US4786718A
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Method of preparing antibodies to characterize oncogenes
Assignee: Massachusetts Institute of Technology
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Filing: 1985-08-08
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Granted: 1988-11-22
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Expires: 2005-08-08
Abstract
Abstract Experiments designed to define the differences between the 21 oncogene of DNA isolated from human bladder cancer cells and its corresponding proto-oncogene are described herein. By a series of in vitro recombinations, the difference was initially isolated to a 350 kb segment of DNA; sequencing defined the difference as a change in the Gly 12 codon causing the p21 protein of the oncogene to contain valine at a location where the p21 protein of the proto-oncogene contained glycine. Assays for detecting carcinogenesis based on such differences are also described. In one type of assay, a restriction enzyme specific for either the altered or non-altered DNA segment of the genes are employed to detect carcinogenesis. In another type of assay, seralogical reagents, such as antibody specific for either p21 protein expressed from the proto-oncogene or oncogene, or a common site therein, are described.
Inventors
- 👤 Robert A. Weinberg
- 👤 Clifford J. Tabin
- 👤 Scott M. Bradley
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 13:18
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/577; C; C07; C07K; C07K16/00; C07K16/18; C07K16/28; C07K16/30; C07K16/3038; C07K16/32; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; C12Q1/683; C12Q1/6876; C12Q1/6883; C12Q1/6886; G01N33/531; C12Q2600/00; C12Q2600/156; Y; Y10; Y10S; Y10S436/00; Y10S436/811; Y10S436/813; Y10S530/00; Y10S530/808; Y10S530/809; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
88 words
A method of producing antibodies capable of detecting whether a wild-type allele has been mutated to a mutant allele, comprising: a. isolating a segment of DNA which includes a nucleotide substitution which distinguishes the wild-type allele from the mutant allele; b. creating an antigen capable of causing, upon immuniation into a host, production of antibodies specific for protein expressed by the wild-type allele or for protein expressed by the mutant allele, but not by both; and c. immunizing a host under conditions whereby said host produces said antibodies.
Claim 2
Independent Claim
88 words
A method of producing antibodies capable of detecting whether a wild-type allele has been mutated to a mutant allele, comprising: a. isolating a segment of DNA which includes a nucleotide substitution which distinguishes the wild-type allele from the mutant allele; b. creating an antigen capable of causing, upon immuniation into a host, production of antibodies specific for protein expressed by the wild-type allele or for protein expressed by the mutant allele, but not by both; and c. immunizing a host under conditions whereby said host produces said antibodies.
Claim 3
Dependent Claim
19 words
A method of claim 1 wherein said wild-type allele comprises a proto-oncogene and said mutant allele comprises an oncogene.
Claim 4
Dependent Claim
12 words
A method of claim 2 wherein said proto-oncogene comprises a ras proto-oncogene.
Claim 5
Dependent Claim
16 words
A method of claim 3 wherein said ras proto-oncogene comprises a proto-oncogene for human bladder cancer.
Claim 6
Dependent Claim
24 words
A method of claim 4 wherein said segment of DNA includes the codon for Gly 12 in the p21 protein expressed from said proto-oncogene.
Claim 7
Dependent Claim
16 words
A method of claim 5 wherein said antigen comprises an oligopeptide coupled to a polypeptide carrier.
Claim 8
Dependent Claim
20 words
A method of claim 6 wherein an antibody-producing cell is selected from said host and employed to produce monoclonal antibody.
Claim 9
Dependent Claim
33 words
A method of claim 7 wherein said antibody-producing cell selected is fused with a cell having immortality, to produce a fused cell hybrid which has immortality and is capable of producing monoclonal antibody.
Claim 10
Dependent Claim
12 words
Antibodies produced by a method of claim 1, 3, 6 or 8.
Claim 11
Independent Claim
200 words
A method of producing antibody capable of detecting carcinogenesis caused by mutation of a proto-oncogene into an oncogene, comprising: a. isolating a segment of DNA, the nucleotide sequence of which is different in said proto-oncogene from the nucleotide sequence of said oncogene, the difference comprising a nucleotide substitution; b. determining the nucleotide sequence of the proto-oncogene and the nucleotide sequence of the oncogene; c. deducing the amino acid sequence of the protein encoded by the proto-oncogene and the amino acid sequence of the protein encoded by the oncogene; d. determining the segment of the amino acid sequence which is different in the two encoded proteins and is critical for their difference in function; e. synthesizing an oligopeptide which includes an amino acid sequence present in the segment of the protein encoded by the oncogene but not present in the corresponding segment of the protein encoded by the proto-oncogene and critical for the difference in function of the two proteins; f. synthesizing an oligopeptide of the corresponding region of the oncogene-encoded protein; g. immunizing a host with the oligopeptide corresponding to an amino acid sequence encoded by said oncogene, under conditions whereby cells of said host produce antibody against said segment.
