SU1477439A1 - Method of producing modified silica vehicle for immobilizing biospesific ligands - Google Patents

Abstract

The present invention relates to methods for producing a modified silica carrier for immobilizing biospecific ligands and reduces nonspecific protein sorption and simplifies the method. Macroporous silica with a pore diameter of 50-230 nm is moistened with a 3-10% solution of comonomers in an organic solvent (6-22% by weight of silica), while the comonomers are N-nitrophenyl acrylate and acrylamide in a molar ratio of 1: 3 to 3: 1. Oligoethylene glycol dimethacrylate (TGM-3, TGM-13) is added to the mixture in an amount of 5-10% by weight of monomers and lauryl peroxide in an amount of 0.5-2% by weight of monomers. The reaction mixture is stirred, evacuated, heated to 70 ° C and kept for 3 hours. The product is washed with dimethylformamide, ethanol and dried. 1 tab.

Description

The invention relates to a process for the preparation of polymerization-modified silicas and can be used to immobilize bio-specific ligands.

The purpose of the invention is to reduce non-specific protein sorption and simplify the method.

Example 1. 7.1 g of acrylamide (0.1 mol) are dissolved in 5 g of dry dimethylformamide, 0.318 g of lauryl peroxide and 19.3 g (0.1 mol) of p- nitrophenyl acrylate. The solutions are mixed with stirring. Then 1.3 ml of TGM-3 (5% by weight of the monomers) are added to this solution. The prepared mixture is placed in pre-loaded 180 g of macroporous glass with a pore diameter of 2300 X and a specific surface of 28 m / g of a reactor with a volume of 0.01 m3. The reaction mixture is stirred for 1 5 minutes. Vacuum the solution and heat the reaction mixture to 70 V. R for 3 hours is carried out by modifying the silica with a copolymer. After the grafting of silica promn.kp was completed, hot dimethylformamide, Hbcc, was prepared with buffer pH 4.5, ethanol, h hrc. m and dried

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on lyophilic boil. The subsequent treatment of the modified criss-cease with a 25% -nm solution of aqueous ammonia gives a content of 40 μmol of p-nitrophenyl acrylate groups per 1 g of the terpentine phase. The course of the reaction is monitored spectrophotometrically at YaMax 405 nm, fixing the amount of yupogos nitrophenol.

II p m m e p 2. Synthesis of the carrier is carried out as in Example 1, but 21.3 g of acrylamide (0.3 mol) are taken for 19.3 g of p-nitrophenyl acrylate (0.1 mol), and lauryl peroxide is taken in the amount of 0 , 8 g, the content of p-nitrophenylacrylate groups is 20 µmol per 1 g of the solid phase.

Example 3. Synthesis of the carrier is carried out as in Example 1, but for 19.3 g, p-nitrophenyl acrylate (0.1 mol), 2.4 g (0.033 mol) of acrylamide are taken, and TGM-3 is added in the amount of 2.2 g (10% by weight of monomers). The content of n-nitrophenyl acrylate groups is 60 µmol per 1 g of the solid phase.

PRI me R 4. Synthesis of a carrier wire as in Example 1, but 35.9 5 g of acrylamide are taken for 19.3 g of p-nitrophenyl acrylate (0.1 mol). The content of n-nitrophenyl acrylate groups is 2 µmol per 1 g of the solid phase.

PRI me R 5. Synthesis of the carrier is carried out as in example 1, but 19.3 g

p-nitrophenyl acrylate (0.1 mol) without

root 1.4 g acrylamide. The content of p-nitrophenyl acrylate groups is 10 µmol per 1 g of the solid phase.

Example 6: Synthesis of the matrix was carried out as in Example 1, but taking 3.5 g of acrylamide and 19.6 g of p-nitrophenylacrylate, and instead of 1 ml of THM-3, 1 ml of THM-13 was added. The content of n-nitrophenyl acrylate groups is 80 µmol per 1 g of the solid phase.

Example 7. Modification of silica with acrylamide and p-nitrophenyl acrylate copolymer with a comonomer ratio of 3: 1.

The synthesis is carried out according to Example 2, but TGM-3 is added in an amount of 6 g (15% by weight of the monomers). The content of n-nitrophenyl acrylate groups is 30 µmol per gram of solid.

Example 8: In order to block the activated groups and then determine the non-specific protein sorption, 1 g of the carrier obtained by the proposed method is added

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and 10 ml of a 5% solution of ethanolamine in dimethylformamide and stirred for 24 hours with shaking. The ethanolamine solution is decanted; the inactivated H.HTPTPL is washed 3 times with 50 ml of 0.05 M sodium phosphate buffer (pH 7.0) containing O, M NaCl and placed in a column of size cm. Nonspecific sorption of α-chymotrypsin and albumin is determined in the indicated buffer while applying 3 mg of protein in 0.3 ml of buffer. Zoned at a rate of 0.5 ml / min. The sorption of chromium-C is determined under the conditions of a known method: 0.02 M phosphate buffer, pH 9.0. Column cm, protein sample 1 mg in 0.1 ml buffer. The content of the bed in the eluate is determined according to Coomassie, Table B presents the comparative results.

Example 9. Determination of the capacity of the activated carrier according to immunoglobulin G. To 1 ml of a 10% human immunoglobulin G solution was added 4 ml of bicarbonate buffer (0.3 M NaHTOj solution containing 0.1 M NaCl). To the resulting solution was added 1 g of the activated carrier, shaken and incubated for 24 hours at 4 ° C. The carrier is then washed on a porous filter with 50 ml of bicarbonate buffer, the filtrate is dialyzed against the same buffer, after which its volume and optical density are measured at А 280 nm (D,). 1 ml of a 10% immunoglobulin solution is diluted with bicarbonate buffer to a volume of filtrate and absorbance is measured at L 280 nm (Pd). The capacity of the activated carrier is calculated by the formula

with vl. yo

D

mr

five

The values of non-specific protein sorption on carriers according to examples 1-7 and the capacity of carriers for immunoglobulin G are presented in the table.

The carrier obtained by the proposed method allows immobilization of significant amounts of protein (up to 65 mg / g of carrier), has at the same time low non-specific protein adsorption (0.2-0.3 mg / g of carrier) and can serve as an initial basis for obtaining a large amount of protein. amounts of affinity sorbents. In addition, the carrier has all the advantages of hard K

Before scaffolds of chromatographic materials - high mechanical strength, non-swellability in organic solvents, resistance to the action of microorganisms and, therefore, can be used in massive columns with high pressure and high flow rate of eluent.

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when their molar ratio is (1-3):: (3-1) in the presence of a crosslinking agent — oligoethylene glycol dimethacrylate, and lauryl peroxide is used as the polymerization initiator.

Claims (1)

Invention Formula A method of preparing a modified silica carrier for immobilizing biospecific ligands, comprising treating the macroporous silica carrier with a solution of the comonomers of n-nitrophenylacrylate and acrylic acid amide followed by polymerization of the comonomers in the presence of an initiator, in order to reduce the specificity of the non-syphonic with the monomer. process simplification, silica treated with comonomer solution Editor N. Tupitsa Compiled by T. Chilikina Tehred M. Didyk Order 2188/10 Circulation 600 VNISh of the State Committee on Inventions and Discoveries at the State Committee on Science and Technology of the USSR 113035, Moscow, Zh-35, 4/5, Raushsk nab. Proofreader E. Lonchakova Subscription