RU2040274C1 - METHOD FOR PRODUCTION OF IMMUNOSORBENT FOR BINDING α- GROUP SPECIFIC AND β-AGGLUTININES - Google Patents

Abstract

FIELD: medicinal chemistry, immunology. SUBSTANCE: aminopropylated carrier is treated initially by poly(4-nitrophenylacrylate) or by poly(4-nitrophenylmethacrylate), then it is treated by aminoalkylglycoside of A(B) disaccharide; their quantities being 40-50 mg per 1 g of carrier and 2-10 micromoles respectively. EFFECT: simplifies the method, improves quality of sorbent.

Description

 The invention relates to medical chemistry and immunology, specifically to biospecific sorbents, which are solid carriers with synthetic oligosaccharides immobilized on them. Such sorbents are designed to remove antibodies with group specificity A and B / α and β-agglutinins / from blood, plasma, serum, and blood products, for example, when receiving anti-Rhesus diagnostic sera.

 The purpose of the invention is to simplify the method and improve the quality of the sorbent.

 PRI me R 1. Obtaining sorbent B.

 To 1 g of aminopropylated macroporous glass (MPS), a solution of 40 ml of poly / 4-nitrophenylacrylate / in 2 ml of DMF is added, the mixture is incubated, periodically shaking for 15 hours, washed with DMF. A solution of 3 mg / 7.5 μmol / 3-aminopropyl / -3-0- / α-D-galactopyranosyl / β-D-galactopyranoside in 2 ml of DMF is added to the obtained activated carrier, incubated for one day, periodically shaking, then 0.4 ml of ethanolamine is added, after 2 hours the DMF sorbent is washed, then with ethanol, ether and dried. The resulting sorbent contains 7.5 μmol, disaccharide ligand per 1 g of MPS. Before use, the resulting sorbent is washed with water, then with a working buffer solution.

 PRI me R 2. Obtaining sorbent A.

 A solution of 50 mg of poly / 4-nitrophenylmethacrylate / MM 4 kD in 2 ml of DMF is added to 1 g of aminopropylated MPS, it is kept, periodically shaking for 15 hours, washed with DMF. A solution of 4 mg / 7.5 μmol / 3-aminopropyl / -3-0- / α -DN-acetylgalactosaminyl / β-D-galactopyranoside in 2 ml of water is added to the obtained activated carrier, incubated for one day, shaking periodically, then 1 ml of a 20% ammonia solution is added, after 2 hours the sorbent is washed with water, then with acetone, hexane, and dried. The resulting sorbent contains 10 μmol of disaccharide ligand A per 1 g of MPS.

 PRI me R 3. Obtaining sorbent B.

 A solution of 45 mg of poly / 4-nitrophenylacrylate / MM 80 kD in 2 ml of DMF is added to 1 g of aminopropylated MPS, and it is kept, periodically shaking for 15 h, washed with DMSO. A solution of 0.8 mg / 2 μmol / / 3-aminopropyl / -3-0- / α-D-galactopyranosyl / β-D-galactopyranoside in 2 ml DMSO is added to the obtained activated carrier, incubated for 24 hours, periodically shaken, then add 0.5 g of Tris / base /, stand, shaking, 15 h, washed with DMSO, acetone, pentane, dried. The resulting sorbent contains 2 μmol of disaccharide ligand B per 1 g of MPS.

 PRI me R 4. The determination of the degree of desorption of the ligand from the sorbent.

 Polymer-free option / sorbent F /.

To 0.5 g of aminopropyl MEAs in 5 ml of 0.1 M bicarbonate buffer / pH 8.4 / was added a solution of 1.2 mg of FITC in 0.1 ml DMF was stirred for 15 hours at ²⁰ ° C, washed with water.

 Polymer option / sorbent FP /.

To 0.5 activated polymer MEAs capacity 100 pmol / g in 5 ml of 0.1 M bicarbonate buffer solution was added 1.2 mg of FITC in DMF was kept for 15 hours at ²⁰ ° C, is added 0.2 ml of ethanolamine after 2 h washed with water.

