US4030995A
Expired / Lapsed
US
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Alkaline phosphatase isoenzyme determination
Assignee: Sigma Chemical Co
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Filing: 1976-07-13
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Granted: 1977-06-21
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Expires: 1996-07-13
Abstract
Abstract Alkaline phosphatase isoenzymes are separated electrophoretically in agarose gels, or cellulose acetate membranes, equilibrated in a solution containing a low ionic strength buffer and a non-ionic detergent, such as Triton X-100. The electrophoretically separated isoenzymes are developed colorimetrically using 5-bromo-4-chloro-3-indolyl phosphate with a transphosphorylating buffer. Zones of enzyme activity appear as sharply-defined brilliant blue bands. The method is capable of resolving liver, bone, placental, intestinal and bile isoenzymes.
Inventors
- 👤 William H. Starkweather
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-27 13:53
CPC Classifications
G; G01; G01N; G01N27/00; G01N27/26; G01N27/416; G01N27/447; C; C12; C12Q; C12Q1/00; C12Q1/007; Y; Y10; Y10S; Y10S435/00; Y10S435/814
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
79 words
In an electrophoretic process of determining alkaline phosphatase isoenzymes in a sample comprising a step of applying said sample to a support medium, a step of subjecting the support medium to a differential electric potential to separate said isoenzymes, and a step of rendering said isoenzymes detectable, the improvement wherein said support medium is chosen from the group consisting of agarose gels and cellulose acetate membranes, said support medium containing a buffer and a non-ionic detergent diffused through it.
Claim 2
Dependent Claim
79 words
In an electrophoretic process of determining alkaline phosphatase isoenzymes in a sample comprising a step of applying said sample to a support medium, a step of subjecting the support medium to a differential electric potential to separate said isoenzymes, and a step of rendering said isoenzymes detectable, the improvement wherein said support medium is chosen from the group consisting of agarose gels and cellulose acetate membranes, said support medium containing a buffer and a non-ionic detergent diffused through it.
Claim 3
Dependent Claim
20 words
The process of claim 1 wherein said non-ionic detergent has a molecular weight of from about 600 to about 2000.
Claim 4
Dependent Claim
35 words
The process of claim 1 wherein said buffer is a low ionic strength buffer, having an ionic strength of less than 0.05, and buffering said support medium to a pH of from 8 to 9.
Claim 5
Dependent Claim
18 words
The process of claim 1 wherein said buffer has an ionic strength from about 0.020 to about 0.025.
Claim 6
Dependent Claim
20 words
The process of claim 1 wherein said support medium is an agarose gel containing less than 3% agarose by weight.
Claim 7
Dependent Claim
21 words
The process of claim 5 wherein said agarose gel is equilibrated in a solution of said buffer and said non-ionic detergent.
Claim 8
Dependent Claim
25 words
The process of claim 1 wherein said support medium is a cellulose acetate gel equilibrated in a solution of said buffer and said non-ionic detergent.
Claim 9
Dependent Claim
92 words
An electrophoretic method capable of separating into well-defined, easily observed, discrete fractions at least the liver, bone, placental, intestinal and bile alkaline phosphatase isoenzymes in a biological fluid, said method comprising a step of applying a sample of said fluid to a support medium containing a buffer and a non-ionic detergent; imposing a differential electric potential across the support medium; and a subsequent dyeing step, said dyeing step comprising the addition of a phosphate ester of an indigo congener stain, an activating amount of a soluble magnesium salt and a transphosphorylating buffer.
Claim 10
Dependent Claim
92 words
An electrophoretic method capable of separating into well-defined, easily observed, discrete fractions at least the liver, bone, placental, intestinal and bile alkaline phosphatase isoenzymes in a biological fluid, said method comprising a step of applying a sample of said fluid to a support medium containing a buffer and a non-ionic detergent; imposing a differential electric potential across the support medium; and a subsequent dyeing step, said dyeing step comprising the addition of a phosphate ester of an indigo congener stain, an activating amount of a soluble magnesium salt and a transphosphorylating buffer.
Claim 11
Dependent Claim
22 words
The method of claim 8 wherein said support medium is chosen from the group consisting of agarose gels and cellulose acetate membranes.
Claim 12
Dependent Claim
21 words
The method of claim 9 wherein said medium is prepared by equilibration in a solution containing said buffer and said detergent.
Claim 13
Dependent Claim
20 words
The method of claim 9 wherein said non-ionic detergent has a molecular weight of from about 600 to about 2,000.
Claim 14
Dependent Claim
18 words
The method of claim 11 wherein said buffer has an ionic strength from about 0.020 to about 0.025.
Claim 15
Dependent Claim
17 words
The method of claim 9 wherein said method is also capable of separating placental alkaline phosphatase phenotypes.
Claim 16
Dependent Claim
13 words
The method of claim 9 wherein said transphosphorylating buffer is an amino alcohol.
Claim 17
Dependent Claim
21 words
The method of claim 14 wherein said transphosphorylating buffer is chosen from the group consisting of AMP, DEA, EAE and AMPD.
Claim 18
Dependent Claim
22 words
The method of claim 9 wherein said stain is chosen from the group consisting of soluble salts of 3-indolylphosphate and derivatives thereof.
Claim 19
Dependent Claim
17 words
The method of claim 16 wherein said stain is a soluble salt of a halogenated 3-indolyl phosphate.
Claim 20
Dependent Claim
26 words
The method of claim 9 wherein said medium is an agarose gel equilibrated in a solution containing a low ionic strength buffer and a non-ionic detergent.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1980-06-20 | 1980-12-20 | 1981-06-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1984-06-20 | 1984-12-20 | 1985-06-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1988-06-20 | 1988-12-20 | 1989-06-21 | $7700.00 | Window Closed / Lapsed |