T. Marchesi
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 14:09
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/68; C; C07; C07K; C07K16/00; C07K16/18; Y; Y10; Y10S; Y10S435/00; Y10S435/962; Y10S436/00; Y10S436/819; Y10S530/00; Y10S530/808
Claims Analysis
Total Claims: 72
Claim 1
Independent Claim
45 words
A method for binding a particular native protein with an antibody reagent comprising the steps of treating the protein to denature a significant amount thereof and contacting the denatured protein with an antibody reagent specific for binding a linear peptide epitope in such particular protein.
Claim 2
Independent Claim
45 words
A method for binding a particular native protein with an antibody reagent comprising the steps of treating the protein to denature a significant amount thereof and contacting the denatured protein with an antibody reagent specific for binding a linear peptide epitope in such particular protein.
Claim 3
Dependent Claim
19 words
The method of claim 1 wherein the linear peptide epitope comprises a sequence of at least two amino acids.
Claim 4
Dependent Claim
19 words
The method of claim 2 wherein the linear peptide epitope comprises a sequence of less than 15 amino acids.
Claim 5
Dependent Claim
21 words
The method of claim 1 wherein the epitope appears at an N- or C-terminus of a peptide chain in such protein.
Claim 6
Dependent Claim
12 words
The method of claim 4 wherein the epitope comprises a non-peptide group.
Claim 7
Dependent Claim
17 words
The method of claim 1 wherein the epitope appears along an internal peptide chain in such protein.
Claim 8
Dependent Claim
12 words
The method of claim 6 wherein the epitope comprises a non-peptide group.
Claim 9
Dependent Claim
52 words
The method of claim 1 wherein 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 10
Dependent Claim
63 words
The method of claim 1, wherein the linear peptide epitope in the native protein is capable of limited detectable binding by the antibody reagent and the denaturation step is carried out to be sufficient to increase exposure at least in the region of the peptide epitope in the protein such that there is an increase in the detectable binding by the antibody reagent.
Claim 11
Dependent Claim
63 words
An immunoassay method for determining a particular protein analyte in an aqueous test sample, comprising the steps of treating the test sample to denature a significant amount of any such protein in the sample, contacting the denatured sample with an antibody reagent specific for binding a linear peptide epitope in such particular protein, and determining binding of the antibody reagent to such protein.
Claim 12
Dependent Claim
63 words
An immunoassay method for determining a particular protein analyte in an aqueous test sample, comprising the steps of treating the test sample to denature a significant amount of any such protein in the sample, contacting the denatured sample with an antibody reagent specific for binding a linear peptide epitope in such particular protein, and determining binding of the antibody reagent to such protein.
Claim 13
Dependent Claim
19 words
The method of claim 10 wherein the linear peptide epitope comprises a sequence of at least two amino acids.
Claim 14
Dependent Claim
19 words
The method of claim 11 wherein the linear peptide epitope comprises a sequence of less than 15 amino acids.
Claim 15
Dependent Claim
21 words
The method of claim 10 wherein the epitope appears at an N- or C-terminus of a peptide chain in such protein.
Claim 16
Dependent Claim
12 words
The method of claim 13 wherein the epitope comprises a non-peptide group.
Claim 17
Dependent Claim
17 words
The method of claim 10 wherein the epitope appears along an internal peptide chain in such protein.
Claim 18
Dependent Claim
12 words
The method of claim 15 wherein the epitope comprises a non-peptide group.
Claim 19
Dependent Claim
52 words
The method of claim 10 wherein 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 20
Dependent Claim
63 words
The method of claim 10 wherein the linear peptide epitope in the native protein is capable of limited detectable binding by the antibody reagent and the denaturation step is carried out to be sufficient to increase exposure at least in the region of the peptide epitope in the protein such that there is an increase in the detectable binding by the antibody reagent.
Claim 21
Dependent Claim
49 words
The method of claim 10 wherein 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.
Claim 22
Dependent Claim
70 words
The method of claim 19 wherein 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 23
Dependent Claim
20 words
The method of claim 19 wherein the chaotrope is selected from the group consisting of guanidine, sodium dodecylsulfate, and urea.
Claim 24
Dependent Claim
20 words
The method of claim 20 wherein the chaotrope is selected from the group consisting of guanidine, sodium dodecylsulfate and urea.
Claim 25
Dependent Claim
46 words
The method of claim 20 wherein the chaotrope is guanidine and is present in the mixture of the test sample and the chaotropic aqueous solution at a concentration greater than about 3 molar and in the diluted mixture at a concentration less than about 1 molar.
Claim 26
Dependent Claim
35 words
The method of claim 10 wherein 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 27
Dependent Claim
15 words
The method of claim 24 wherein the antibody reagent is obtained by a monoclonal technique.
Claim 28
Dependent Claim
14 words
The method of claim 10 wherein the aqueous test sample is a biological fluid.
Claim 29
Dependent Claim
13 words
The method of claim 26 wherein the protein analyte is a glycosylated protein.
Claim 30
Dependent Claim
15 words
The method of claim 27 wherein such glycosylated protein is a glucosylated hemoglobin or albumin.
Claim 31
Dependent Claim
47 words
The method of claim 10 according to a competitive binding technique wherein the test sample is also contacted with a labeled reagent capable of being bound by said antibody reagent, and the amount of resulting labeled reagent bound to the protein analyte or unbound thereto is determined.
Claim 32
Dependent Claim
18 words
The method of claim 29 wherein the labeled reagent is a labeled form of said linear peptide epitope.