 Elution.

 The following solutions are passed through sorbents Ф and ФП during the day in a closed loop: phosphate-saline buffer / pH 7.4 /; 1% ammonia solution; glycine-hydrochloric acid buffer / pH 2.8 /.

 Equal volumes of eluates were selected, and the fluorescence of the resulting solutions was measured. At all pH values, the sorbent AF showed less fluorescence, the difference was 10-50 times.

 PRI me R 5. Removal of antibodies with group specificity In from the blood serum of donors.

5 mg of dry sorbent prepared according to Example 1, washed with water, then phosphate-buffered saline / FSB /, maintained at 4-20 ^{° C} with 0.5 ml of donor serum of blood group A or 0 for 1-3 hours. Titer serum was determined before and after sorption by the standard method of salt agglutination.

 Antibody desorption is carried out by washing several times the sorbent with a buffer solution of 1% ammonia / pH 9 / or glycine-hydrochloric acid buffer / pH 3 /, then washing with water, alcohol, and the sorbent is dried. After that, the sorbent can be stored or reused for sorption of antibodies. Nonspecific sorption was not observed at all or amounted to 1 step.

 PRI me R 6. Removal of antibodies with group specificity And from the blood serum of donors.

50 mg of dry sorbent A obtained in Example 2, washed with water, then FSB, maintained at 4-20 ^{° C} with 0.5 ml of blood donors of blood group B or O serum for 1-3 hours. The serum titer was determined before and after sorption by the standard method of salt agglutination. Desorption was carried out as described in example 5.

 The principal advantage of the proposed method from the prototype is that the ligands are not attached directly to the carrier, but with the help of a polymer, which is flexible polymer, which made it possible to replace trisaccharides with disaccharides, which are much easier to synthesize (9 stages instead of 15).

 Unlike used in the prototype of acyl azides, aminoalkyl glycosides of disaccharides are stable. Due to the multipoint binding, the polymer (and with it the ligand covalently attached to it) is approximately two orders of magnitude stronger attached to the carrier than when attached without a polymer. This is shown in specially conducted experiments with a fluorescently labeled ligand attached by two compared methods / many hours of elution in a closed loop was carried out at various pH values /. The polymer molecules uniformly distributed on the surface of the MPS significantly prevent the destruction of blood elements during sorption. In addition, the sorbent obtained by the proposed method can be used repeatedly, more than 9 times, after regeneration with an acidic / pH 3 / or alkaline / pH 9 / buffer solution. Sorbents withstand long-term storage of more than 1.5 years in dry form, sterilization with alcohol. The synthesis is stably reproduced.

 A direct comparison of the sorbents obtained by this method with the sorbents (Canada), i.e., the prototype, showed their better sorption activity, less non-specific sorption, greater durability / number of regeneration cycles /.

Nonspecific sorption, i.e., sorption of anti-A antibodies by sorbent B / or vice versa / was practically not observed. At the same time, specific sorption is 3-4 steps / step-logarithm of ₂ serum titers, i.e. a decrease in titer by 4 steps means a decrease in antibody concentration by 16 times /, in the prototype method, specific sorption is 2-3 steps.

Claims (1)

 METHOD FOR PRODUCING AN IMMUNOSORBENT FOR BINDING GROUP-SPECIFIC ANTIBODIES of α- and β-AHGLUTININS by attaching a group-specific blood oligosaccharide A (B) to a macroporous carrier, characterized in that, in order to simplify the method and improve the quality of the aminoporophenyl sorbent 4 sorbent ) or poly (4-nitrophenylmethacrylate) mol. m. 4 80 kDa in an amount of 40 to 50 mg per 1 g of carrier in dimethylformamide or dimethyl sulfoxide, and then with an aminoalkyl glycoside of disaccharide A (B) in an amount of 2 10 μmol of disaccharide per 1 g of carrier in the same solvents or water, then a hydrophilic amine or ammonia.