US4670390A
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Process for the immobilization of compounds comprising nucleophilic groups
Assignee: Reanal Finomvegyszergyar Rt
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Filing: 1983-08-24
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Granted: 1987-06-02
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Expires: 2003-08-24
Abstract
Abstract The invention relates to a process for the immobilization of compounds comprising nucleophilic groups. According to the process of the invention a polymer comprising an amido group is swollen in a buffer. For this purpose any buffer is suitable which contains no amino, sulfhydryl and/or hydroxy groups in the molecule. To the swollen polymer a solution of a quinone - preferably p-benzoquinone - in a water miscible organic solvent is added and the mixture is activated at 273°-343° K. for 0.5-48 hours - preferably for 24 hours. The unreacted quinone is washed out with an aqueous solution of a water miscible organic solvent and thereafter water and/or a buffer. To the purified gel at a pH value between 3 and 11 - preferably between 6 and 8 - a buffered solution of a compound comprising a nucleophilic group is added, the suspension is incubated at 260°-313° K. for 24 hours, the gel is separated and the non-bound compound comprising a nucleophilic group is removed by washing.
Inventors
- 👤 Zsuzsanna Antal nee Magyar
- 👤 Eva Beller
- 👤 Laszlo Boross
- 👤 Ivan Daroczi
- 👤 Miklos Kalman
- 👤 Imre Suto
- 👤 Bela Szajani
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 19:36
CPC Classifications
C; C07; C07H; C07H3/00; C08; C08F; C08F8/00; C08F8/28; C08F8/30; C08F8/32; C12; C12N; C12N11/00; C12N11/02; C12N11/08; C12N11/082; C12N11/087
Claims Analysis
Total Claims: 10
Claim 1
Independent Claim
263 words
A process for the immobilization of a macromolecular compound comprising a nucleophilic group which comprises the steps of: (a) swelling a polymer which contains an amido group in a buffer having a pH value between 3 and 11 wherein said buffer contains no amino, sulfhydryl or hydroxy group; (b) activating said polymer to react with said macromolecular compound by adding to said polymer a solution of a benzoquinone in an inert water-miscible organic solvent in such an amount that the final concentration of the benzoquinone is 2-100 millimole to form a covalent bond between the amido group of said polymer and said benzoquinone; (c) allowing the polymer treated according to step (b) with the solution of the benzoquinone in the inert water-miscible organic solvent to stand for a period of 0.5 to 48 hours at 273° to 343° K. to form a gel; (d) removing any non-reacted benzoquinone from the gel by washing the latter with a mixture of water and an inert water-miscible organic solvent and thereafter with distilled water, with a buffer or with both; (e) adding to the gel at a pH value between 3 and 11 a 3 to 100 mg/ml solution of said macromolecular compound to form a suspension; (f) incubating the suspension at 260° to 313° K. for 24 hours to immobilize said macromolecular compound; and (g) washing the suspension with a 0.1 molar sodium acetate buffer comprising 1.0 mole of sodium chloride and then with a 0.1 molar sodium bicarbonate solution to remove any unreacted macromolecular compound from said immobilized macromolecular compound prepared as a gel.
Claim 2
Dependent Claim
263 words
A process for the immobilization of a macromolecular compound comprising a nucleophilic group which comprises the steps of: (a) swelling a polymer which contains an amido group in a buffer having a pH value between 3 and 11 wherein said buffer contains no amino, sulfhydryl or hydroxy group; (b) activating said polymer to react with said macromolecular compound by adding to said polymer a solution of a benzoquinone in an inert water-miscible organic solvent in such an amount that the final concentration of the benzoquinone is 2-100 millimole to form a covalent bond between the amido group of said polymer and said benzoquinone; (c) allowing the polymer treated according to step (b) with the solution of the benzoquinone in the inert water-miscible organic solvent to stand for a period of 0.5 to 48 hours at 273° to 343° K. to form a gel; (d) removing any non-reacted benzoquinone from the gel by washing the latter with a mixture of water and an inert water-miscible organic solvent and thereafter with distilled water, with a buffer or with both; (e) adding to the gel at a pH value between 3 and 11 a 3 to 100 mg/ml solution of said macromolecular compound to form a suspension; (f) incubating the suspension at 260° to 313° K. for 24 hours to immobilize said macromolecular compound; and (g) washing the suspension with a 0.1 molar sodium acetate buffer comprising 1.0 mole of sodium chloride and then with a 0.1 molar sodium bicarbonate solution to remove any unreacted macromolecular compound from said immobilized macromolecular compound prepared as a gel.