Claim 33
Dependent Claim
72 words
The method of claim 10 according to a sandwich technique wherein the test sample is contacted with first and second antibody reagents specific for binding mutually exclusive linear peptide epitopes respectively, one of such antibody reagents being labeled, and the amount of the labeled one of the two antibody reagents that is present in the complex of the protein analyte which is bound to each of the two antibody reagents is determined.
Claim 34
Dependent Claim
51 words
A reagent system for the immunoassay determination of a particular protein analyte in an aqueous test sample, comprising: (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 35
Dependent Claim
51 words
A reagent system for the immunoassay determination of a particular protein analyte in an aqueous test sample, comprising: (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 36
Dependent Claim
20 words
The reagent system of claim 32 wherein the linear peptide epitope comprises a sequence of at least two amino acids.
Claim 37
Dependent Claim
20 words
The reagent system of claim 33 wherein the linear peptide epitope comprises a sequence of less than 15 amino acids.
Claim 38
Dependent Claim
22 words
The reagent system of claim 32 wherein the epitope appears at an N- or C-terminus of a peptide chain in such protein.
Claim 39
Dependent Claim
13 words
The reagent system of claim 35 wherein the epitope comprises a non-peptide group.
Claim 40
Dependent Claim
18 words
The reagent system of claim 32 wherein the epitope appears along an internal peptide chain in such protein.
Claim 41
Dependent Claim
13 words
The reagent system of claim 37 wherein the epitope comprises a non-peptide group.
Claim 42
Dependent Claim
13 words
The reagent system of claim 32 wherein the chemical agent is a chaotrope.
Claim 43
Dependent Claim
21 words
The reagent system of claim 39 wherein the chaotrope is selected from the group consisting of guanidine, sodium dodecylsulfate, and urea.
Claim 44
Dependent Claim
36 words
The reagent system of claim 32 wherein 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 45
Dependent Claim
16 words
The reagent system of claim 41 wherein the antibody reagent is obtained by a monoclonal technique.
Claim 46
Dependent Claim
15 words
The reagent system of claim 32 wherein the aqueous test sample is a biological fluid.
Claim 47
Dependent Claim
14 words
The reagent system of claim 43 wherein the protein analyte is a glycosylated protein.
Claim 48
Dependent Claim
15 words
The reagent system of claim 44 wherein such glycosylated protein is glucosylated hemoglobin or albumin.
Claim 49
Dependent Claim
20 words
The reagent system of claim 32 which additionally comprises a labeled reagent capable of being bound by said antibody reagent.
Claim 50
Dependent Claim
18 words
The reagent system of claim 46 wherein the labeled reagent is a labeled form of said linear epitope.
Claim 51
Dependent Claim
29 words
The reagent system of claim 32 which comprises first and second antibody reagents specific for binding mutually exclusive linear peptide epitopes respectively, one of such antibody reagents being labeled.
Claim 52
Dependent Claim
16 words
A method of claim 1 wherein the protein is denatured by addition of a chaotropic agent.
Claim 53
Dependent Claim
19 words
A method of claim 49 wherein the chaotropic agent is selected from the group consisting of guanidine and urea.
Claim 54
Dependent Claim
12 words
A method of claim 49 wherein the chaotropic agent is a detergent.
Claim 55
Dependent Claim
16 words
A method of claim 1 wherein the protein is denatured by high pH or low pH.
Claim 56
Dependent Claim
16 words
A method of claim 1 wherein the protein is denatured by digestion with a proteolytic enzyme.
Claim 57
Dependent Claim
12 words
A method of claim 1 wherein the protein is denatured by heating.
Claim 58
Dependent Claim
12 words
A method of claim 1 wherein the protein is denatured by sonication.
Claim 59
Dependent Claim
35 words
A method of claim 1 wherein 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 60
Dependent Claim
15 words
A method of claim 56 wherein the antibody reagent is obtained by a monoclonal technique.
Claim 61
Dependent Claim
32 words
A method of claim 1 wherein the antibody reagent is derived from immunization of an animal with a denatured form of the protein or a fragment thereof comprising the linear peptide epitope.
Claim 62
Dependent Claim
16 words
A method of claim 10 wherein the protein is denatured by addition of a chaotropic agent.
Claim 63
Dependent Claim
19 words
A method of claim 59 wherein the chaotropic agent is selected from the group consisting of guanidine and urea.
Claim 64
Dependent Claim
12 words
A method of claim 59 wherein the chaotropic agent is a detergent.
Claim 65
Dependent Claim
16 words
A method of claim 10 wherein the protein is denatured by high pH or low pH.
Claim 66
Dependent Claim
16 words
A method of claim 10 wherein the protein is denatured by digestion with a proteolytic enzyme.
Claim 67
Dependent Claim
12 words
A method of claim 10 wherein the protein is denatured by heating.
Claim 68
Dependent Claim
12 words
A method of claim 10 wherein the protein is denatured by sonication.
Claim 69
Dependent Claim
32 words
A method of claim 10 wherein the antibody reagent is derived from immunization of an animal with a denatured form of the protein or a fragment thereof comprising the linear peptide epitope.
Claim 70
Dependent Claim
13 words
A reagent system of claim 32 wherein the chemical agent is a detergent.
Claim 71
Dependent Claim
14 words
A reagent system of claim 32 wherein the chemical agent is a proteolytic enzyme.
Claim 72
Dependent Claim
33 words
A reagent system of claim 32 wherein the antibody reagent is derived from immunization of an animal with a denatured form of the protein or a fragment thereof comprising the linear peptide epitope.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1990-04-13 | 1990-10-13 | 1991-04-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-04-13 | 1994-10-13 | 1995-04-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-04-13 | 1998-10-13 | 1999-04-14 | $7700.00 | Window Closed / Lapsed |