US4446237A
Expired / Lapsed
US
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Method for detection of a suspect viral deoxyribonucleic acid in an acellular biological fluid
Assignee: Life Technologies Inc
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Filing: 1981-03-27
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Granted: 1984-05-01
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Expires: 2001-03-27
Abstract
Abstract There is disclosed a method for evaluating acellular biological fluid for viral DNA, such as DNA of the Hepatitus-B virus, whereby the acellular biological fluid is treated to immobilize in denatured form the DNAs including the suspect viral DNA on a solid support or substrate with the resulting solid substrate being contacted with a solution including radioisotropically-labeled viral denatured DNA followed by analysis of solid substrate for radioisotopically-labeled suspect viral renatured DNA.
Inventors
- 👤 Mark S. Berninger
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 04:07
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/58; G01N33/60; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/70
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
103 words
A method for evaluating an acellular biological fluid as drawn from a subject for the presence of a suspect viral DNA thereby obviating in vivo or tissue culture techniques, consisting essentially of: (a) treating said acellular biological fluid to immobilize on a solid substrate in denatured form said suspect viral DNA present in said acellular biological fluid; (b) contacting said solid substrate with a radioisotopically-labeled suspect viral denatured DNA probe to renature said immobilized suspect viral DNA present on said substrate to form a radioisotopically-labeled renatured DNA; and (c) evaluating the product of step (b) for the presence of said radioisotopically-labeled renatured DNA.
Claim 2
Independent Claim
103 words
A method for evaluating an acellular biological fluid as drawn from a subject for the presence of a suspect viral DNA thereby obviating in vivo or tissue culture techniques, consisting essentially of: (a) treating said acellular biological fluid to immobilize on a solid substrate in denatured form said suspect viral DNA present in said acellular biological fluid; (b) contacting said solid substrate with a radioisotopically-labeled suspect viral denatured DNA probe to renature said immobilized suspect viral DNA present on said substrate to form a radioisotopically-labeled renatured DNA; and (c) evaluating the product of step (b) for the presence of said radioisotopically-labeled renatured DNA.
Claim 3
Dependent Claim
15 words
The method as defined in claim 1 wherein said acellular biological fluid is human serum.
Claim 4
Dependent Claim
16 words
The method as defined in claim 1 wherein said acellular biological fluid is cerebral spinal fluid.
Claim 5
Independent Claim
21 words
The method as defined in claims 1 or 2 wherein said suspect viral DNA is DNA of the infectious agent Hepatitis-B.
Claim 6
Independent Claim
20 words
The method as defined in claims 1 or 3 wherein said suspect viral DNA is Herpes Simplex Virus Type 1.
Claim 7
Independent Claim
33 words
The method as defined in claims 1, 2 or 3 wherein said acellular biological fluid is treated with an organic solvent prior to step (a) to remove protein matter in an organic phase.
Claim 8
Dependent Claim
19 words
The method as defined in claim 6 wherein said evaluation of step (b) is effected by scintillation spectrometric technique.
Claim 9
Dependent Claim
18 words
The method as defined in claim 6 wherein said evaluation of step (c) is effected by autoradiographic technique.
Claim 10
Dependent Claim
14 words
The method as defined in claim 6 wherein said solid substrate is pure nitrocellulose.
Claim 11
Dependent Claim
20 words
The method as defined in claim 6 wherein step (a) includes the use of a slight vacuum to effect immobilization.
Claim 12
Independent Claim
191 words
The method for evaluating an acellular biological fluid as drawn from a subject having normal DNA for the presence of a suspect viral DNA, consisting essentially of: (a) contacting said acellular biological fluid with a detergent and proteolytic enzyme; (b) extracting the product of step (a) with an organic solvent to form an essentially protein-free aqueous phase containing said normal DNA and suspect viral DNA present in said acellular biological fluid; (c) contacting said aqueous phase of step (b) with an alkaline to denature said normal DNA and suspect viral DNA present in said acellular biological fluid; (d) neutralizing the resulting solution of step (c); (e) applying the solution of step d) to a solid substrate to immobilize said denatured normal DNA and suspect viral DNA present in said acellular biological fluid thereto; (f) contacting said solid substrate of step (e) with a solution of radiolabeled suspect viral denatured DNA probe to renature said suspect viral denatured DNA present in said acellular biological fluid and immobilized on said solid substrate to form a radiolabeled suspect viral renatured DNA; and (g) evaluating said solid substrate for said radio-labeled suspect viral renatured DNA.
Claim 13
Independent Claim
191 words
The method for evaluating an acellular biological fluid as drawn from a subject having normal DNA for the presence of a suspect viral DNA, consisting essentially of: (a) contacting said acellular biological fluid with a detergent and proteolytic enzyme; (b) extracting the product of step (a) with an organic solvent to form an essentially protein-free aqueous phase containing said normal DNA and suspect viral DNA present in said acellular biological fluid; (c) contacting said aqueous phase of step (b) with an alkaline to denature said normal DNA and suspect viral DNA present in said acellular biological fluid; (d) neutralizing the resulting solution of step (c); (e) applying the solution of step d) to a solid substrate to immobilize said denatured normal DNA and suspect viral DNA present in said acellular biological fluid thereto; (f) contacting said solid substrate of step (e) with a solution of radiolabeled suspect viral denatured DNA probe to renature said suspect viral denatured DNA present in said acellular biological fluid and immobilized on said solid substrate to form a radiolabeled suspect viral renatured DNA; and (g) evaluating said solid substrate for said radio-labeled suspect viral renatured DNA.
Claim 14
Dependent Claim
31 words
The method as defined in claim 11 wherein step (a) is effected at a temperature of from 60° and 80° C. for a time period of from 1 to 10 hours.
Claim 15
Dependent Claim
19 words
The method as defined in claim 12 wherein temperature is 70° C. for a time period of 2 hours.
Claim 16
Dependent Claim
20 words
The method as defined in claim 11 wherein step (c) is effected at a pH of from 10 to 12.5.
Claim 17
Dependent Claim
12 words
The method as defined in claim 14 wherein said pH is 11.5.
Claim 18
Dependent Claim
35 words
The method as defined in claim 11 wherein said solid substrate of step (e) is baked at a temperature of from 60° to 100° C. for from 1 to 6 hours prior to step (f).
Claim 19
Dependent Claim
23 words
The method as defined in claim 11 or 16 wherein said solid substrate is treated to prevent further binding of nucleic acid thereto.
Claim 20
Dependent Claim
29 words
The method as defined in claim 11 wherein said solid substrate of step (f) is washed prior to step (g) to remove nonspecifically bound radiolabeled suspect viral denatured DNA.
Claim 21
Dependent Claim
15 words
The method as defined in claim 11 wherein step (g) is effected by scintillation spectrometry.
Claim 22
Dependent Claim
14 words
The method as defined in claim 11 wherein step (g) is effected by autoradiography.
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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) | 1987-05-01 | 1987-10-31 | 1988-05-01 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1991-05-01 | 1991-10-31 | 1992-05-01 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1995-05-01 | 1995-10-31 | 1996-05-01 | $7700.00 | Window Closed / Lapsed |