Claim 12
Independent Claim
200 words
A method of producing antibody capable of detecting carcinogenesis caused by mutation of a proto-oncogene into an oncogene, comprising: a. isolating a segment of DNA, the nucleotide sequence of which is different in said proto-oncogene from the nucleotide sequence of said oncogene, the difference comprising a nucleotide substitution; b. determining the nucleotide sequence of the proto-oncogene and the nucleotide sequence of the oncogene; c. deducing the amino acid sequence of the protein encoded by the proto-oncogene and the amino acid sequence of the protein encoded by the oncogene; d. determining the segment of the amino acid sequence which is different in the two encoded proteins and is critical for their difference in function; e. synthesizing an oligopeptide which includes an amino acid sequence present in the segment of the protein encoded by the oncogene but not present in the corresponding segment of the protein encoded by the proto-oncogene and critical for the difference in function of the two proteins; f. synthesizing an oligopeptide of the corresponding region of the oncogene-encoded protein; g. immunizing a host with the oligopeptide corresponding to an amino acid sequence encoded by said oncogene, under conditions whereby cells of said host produce antibody against said segment.
Claim 13
Independent Claim
120 words
A method of producing antibodies specific for a protein encoded by an oncogene or a protein encoded by its corresponding proto-oncogene, but not for both, comprising the steps of: a. isolating a nucleotide sequence present in the oncogene or present in its corresponding proto-oncogene, but not in both the oncogene and the corresponding proto-oncogene; b. producing an antigen encoded by the nucleotide sequence present in the oncogene or by the nucleotide sequence present in the corresponding proto-oncogene; and c. immunizing an appropriate host with the antigen encoded by the nucleotide sequence present in the oncogene or with the antigen encoded by the nucleotide sequence present in the corresponding proto-oncogene, under conditions whereby the host produces antibodies specific for the antigen.
Claim 14
Independent Claim
120 words
A method of producing antibodies specific for a protein encoded by an oncogene or a protein encoded by its corresponding proto-oncogene, but not for both, comprising the steps of: a. isolating a nucleotide sequence present in the oncogene or present in its corresponding proto-oncogene, but not in both the oncogene and the corresponding proto-oncogene; b. producing an antigen encoded by the nucleotide sequence present in the oncogene or by the nucleotide sequence present in the corresponding proto-oncogene; and c. immunizing an appropriate host with the antigen encoded by the nucleotide sequence present in the oncogene or with the antigen encoded by the nucleotide sequence present in the corresponding proto-oncogene, under conditions whereby the host produces antibodies specific for the antigen.
Claim 15
Independent Claim
95 words
A method of producing antibodies capable of determining whether a proto-oncogene has been mutated to its corresponding oncogene, comprising the steps of: a. producing a DNA fragment having the nucleotide sequence of DNA of the proto-oncogene which is mutated to produce the nucleotide sequence of the oncogene; b. producing a protein encoded by the nucleotide sequence of the oncogene, the protein capable of causing, upon introduction into an appropriate host, production of antibodies specific for protein encoded by the oncogene, but not for both; and c. immunizing a host, under conditions appropriate for producing antibodies.
Claim 16
Independent Claim
95 words
A method of producing antibodies capable of determining whether a proto-oncogene has been mutated to its corresponding oncogene, comprising the steps of: a. producing a DNA fragment having the nucleotide sequence of DNA of the proto-oncogene which is mutated to produce the nucleotide sequence of the oncogene; b. producing a protein encoded by the nucleotide sequence of the oncogene, the protein capable of causing, upon introduction into an appropriate host, production of antibodies specific for protein encoded by the oncogene, but not for both; and c. immunizing a host, under conditions appropriate for producing antibodies.
Claim 17
Dependent Claim
21 words
A method of claim 12 wherein a DNA fragment having the nucleotide sequence of the ras oncogene is produced in (a).
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-11-22 | 1992-05-23 | 1992-11-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-11-22 | 1996-05-23 | 1996-11-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-11-22 | 2000-05-23 | 2000-11-22 | $7700.00 | Window Closed / Lapsed |