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US4276378A
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High sensitivity assays for angiotensin converting enzyme
Assignee: VENTREX LABORATORIES Inc A CORP OF DE
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Filing: 1977-11-25
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Granted: 1981-06-30
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Expires: 1997-11-25
Abstract
Abstract A sensitive method for measuring angiotensin converting enzyme in clinical material such as serum, urine, and tissue biopsies. The method employs Hip His Leu as the substrate. The method exploits the finding that sulfate ion in the range of 0.75 through 1.1 M lowers the apparent Km for Hip His Leu from about 2.6×10 -3 M to about 1.8×10 -4 M. This 14-fold decrease in Km effectively permits a more sensitive assay with Hip His Leu. The assay may be fluorimetric or radioactive means to measure the presence of eznymatic reaction.
Inventors
- 👤 James W. Ryan
- 👤 Alfred Chung
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 16:01
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/37; Y; Y10; Y10S; Y10S530/00; Y10S530/802
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
111 words
A method for measuring the activity of angiotensin converting enzyme in a sample of clinical material, comprising: diluting said sample in a buffer comprising a stimulatory amount of sulfate ion, where said sulfate ion is in the concentration range of 0.1 M-1.1 M and stimulates the angiotensin converting enzyme hydrolysis of HipHisLeu; adding sufficient HipHisLeu to the buffer-diluted sample to provide a reaction mixture having a concentration of HipHisLeu prior to incubation in the range of 1×10 -10 M-2×10 -3 M; incubating the reaction mixture for a time sufficient to permit a measurable reaction to occur; and, measuring the amount of HisLeu produced by angiotensin converting enzyme catalyzed hydrolysis of HipHisLeu.
Claim 2
Independent Claim
111 words
A method for measuring the activity of angiotensin converting enzyme in a sample of clinical material, comprising: diluting said sample in a buffer comprising a stimulatory amount of sulfate ion, where said sulfate ion is in the concentration range of 0.1 M-1.1 M and stimulates the angiotensin converting enzyme hydrolysis of HipHisLeu; adding sufficient HipHisLeu to the buffer-diluted sample to provide a reaction mixture having a concentration of HipHisLeu prior to incubation in the range of 1×10 -10 M-2×10 -3 M; incubating the reaction mixture for a time sufficient to permit a measurable reaction to occur; and, measuring the amount of HisLeu produced by angiotensin converting enzyme catalyzed hydrolysis of HipHisLeu.
Claim 3
Dependent Claim
14 words
A method as in claim 1 wherein the sample of clinical material is urine.
Claim 4
Dependent Claim
19 words
A method according to claim 1 wherein the HipHisLeu concentration is in the range 1×10 -9 M-5×10 -4 M.
Claim 5
Dependent Claim
21 words
A method according to claim 1 wherein the sulfate ion concentration is in the range of 0.7 M to 1.1 M.
Claim 6
Dependent Claim
14 words
A method according to claim 1 wherein the buffer contains 0.1 M chloride ions.
Claim 7
Dependent Claim
35 words
A method according to claim 1 wherein the step of measuring the HisLeu comprises adding a fluorescence--inducing agent, then measuring the fluorescence intensity which is a function of the concentration of the HisLeu reaction product.
Claim 8
Dependent Claim
60 words
A method according to claim 1 wherein the HipHisLeu concentration is in the range from 1×10 -5 M to 2×10 -3 M, the sulfate concentration is 0.75 M, the buffer contains 0.1 M chloride ions, and the step of measuring the HisLeu comprises adding o-phthaldialdehyde and measuring the fluorescence intensity which is a function of the concentration of the HisLeu.
Claim 9
Independent Claim
123 words
A method for measuring the activity of angiotensin converting enzyme in a sample of clinical material, comprising: providing a reaction buffer comprising a stimulatory amount of sulfate ion, where said sulfate ion is in the concentration range of 0.1 M-1.1 M and stimulates the angiotensin converting enzyme hydrolysis of HipHisLeu; mixing said sample together with HipHisLeu in the reaction buffer to provide a reaction mixture, the HipHisLeu having a final concentration in the range of 1×10 -10 M-2×10 -3 M; incubating the reaction mixture in order to permit an angiotensin converting enzyme catalyzed reaction to proceed to a measurable extent; stopping the reaction by acidifying the reaction mixture; and, measuring the amount of Hip produced by angiotensin converting enzyme catalyzed hydrolysis of HipHisLeu.
