RU2288008C1 - Method for obtaining secretory immunoglobulin a out of biological liquid in animals - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: the present innovation deals with obtaining secretory immunoglobulin A (S-IgA) applied for evaluating immune status in animals. The method deals with biochemical treatment of animals' biological liquid, separating a target product due to gel-filtration followed by freezing, moreover, biological liquid should be centrifuged at 1000-1700 g for about 15-30 min followed by dialysis against 0.02 M Tris-HCL pH 8.0-8.2 for about 1.5-2.5 h and gel-filtration upon Sephacryl S-400 with 0.015-0.02 M Tris-HCL buffer at pH being 8.0-8.2 and with 0.4-0.9 M sodium chloride due to separating the second fraction of proteins under the control of immunoelectrophoresis at applying antisera to animal proteins, as for the target product it should be obtained due to concentrating the fractions of proteins in polyethylene glycol-40000 to increase the content of immunoglobulin A in the target product by 3-6-fold in comparison to its content in initial biological liquid in animals. The innovation provides higher output and quality of the target product and more accelerated rate of method's implementation.

EFFECT: higher efficiency.

3 ex

Description

The invention relates to veterinary medicine and can be used to obtain secretory immunoglobulin A (S-IgA), used to assess the immune status of the animal.

A known method of producing IgA from animal biological fluid by biochemical treatment of biological fluid, separation of the target product by gel filtration in combination with ion exchange chromatography (Transactions of VIEV, 2003, vol. 73, pp. 197-200). According to a known method, 3-fold salting out is carried out with ammonium sulfate and treatment with zinc sulfate to obtain a gamma globulin fraction, which is subjected to gel filtration and ion exchange chromatography. Gel filtration was carried out on Sephacryl S-300 in 0.05 M Tris-HCl buffer pH 8.0. Analysis of fractions for the content of IgA is carried out by immunoelectrophoresis using antisera to all cattle and sheep proteins. The fractions of the second and ascending part of the third peaks containing IgA are combined, concentrated and subjected to ion exchange chromatography in a gradient of pH and ionic strength (from 0.01 M pH 7.4 to 0.13 M pH 6.0 phosphate buffer). IgA was obtained in the eluate of 3 peaks while passing 0.06 M phosphate buffer pH 6.5. Fractions containing IgA are concentrated and examined for protein content and immunochemical purity by immunoelectrophoresis and SDS-PAGE.

The disadvantages of this method are the low yield and quality of the target product, the duration and difficulty of obtaining immunoglobulin A from the biological fluid of animals.

The objective of the claimed technical solution is to increase the yield and quality of the target product, as well as speeding up the method.

The problem is solved in a method for producing immunoglobulin A from animal biological fluid, separation of the target product by gel filtration, followed by freezing of the biological fluid by centrifuging at 1000-1700 g for 15-30 minutes, dialysis against 0.02 M Tris-HCl buffer with a pH of 8.0-8.2 for 1.5-2.5 hours and gel filtration on a Sephacryl S-400 with 0.015-0.02 M Tris-HCl buffer with a pH of 8.0-8.2 and with 0.4-0.9 M sodium chloride, separating the second fraction of proteins under the control of immunoelectrophoresis using antisera to animal proteins, and the whole Eva product is obtained by concentrated protein fractions in polyethylene glycol-40,000 to the content of immunoglobulin A in the target product in comparison with its content in the initial biological fluid of animals 3-6 times.

In the patent and scientific literature, technical solutions are not known that contain S-IgA production regimes similar to those claimed, i.e. proposal meets the criterion of "novelty."

All modes of the method are feasible in laboratory conditions, aimed at solving a real technical problem, i.e. the proposal is “industrially applicable”.

For the first time, a method for producing S-IgA has been proposed, in which animal biological fluid without preliminary purification from ballast by sequential precipitation with ammonium sulfate is centrifuged, dialysed and the target product is isolated from the test biological fluid by gel filtration on Sephacryl S-400 (adding chloride to Tris-HCl buffer sodium only in certain concentrations allows you to get a non-obvious positive effect - increasing the yield and quality of the target product). In addition, the exclusion of a number of steps in the method for producing secretory immunoglobulin A from animal biological fluid with an increase in the yield and quality of the target product made it possible to significantly simplify and accelerate the process of its preparation, i.e. the proposal meets the criteria of "novelty" and "inventive step". The method is illustrated in the following examples.

