RU2000809C1 - Process for isolating pregnancy-associated alfa-macroglobuline and alfa-glycoprotein from blood plasma - Google Patents

Abstract

Use in biology and medicine The essence of the method is first carried out by affinity chromatography on iminodiacetic acid agarose with a spacer and a-macrogobobulin is eluted from it, and then the material not adsorbed on said agarose is passed through iminodiacetic acid agarose without spacer; by its negative immunoaffinity chromatography on 4-beta-hydroxyethylsulfonip-2-amino-nizol-agarose with immobilized antibodies against serum proteins of men

Description

The invention relates to biological chemistry, in particular to biotechnology, and can be used in biology and medicine. Pregnancy-associated alpha-2-glycoprotein (ABH) and alpha-macroglobulin (MG) are homologous proteins, have the ability to suppress proteinase activity and potent immunomodulatory effects.

The purpose of the invention is to simplify the method of sequential extraction of MG and ABH from the blood plasma of pregnant women by reducing the number of cleaning steps and their overall duration, as well as obtaining high purity proteins.

The method is carried out as follows. At the first stage of purification, zinc-chelate chromatography was carried out on an iminodiacetic acid agaroea with a co-spacer consisting of 6 carbon atoms (Estar, a small enterprise in the Estonian SSR). ABH does not interact with this gel at all, while all MG is adsorbed on it. MG elution was carried out under a pH gradient of 7.0-5.0. After that, its purity exceeds 95%, and the yield is 39% (1.1 g from 1 l of blood plasma), which is significantly higher than in the prototype method. Proteins that freely pass through iminodiacetic agarose with a spacer, including ABH without dialysis, are passed through iminodiacetic agarose without a spacer. Elution of ABH is carried out in the same way as MG. ABH-containing fractions were passed without concentration through a column of 4- / 3-hydroxyethylsulfonyl-2-aminoanisolagarose with immobilized anti-male serum antibodies. The final yield of ABH having a purity of more than 95% is 30% (0.18 g from 1 liter of blood plasma), which is significantly greater than in the method; plototype

Example 1. Dry sodium chloride was added to 100 ml of blood plasma of pregnant women to a concentration of 1 m and it was titrated to pH 8.0. Plasma is then applied to a column (2.6 cm x 10 cm) containing 50 ml of iminodiacetic acid agarose with a spacer (6 carbon atoms). Iminodiacetic acid agarose is preliminarily converted to the zinc form, 200 ml of a 0.05 M solution of Trilon B, 200 ml of distilled water, 200 ml of a 0.05 M solution of zinc sulfate, 200 ml of distilled water, 200 ml 0 are passed successively through it. 02 M phosphate buffer. pH 8.0 containing 1 M sodium chloride. Then, after the introduction of blood plasma, the column is washed with the starting buffer and elution is carried out in a pH gradient of 7.0 -5 0.

0.02 M phosphate buffer pH 7.0 containing 1 M sodium chloride and 0.02 M phosphate-citrate buffer, pH 5.0, as well as 1 M sodium chloride. The total volume of both buffers is 300 ml. 5 ml fractions were determined by missile immunoelectrophoresis and then collected. Concentrated MG preparations are generally not administered, since the protein is removed from the iminodiacetic acid agarose by a fairly sharp peak.

Protein material unrelated to the specialized iminodiacetic agarose is applied without dialysis to a column (2.6 cm x 10 cm) containing 50 ml of iminodiacetic agarose without a spacer (Estar, a small enterprise in the Estonian SSR), which is in zinc form and pre-equilibrated with 0.02 M phosphate buffer, pH 8.0 with 1 M sodium chloride. The impurity proteins were removed from the gel with start buffer, then elution of agarose-bound proteins was carried out in a pH gradient of 7.0-5.0 by the described method. Fractions containing ABG were found by rocket immunoelectrophoresis, poured and applied to a column (1 cm x 20 cm) filled with 14 ml of 4- / -oxyethylsulfonyl-2-amino-anisole-agarose with attached affinity-purified goat antibodies against proteins blood serum of men. For the synthesis of the last sorbent, a mixture of male blood serum with a low level of ABH is used. Proteins from this mixture are attached to 4-fl hydroxyethylsulfonyl-2-aminoanisole-agarose by diazotization using the obtained sorbent antibodies are removed from the goat antiserum against all human serum proteins, these antibodies are immobilized on 4- / -oxyethylsulfonyl-2 - aminoanisole agarose by diazotirovani.