Claim 10
Independent Claim
123 words
A method for measuring the activity of angiotensin converting enzyme in a sample of clinical material, comprising: providing a reaction buffer comprising a stimulatory amount of sulfate ion, where said sulfate ion is in the concentration range of 0.1 M-1.1 M and stimulates the angiotensin converting enzyme hydrolysis of HipHisLeu; mixing said sample together with HipHisLeu in the reaction buffer to provide a reaction mixture, the HipHisLeu having a final concentration in the range of 1×10 -10 M-2×10 -3 M; incubating the reaction mixture in order to permit an angiotensin converting enzyme catalyzed reaction to proceed to a measurable extent; stopping the reaction by acidifying the reaction mixture; and, measuring the amount of Hip produced by angiotensin converting enzyme catalyzed hydrolysis of HipHisLeu.
Claim 11
Dependent Claim
23 words
A method according to claim 8 wherein the concentration of HipHisLeu is in the range from 1×10 -9 M to 5×10 -4 M.
Claim 12
Dependent Claim
21 words
A method according to claim 8 wherein the sulfate ion concentration is in the range of 0.7 M to 1.1 M.
Claim 13
Dependent Claim
15 words
A method according to claim 8 wherein the reaction buffer contains 0.1 M chloride ions.
Claim 14
Dependent Claim
72 words
A method according to claim 8 wherein at least a portion of the HipHisLeu molecules are radioactively labeled in the Hip moiety, the Hip produced by angiotensin converting enzyme catalyzed hydrolysis of HipHisLeu is separated from the HisLeu produced by said hydrolysis and unreacted HipHisLeu in the reaction mixture, and the radioactivity of the Hip is measured in order to measure the activity of any angiotensin converting enzyme present in said sample.
Claim 15
Dependent Claim
104 words
A method according to claim 8 wherein the HipHisLeu concentration is in the range of 1×10 -9 M-5×10 -4 M, the sulfate ion concentration is 0.75 M, the reaction buffer contains 0.1 M chloride ion, at least a portion of the HipHisLeu molecules are radioactively labeled in the Hip moiety, and after stopping the reaction the Hip produced by angiotensin converting enzyme catalyzed hydrolysis of HipHisLeu is separated from the HisLeu produced by said hydrolysis and unreacted HipHisLeu in the reaction mixture and the radioactivity of the Hip is measured in order to measure the activity of any angiotensin converting enzyme in said sample.
Claim 16
Dependent Claim
12 words
A method according to claim 13 wherein the clinical material is urine.
Claim 17
Independent Claim
129 words
A method for measuring angiotensin converting enzyme activity in a sample of clinical material comprising: providing HipHisLeu as the substrate for angiotensin converting enzyme; providing a buffer adjusted for optimal angiotensin converting enzyme activity, said buffer containing added chloride ion to provide a final concentration in the range of 0.0 M-1.0 M and also containing an added stimulatory sulfate ion to provide a final concentration in the range of 0.1 M-1.1 M, said sulfate ion stimulates the angiotensin converting enzyme hydrolysis of HipHisLeu; producing a reaction mixture by mixing together the substrate and said sample in the buffer; incubating the reaction mixture to permit an angiotensin converting enzyme catalyzed reaction to occur; and, measuring the amount of any reaction product produced by angiotensin converting enzyme catalyzed hydrolysis of HipHisLeu.
Claim 18
Independent Claim
129 words
A method for measuring angiotensin converting enzyme activity in a sample of clinical material comprising: providing HipHisLeu as the substrate for angiotensin converting enzyme; providing a buffer adjusted for optimal angiotensin converting enzyme activity, said buffer containing added chloride ion to provide a final concentration in the range of 0.0 M-1.0 M and also containing an added stimulatory sulfate ion to provide a final concentration in the range of 0.1 M-1.1 M, said sulfate ion stimulates the angiotensin converting enzyme hydrolysis of HipHisLeu; producing a reaction mixture by mixing together the substrate and said sample in the buffer; incubating the reaction mixture to permit an angiotensin converting enzyme catalyzed reaction to occur; and, measuring the amount of any reaction product produced by angiotensin converting enzyme catalyzed hydrolysis of HipHisLeu.
Claim 19
Dependent Claim
29 words
A method according to claim 15 wherein the concentration of chloride ion is 0.0 M and the concentration of sulfate ion is in the range of 0.75 M-1.1 M.
Claim 20
Dependent Claim
14 words
A method according to claim 15 wherein the concentration of chloride is 0.1 M.
References Cited (Backward Citations)
US3832337A
Cited
US4137225A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1984-06-29 | 1984-12-29 | 1985-06-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1988-06-29 | 1988-12-29 | 1989-06-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1992-06-29 | 1992-12-29 | 1993-06-30 | $7700.00 | Window Closed / Lapsed |