Example 1. Obtaining S-IgA from the nasal secretions of cattle. 5.5 ml of nasal secretion is centrifuged at 1700 g for 15 minutes and dialyzed against 0.02 M Tris-HCl buffer with a pH of 8.0 for 1.5 hours. Then the dialysate in a volume of 5 ml is subjected to fractionation on a Sephacryl S-400 on a column of 12 × 400 mm. As an eluent, 0.015 M Tris-HCl buffer pH 8.0 with 0.4 M NaCl is used. The optical density of the fractions exiting the column and collected in a volume of 6 ml was determined on a spectrophotometer at 280 nm. The protein eluted from the column in the 2nd fraction contained immunochemically pure class A immunoglobulins (one line of precipitation corresponding to S-IgA), which was confirmed by immunoelectrophoretic analysis of protein fractions.

Concentrate in PEG (40,000) to an IgA content of 10.5 mg / ml, which is 4 times more than its content in the original biological fluid of animals. The target product is frozen at -20 ° C. S-IgA production time is 24 hours. At the same time, in a known manner, the time to obtain S-IgA from nasal secretions is 3 days (3 times slower than the claimed method), and the amount of IgA obtained is 3.5 mg / ml (i.e., 67% less).

Example 2. Obtaining S-IgA from the salivary fluid of calves. In the first stage, 7 ml of calf salivary fluid is centrifuged at 1350 g for 30 minutes, the precipitate is discarded, and the supernatant is dialyzed against 0.02 M Tris-HCl buffer with a pH of 8.1 for 2.0 hours. Then the dialysate in a volume of 6 ml is subjected to fractionation on a Sephacryl S-400 on a column of 12 × 400 mm. As an eluent, 0.02 M Tris-HCL buffer pH 8.2 with 0.9 M NaCl was used. The optical density of the fractions leaving the column and collected in a volume of 5 ml was determined on a spectrophotometer at 280 nm. The protein eluted from the column in the 2nd fraction contained immunochemically pure class A immunoglobulins (one line of precipitation, corresponding to S-IgA), which was confirmed by immunoelectrophoretic analysis of protein fractions.

Concentrate in PEG (40,000) to a S-IgA content of 2.0 mg / ml, which is 6 times more than the content in the original biological fluid of animals, and freeze at -20 ° C. At the same time, in a known manner, the time for obtaining S-IgA from calves saliva is 3 days (3 times slower than the claimed method), and the amount of IgA obtained is 0.5 mg / ml (i.e., 75.0% less )

Example 3. Obtaining S-IgA from the lacrimal secretions of calves. 6 ml of tear fluid are centrifuged at 1000 g for 22.5 minutes, the supernatant is dialyzed against 0.02 M Tris-HCL buffer with a pH of 8.2 for 2.5 hours . The 5.5 ml dialysate is then fractionated on a Sephacryl S-400 on a 12 x 400 mm column. As an eluent, 0.0175 M Tris-HCl buffer pH 8.1 with 0.65 M NaCl is used. The optical density of the fractions leaving the column and collected in a volume of 5 ml was determined on a spectrophotometer at 280 nm. The protein eluted from the column in the 2nd fraction contained immunochemically pure class A immunoglobulins (one line of precipitation corresponding to S-IgA), which was confirmed by immunoelectrophoretic analysis of protein fractions. Concentrate in PEG (40,000) to an S-IgA content of 7.5 mg / ml, which is 3 times more in comparison with its content in the original biological fluid of animals, and freeze at -20 ° C. At the same time, in a known manner, the time to obtain S-IgA from tear secrets is 3 days (3 times slower than the claimed method), and the amount of S-IgA obtained is 3.5 mg (i.e., 54.0% less )

As shown by the results of experimental studies, according to examples 1-3, the proposed method allows to increase the yield of the target product with preservation of its biological activity by 54.0% -75.0%, and also accelerate the process of obtaining S-IgA by 3 times.

Claims (1)

The method of obtaining secretory immunoglobulin A from animal biological fluid by biochemical treatment of the biological fluid, separation of the target product by gel filtration, followed by freezing, characterized in that the biological fluid is centrifuged at 1000-1700 g for 15-30 minutes, subjected to dialysis against 0.02 M Tris-HCL pH 8.0-8.2 for 1.5-2.5 hours and gel filtration on a Sephacryl S-400 with 0.015-0.02 M Tris-HCL buffer with pH 8.0-8, 2 and with 0.4-0.9 M sodium chloride, separating the second fraction of proteins under the control of immunoelectrophoresis using serum to animal proteins, and the target product is obtained by concentration of protein fractions in polyethylene glycol-40,000 to the content of immunoglobulin A in the target product in comparison with its content in the original biological fluid of animals 3-6 times.