US4405601A
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US
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Extraction and purification of biologically active lymphokines
Assignee: PAPERMASTER BENJAMIN W
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Filing: 1981-05-21
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Granted: 1983-09-20
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Expires: 2001-05-21
Abstract
Abstract A biologically active lymphokine fraction which exhibits macrophage activation activity and lymphotoxin activity is prepared from large scale supernatant culture fluids of human lymphoblastoid cultured cell lines grown in the presence of human serum to minimize antigenicity. Growth of lymphoblastoid cells from human lymphoblastoid cultured cell lines is effected and the supernatant culture fluids are harvested, concentrated and clarified. The supernatant culture fluids are then extracted with trichloroacetic acid, guanidine hydrochloride, sodium dodecyl sulfate or perchloric acid as the extracting solvent followed by removal of the solvent. Further purification of the lymphokine fraction can be achieved by treatment with guanidine hydrochloride or sodium dodecyl sulfate as a dissociating solvent and by subjecting the lymphokine fraction to gel filtration, sodium dodecyl sulfate gel electrophoresis and/or high pressure liquid chromatography.
Inventors
- 👤 John E. McEntire
- 👤 Ben W. Papermaster
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 09:46
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/555; C07K14/57; C07K1/00; C07K1/107; C07K1/113; C07K1/1136; C07K14/525; A; A61; A61K; A61K38/00; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/948
Claims Analysis
Total Claims: 28
Claim 1
Independent Claim
95 words
In a method for the extraction and purification of biologically active lymphokines from large scale culture supernatants comprising the steps of effecting the growth of lymphoblastoid cells from human lymphoblastoid cultured cell lines, and thereafter harvesting, concentrating and clarifying the resulting supernatant culture fluids, the improvement which comprises extracting said supernatant culture fluids with a solvent selected from the group consisting of trichloroacetic acid, guanidine hydrochloride, sodium dodecyl sulfate and perchloric acid and removing said solvent to produce a lymphokine fraction which exhibits the capability of inducing cell-mediated immunity reactions and tumor regression in mammals.
Claim 2
Dependent Claim
95 words
In a method for the extraction and purification of biologically active lymphokines from large scale culture supernatants comprising the steps of effecting the growth of lymphoblastoid cells from human lymphoblastoid cultured cell lines, and thereafter harvesting, concentrating and clarifying the resulting supernatant culture fluids, the improvement which comprises extracting said supernatant culture fluids with a solvent selected from the group consisting of trichloroacetic acid, guanidine hydrochloride, sodium dodecyl sulfate and perchloric acid and removing said solvent to produce a lymphokine fraction which exhibits the capability of inducing cell-mediated immunity reactions and tumor regression in mammals.
Claim 3
Dependent Claim
33 words
The method as set forth in claim 1 wherein said lymphokine fraction is further purified by treatment with a dissociating solvent selected from the group consisting of guanidine hydrochloride and sodium dodecyl sulfate.
Claim 4
Dependent Claim
16 words
The method as set forth in claim 1 wherein said solvent is removed by gel filtration.
Claim 5
Dependent Claim
38 words
The method as set forth in claim 1 wherein said lymphokine fraction is subjected to further purification by a procedure selected from the group consisting of gel filtration, sodium dodecyl sulfate gel electrophoresis and high pressure liquid chromatography.
Claim 6
Dependent Claim
18 words
The method as set forth in claim 1 wherein the concentration of trichloroacetic acid is approximately 10% w/v.
Claim 7
Dependent Claim
17 words
The method as set forth in claim 2 wherein said dissociating solvent is 6 molar guanidine hydrochloride.
Claim 8
Dependent Claim
19 words
The method as set forth in claim 2 wherein said dissociating solvent is a 1% sodium dodecyl sulfate solution.
Claim 9
Independent Claim
86 words
The method of producing lymphokine fraction which exhibits the capability of inducing cell-mediated immunity reactions and tumor regression in mammals by the extraction and purification of biologically active lymphokines from large scale culture supernatants comprising the steps of effecting the growth of lymphoblastoid cells from human lymphoblastoid cultured cell lines, harvesting, concentrating and clarifying the resulting supernatant culture fluids, extracting said supernatant fluids with a solvent selected from the group consisting of trichloroacetic acid, guanidine hydrochloride, sodium dodecyl sulfate and perchloric acid, and removing said solvent.