The final MG release after 10 experiments ranged from 38% to 42%, on average 39% (1.1 g from 1 liter of blood plasma), and ABH from 28% to 31%. an average of 30% (0.18 g of 1 liter of blood plasma). A complex of electrophoretic methods and various types of immunoelectrophoresis were used to assess the purity and natianity of the obtained ABG and MG preparations. amino acid analysis, reactions of proteins with various enzymes were emitted. According to the data of electrophoresis in the pore gradient of a polyacrylamide gel mol. m. MG is 720 KDa, ABG 360 KDa. In the study of protein preparations obtained by polyacrylamide gel electrophoresis in reducing

conditions with sodium dodecyl sulfate it is determined that mol. m of polypeptide chains, both MG and ABG, is 180 KDa. When staining polyacrylamide gels using highly sensitive methods, in particular impoegnioan silver, impurity proteins were not found in ABG and MG preparations. When the ampholytes have isoelectric focusing in an egarose gel-isoelectric point, the point of both proteins is in the pH zone 4.7, which corresponds to known data. In accordance with them are also found characteristics of amino acid and carbohydrate compositions. Testing of ABG and MG drugs using cross-sectional immunoelectrophoresis showed that the isolated proteins have alpha2-globulin mobility and do not contain impurities of other proteins. Recent data were obtained using heterological antisera (goat, ram, rabbit). The isolated proteins inhibited the proteolytic activity of plesmin, thrombin, collagenase, trypsin, and other proteinases in ratios close to equimolar, which indicates their high nativeness. Repeated immunization of rabbits with the preparations of these proteins during 1 year demonstrated their high purity: after each immunization, rabbits produced monospecific antibodies against these proteins. When exploring. research by methods of cross immunoelectrophoresis and immunodiffusion of the drug

ABH and anti-MG antiserum, as well as MG drug and anti-ABH antiserum, did not show any cross-reactivity. Recent results indicate the absence of any contamination of ABH and MG with each other. Thus, the obtained data indicate that the purity of ABG and MG exceeds 95% and their nativeness remains practically unchanged during the purification.

Example 2. To 100 ml of serum of retro-placental blood (retro-placental blood., Is the blood of pregnant women, which flows freely during childbirth and may contain small impurities of the blood of the fetus and amniotic fluid), dry sodium chloride is added to concentration of 1 M and titrated with a solution of trisubstituted sodium phosphate to a pH of 8.0. The further purification steps are also similar to those in Example 1. The MG yield and in 10 experiments ranged from 37 to 43%, primarily 39% (1.1 g out of 1 liter). ABG yield from 29% to 31%, an average of 30% (0.18 g per 1 liter). The entire complex of immunochemical and biochemical methods listed in Example 1 testified to the high native ability of ABH and MG, as well as to that. that their purity exceeded 95%.

(56) Sand O and ect Characterlzarion of human pregnancy zone protein, Comparison with Ruman alpha2-macroglobulln IIJ. Blol. Chemistry.-1985.-Vol.260.-P.15723-15735.

Claims (1)

The claims METHOD FOR ISOLATING ALPHA-MACROGLOBULIN AND ALPHA-GLYCOPROTEIN ASSOCIATED WITH PREGNANCY FROM BLOOD PLASMA By chromatographic separation, characterized by the fact that affinity chromatography is carried out on iminodiacetic acid, agarose albumin sparse, agarose, sparse, and they additionally pass through imino-diacetic acid agarose without a spacer, the alpha-2 glycoprotein associated with pregnancy is eluted with it, followed by purification of 4-betaoxyethylsulfonyl-2-aminoanoeol agarose with immobilized antibodies against male serum proteins.