Claim 3
Dependent Claim
17 words
A process defined in claim 1 wherein said macromolecular compound comprising a nucleophilic group is a protein.
Claim 4
Dependent Claim
27 words
The process defined in claim 1 wherein said macromolecular compound comprising a nucleophilic group is selected from the group consisting of bovine serum albumin, glucooxidase, and catalase.
Claim 5
Dependent Claim
19 words
The process defined in claim 1, wherein the step (a), the polymer containing an amido group is acrylamide-N,N'-methylene-bis-acrylamide copolymer.
Claim 6
Dependent Claim
280 words
A process for the immobilization of a low molecular weight compound comprising a nucleophilic group which comprises the steps of: (a) swelling a polymer which contains an amido group in a buffer having a pH value between 3 and 11 wherein said buffer contains no amino, sulfhydryl, or hydroxy group; (b) activating said polymer to react with said low molecular weight compound by adding thereto a solution of a benzoquinone in an inert water-miscible organic solvent in such an amount that the final concentration of the benzoquinone is 2-100 millimoles to form a covalent bond between the amido group of said polymer and said benzoquinone; (c) allowing the polymer treated according to step (b) with the solution of a benzoquinone in the inert water-miscible-organic solvent to stand for a period of 0.5 to 48 hours at 273° to 343° K. to form a gel; (d) removing any non-reacted benzoquinone from the gel by washing the latter with a mixture of water and an inert water-miscible organic solvent and thereafter with distilled water, with a buffer, or with both; (e) adding to the gel at a pH value between 3 and 11 a 0.001 to 1.0 molar solution of said low molecular weight compound comprising a nucleophilic group to form a suspension; (f) incubating the suspension at 260° to 313° K. for 24 hours to immobilize said low molecular weight compound comprising a nucleophilic group; and (g) washing the suspension with a 0.1 molar sodium acetate buffer comprising 1.0 mole of sodium chloride and then with a 0.1 molar sodium bicarbonate solution to remove any unreacted low molecular weight compound from said immobilized low molecular weight compound prepared as a gel.
Claim 7
Dependent Claim
280 words
A process for the immobilization of a low molecular weight compound comprising a nucleophilic group which comprises the steps of: (a) swelling a polymer which contains an amido group in a buffer having a pH value between 3 and 11 wherein said buffer contains no amino, sulfhydryl, or hydroxy group; (b) activating said polymer to react with said low molecular weight compound by adding thereto a solution of a benzoquinone in an inert water-miscible organic solvent in such an amount that the final concentration of the benzoquinone is 2-100 millimoles to form a covalent bond between the amido group of said polymer and said benzoquinone; (c) allowing the polymer treated according to step (b) with the solution of a benzoquinone in the inert water-miscible-organic solvent to stand for a period of 0.5 to 48 hours at 273° to 343° K. to form a gel; (d) removing any non-reacted benzoquinone from the gel by washing the latter with a mixture of water and an inert water-miscible organic solvent and thereafter with distilled water, with a buffer, or with both; (e) adding to the gel at a pH value between 3 and 11 a 0.001 to 1.0 molar solution of said low molecular weight compound comprising a nucleophilic group to form a suspension; (f) incubating the suspension at 260° to 313° K. for 24 hours to immobilize said low molecular weight compound comprising a nucleophilic group; and (g) washing the suspension with a 0.1 molar sodium acetate buffer comprising 1.0 mole of sodium chloride and then with a 0.1 molar sodium bicarbonate solution to remove any unreacted low molecular weight compound from said immobilized low molecular weight compound prepared as a gel.
Claim 8
Dependent Claim
19 words
The process defined in claim 5 wherein said low molecular weight compound is a carbohydrate or an amino acid.
Claim 9
Dependent Claim
17 words
The process defined in claim 6 wherein said low molecular weight compound is glucose or gamma-amino-butyric acid.
Claim 10
Dependent Claim
19 words
The process defined in claim 5 wherein in step (a) the polymer containing an amido group is acrylamide-N,N'-methylene-bis-acrylamide copolymer.
References Cited (Backward Citations)
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1990-06-01 | 1990-12-01 | 1991-06-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1994-06-01 | 1994-12-01 | 1995-06-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1998-06-01 | 1998-12-01 | 1999-06-02 | $7700.00 | Window Closed / Lapsed |