SU1669918A1 - Method of sorbent preparation for affinity chromatography - Google Patents

Abstract

This invention relates to the production of cellulose derivatives that can be used as sorbents for affinity chromatography and biotechnology. The invention makes it possible to increase the mechanical strength of sorbents due to the fact that cellulose is precipitated from an emulsion of a 1% copper – ammonium solution of cellulose in a mixture of chloroform and benzene at their volume ratio of 1: 9, respectively, containing 0.13 - 0.26% polyoxyethylene sorbitan monooleate, acetic acid solution in acetone. The isolated product is treated with a 1.6% solution of sodium borohydride in pyridine for 4 to 8 hours, followed by treatment with an aqueous alkaline solution containing 0.01-0.05% by weight of epichlorohydrin and 0.01-0.04%. % sodium borohydride when heated. The product is then activated with an aqueous solution of sodium periodate and conjugated to the protein. 1 tab.

Description

This invention relates to the production of cellulose derivatives that can be used as sorbents for affinity chromatography used in biotechnology.

The purpose of the invention is to increase the mechanical strength of the sorbent.

Example 1.4.1 of freshly prepared Cu (OH) 2 is dissolved in 50 ml of 25% NHijOH. the solution is added to 5 g of cotton lint cellulose. After 20 hours, the volume of the mixture was adjusted to 500 ml with the same ammonia solution. The solution was clarified by centrifuging for 5 minutes at 4000 rpm, 50 ml of the obtained ammonia solution was placed in a 500 ml vessel containing 200 ml of a mixture of chloroform: benzene 1: 9 containing 0.26 msec.% (0, 6 ml, 08

g / cm polyoxyethylene sorbitan monooleate. A mixer is dipped into the vessel and emulsified for 4-5 minutes at 440 rpm. Emulsified cellulose is released through the lower process into a regenerating solution containing 500 ml of acetone and 50 ml of glacial acetic acid. After 8 minutes, the precipitated balls are separated and washed with 10 volumes of water.

Porous cellulosic beads are fractionated on nylon sieves and fractions containing cellulose particles of 70-200 µm in size are separated.

1 g (100 ml) of porous cellulose beads (PCS) is placed in a 500 ml round-bottom flask, equipped with a reflux condenser, 100 mg is added. pyridine containing 1.6 wt.% borohydride na

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three . and heated in a water bath for 8 hours. After that, the sorbent is washed on a Büchner funnel, first with acetone and then with distilled water until all traces of acetone disappear.

1 g (100 ml) of the PCR was washed once again on a glass filter with distilled water, transferred to a beaker, suspended in 70 ml of distilled water, and 20 ml of a 12% NaOH solution, 0.016% by weight (0.2 ml, , 18 g / cm) epichlorohydrin and 0.01 wt.% (20 mg) No. VSch. The beaker is placed in a water bath and the mixture is heated to 60 ° C for 2 hours, constantly stirring with a mechanical stirrer. After 2 hours, the product is washed with distilled water until neutral pH.

Then the sorbent is activated by treating with a 0.015 M solution of NaJCM for 18 h in the dark and again washed with water.

Next, the sorbent is treated with 0.1 M carbonate-bicarbonate buffer solution with a pH of 9.2 and pre-dialyzed protein is added to the immunosorbent (horse gamma ilobulin) to obtain the immunosorbent. The mixture is stirred for 2 hours at room temperature and incubated for 1 day at 4 ° C. The obtained immunosorbent is washed alternately with a 0.01 M solution of HCl in 0.15 M solution of NaCl and then with a 0.1 M boric borate buffer at pH 8.6. Free aldehyde groups are reduced.

by treating the immunosorbent with 0.1 M MaVSH in 0.15 M NaCI, and then washing it with 0.15 M NaCI solution, Amount of protein attached. Separate by bromophenol blue staining.

The conditions for the preparation and properties of the sorbent with different parameters of the method are presented in the table.

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Claims (1)

Invention Formula A method of producing a sorbent for affinity chromatography, including the precipitation of cellulose from a 1% emulsion copper-ammonia solution of cellulose in a mixture of chloroform and benzene with a volume ratio of 1: 9, respectively, containing 0.13-0.26 wt.% polyoxyethylene sorbitan monooleate, solution of acetic acid acids in acetone and treating the isolated product with a 1.6% solution of sodium borohydride in pyridine for 4-8 hours, followed by activation with an aqueous solution of sodium periodate and conjugation with protein, characterized in that, in order to increase the mechanical strength of the sorbent, before activation, the product is treated with an aqueous alkaline solution containing 0.01-0.05 wt.% epichlorohydrin and 0.01-0.04 wt.% sodium borohydride when heated. further use) The strength of the sorbent is determined by the height. The volume ratio of chloroform: benzene 1 is 86-94%. water column over the sorbent in the column with cm and see: 9, the concentration of sodium borohydride is 1.6 wt.%. Sorbent yield