Claim 10
Independent Claim
86 words
The method of producing lymphokine fraction which exhibits the capability of inducing cell-mediated immunity reactions and tumor regression in mammals by the extraction and purification of biologically active lymphokines from large scale culture supernatants comprising the steps of effecting the growth of lymphoblastoid cells from human lymphoblastoid cultured cell lines, harvesting, concentrating and clarifying the resulting supernatant culture fluids, extracting said supernatant fluids with a solvent selected from the group consisting of trichloroacetic acid, guanidine hydrochloride, sodium dodecyl sulfate and perchloric acid, and removing said solvent.
Claim 11
Dependent Claim
33 words
The method as set forth in claim 8 wherein said lymphokine fraction is further purified by treatment with a dissociating solvent selected from the group consisting of guanidine hydrochloride and sodium dodecyl sulfate.
Claim 12
Dependent Claim
16 words
The method as set forth in claim 8 wherein said solvent is removed by gel filtration.
Claim 13
Dependent Claim
38 words
The method as set forth in claim 8 wherein said lymphokine fraction is subjected to further purification by a procedure selected from the group consisting of gel filtration, sodium dodecyl sulfate gel electrophoresis and high pressure liquid chromatography.
Claim 14
Dependent Claim
18 words
The method as set forth in claim 8 wherein the concentration of trichloroacetic acid is approximately 10% w/v.
Claim 15
Dependent Claim
17 words
The method as set forth in claim 9 wherein said dissociating solvent is 6 molar guanidine hydrochloride.
Claim 16
Dependent Claim
19 words
The method as set forth in claim 9 wherein said dissociating solvent is a 1% sodium dodecyl sulfate solution.
Claim 17
Independent Claim
69 words
The method of inducing cell-mediated immunity reactions and tumor regression in mammals comprising administering thereto a lymphokine fraction produced by effecting the growth of lymphoblastoid cells from human lymphoblastoid cultured cell lines, harvesting, concentrating and clarifying the resulting supernatant culture fluids, extracting aid supernatant culture fluids with a solvent selected from the group consisting of trichloroacetic acid, guanidine hydrochloride, sodium dodecyl sulfate and perchloric acid, and removing said solvent.
Claim 18
Independent Claim
69 words
The method of inducing cell-mediated immunity reactions and tumor regression in mammals comprising administering thereto a lymphokine fraction produced by effecting the growth of lymphoblastoid cells from human lymphoblastoid cultured cell lines, harvesting, concentrating and clarifying the resulting supernatant culture fluids, extracting aid supernatant culture fluids with a solvent selected from the group consisting of trichloroacetic acid, guanidine hydrochloride, sodium dodecyl sulfate and perchloric acid, and removing said solvent.
Claim 19
Dependent Claim
34 words
The method as set forth in claim 15 wherein said lymphokine fraction has been further purified by treatment with a dissociating solvent selected from the group consisting of guanidine hydrochloride and sodium dodecyl sulfate.
Claim 20
Dependent Claim
16 words
The method as set forth in claim 15 wherein said solvent is removed by gel filtration.
Claim 21
Dependent Claim
39 words
The method as set forth in claim 15 wherein said lymphokine fraction has been subjected to further purification by a procedure selected from the group consisting of gel filtration, sodium dodecyl sulfate gel electrophoresis and high pressure liquid chromatography.
Claim 22
Dependent Claim
18 words
The method as set forth in claim 15 wherein the concentration of trichloroacetic acid is approximately 10% w/v.
Claim 23
Dependent Claim
17 words
The method as set forth in claim 16 wherein said dissociating solvent is 6 molar guanidine hydrochloride.
Claim 24
Dependent Claim
19 words
The method as set forth in claim 16 wherein said dissociating solvent is a 1% sodium dodecyl sulfate solution.
Claim 25
Dependent Claim
15 words
The method as set forth in claim 15 wherein said lymphokine fraction is administered intravenously.
Claim 26
Dependent Claim
15 words
The method as set forth in claim 15 wherein said lymphokine fraction is administered intradermally.
Claim 27
Dependent Claim
15 words
The method as set forth in claim 15 wherein said lymphokine fraction is administered subcutaneously.
Claim 28
Dependent Claim
19 words
The method as set forth in claim 15 wherein said lymphokine fraction is administered as an adjuvant to chemotherapy.
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) | 1986-09-19 | 1987-03-21 | 1987-09-20 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-09-19 | 1991-03-21 | 1991-09-20 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-09-19 | 1995-03-21 | 1995-09-20 | $7700.00 | Window Closed / Lapsed |