RU2270021C2 - Method for obtaining transfer-factor in cattle - Google Patents

Abstract

FIELD: veterinary science, veterinary immunology.

SUBSTANCE: the present innovation deals with obtaining biologically active preparations that provide adaptive transfer of immunological information. The method applies cattle lymph nodes to be then homogenized and hydrolyzed with pepsin and DNAase. For protein hydrolysis one should prepare 0.5%-pepsin solution based upon 0.5%-hydrochloric acid solution. Suspension obtained after homogenization should be mixed in glass vial with the given solution at the ratio of 1:10. Simultaneously, for DNA depolymerization one should supplement reagent mixture with MgSO₄ crystals and lyophilized enzyme as DNAase. The vial should be placed into thermostat for 24-28 h at 37-38° C. One should periodically study medium pH and, if necessary, with the help of initial solution of pepsin and hydrochloric acid it is possible to achieve its optimal level being 1.75-2.0. No reaction to protein (biuret reaction) is considered to a control for hydrolysis ending. The obtained transfer-factor should be filtered and conserved with carbolic acid solution up to final concentration of solution being 0.5%. The innovation enables to simplify the technique and obtain more quantity of lymphocytic mass.

EFFECT: higher efficiency.

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Description

The invention relates to the field of veterinary medicine, in particular veterinary immunology, to a method for producing a biologically active preparation, a transfer factor in cattle, which provides adaptive transfer of immunological information to another organism.

A known method of obtaining a transfer factor [1, 2, 3, 4], where a person was used as an object. Experiments on its isolation in animals were, as a rule, unsuccessful [5, 6].

As a prototype, the method of obtaining the transfer factor, proposed by G. Fremel [3], which provides for the following steps:

1. Isolation of white blood cells. Blood is drawn from the donor (500 IU of heparin is added for every 100 ml of blood). Blood is poured into sterile tubes (25 × 155 mm), 2 ml of a solution of dextran (60 g / l) in a 0.15 M solution of sodium chloride or infucolla is added. The tubes are closed, shaken several times and incubated at 37 ° C. Red blood cells precipitate within 30-45 minutes. The duration of sedimentation is 1 hour.

The yellowish or reddish supernatant is transferred using a Pasteur pipette into conical centrifuge tubes, which are centrifuged for 10 min at 1500 g. The plasma is carefully drained. Settled white blood cells form a layer above the erythrocyte sediment. The sediment consists of 70% of lymphocytes. 0.1 ml of a sterile 0.15 M sodium chloride solution is layered on a white blood cell layer. The white blood cells are carefully aspirated with a Pasteur pipette in the form of a suspension in an isotonic sodium chloride solution.

2. Dialysis of white blood cells. For dialysis, complete destruction of the leukocyte mass is necessary. It is achieved by 10-fold freezing and thawing of the suspension. For this purpose, tubes with a suspension of cells are frozen with a mixture of dry ice with ethanol, and thawed in a water bath at 37 ° C. The process is repeated until the liquid becomes viscous. For depolymerization of DNA, several crystals of the lyophilized enzyme DNase and a small amount of Mg ions (several crystals of MgSO ₄ ) are introduced into the solution. Hydrolysis is carried out for 30 min at 37 ° C.

The dialysis process is carried out to remove high molecular weight components. The DNAse-treated leukocyte extract is transferred into a dialysis bag, which is pre-rinsed with a sterile isotonic sodium chloride solution and placed in a vessel with distilled water. 5 ml of distilled water are used per 0.1 ml of extract.

Dialysis takes place for 24 hours at 4 ° C. At the end of dialysis, the solution is filtered through a Millipore filter (0.22 μm pore diameter) under sterile conditions. A sterilizing filtration step is necessary to clean the drug from possible bacterial contamination.

3. Getting the transfer factor. The dialysate of the leukocyte extract is dissolved in phosphate-buffered saline (PBS) with a pH of 7.4 in a ratio of 1:20, if necessary, centrifuged for 20 minutes at 10 thousand rpm.

Isolation of the transfer factor is carried out on chromatographic columns measuring 20 × 530 or 50 × 1000 mm. Columns with Sephadex G-25 are washed before separation with three volumes of 1:20 diluted FSB solution with a pH of 7.4. The leukocyte dialysate solution is added to the column. Upward chromatography was performed in the same buffer at a rate of up to 90 ml / hour. Fractions are collected and preserved by lyophilization or other means.

This method, the closest in technical essence and selected for the prototype, has the following disadvantages:

- high complexity and complexity of the method;

- the use of blood (large volumes) as a source of white blood cells;

- the use of a person as an object for isolating the transfer factor (which makes it impossible to use the drug in veterinary medicine due to its specificity).

The aim of the invention is to remedy these disadvantages. This goal is achieved by the fact that cattle are used as an object for isolating the transfer factor. The source material for the isolation of lymphocytes is regional lymph nodes containing a large number of T and B cells, the most active in the implementation of the immunological transfer reaction [7, 8]. The following are excluded from the technological process of obtaining the transfer factor: 10-fold freezing and thawing, dialysis and subsequent chromatography of the initial suspension of lymphocytes, since all fractions isolated in this case are active [9].

The proposed method for producing transfer factor in cattle is as follows:

1. Obtaining an extract of lymphocytes. Regional lymph nodes (pre-scapular, patellar folds and others) are selected during the slaughter of healthy cattle. They are freed from connective tissue and fat and crushed into small pieces, placed in a homogenizer, distilled water is added in a ratio of 1: 1 and homogenized to a homogeneous consistency at 20 thousand rpm for 2-3 minutes. This mode of treatment of lymphoid tissue provides complete destruction of cellular structures.

2. Hydrolysis of the extract of lymphocytes and obtaining a transfer factor. To implement protein hydrolysis, a 0.5% pepsin solution in a 0.5% hydrochloric acid solution is prepared. The suspension obtained after homogenization of the lymph nodes is mixed in a glass container with this solution in a ratio of 1:10. At the same time, for the depolymerization of DNA, MgSO ₄ crystals in the amount of 0.015-0.017 mg and lyophilized enzyme DNA-ase in the amount of 0.004-0.006 mg are introduced into the reagent mixture. The vessel is placed in a thermostat for 24-28 hours at a temperature of 37-38 ° C. The pH of the medium is periodically checked and, if necessary, adjusted with the initial solution of pepsin and hydrochloric acid to the optimum value of 1.75-2.0. The control of the end of hydrolysis is the lack of response to protein (biuret reaction)

The resulting transfer factor is filtered and canned with a carbolic acid solution to a final solution concentration of 0.5%.

A comparative analysis of the claimed solution with the method selected for the prototype showed that the new solution has the following distinctive features:

- selection of cattle as a transfer factor;

- use as a source material for obtaining lymphocytic mass of regional lymph nodes;

- simplification of the technology for obtaining the drug;

- specificity of the final product for cattle.

The presence of distinctive features from the prototype of the signs ensures that the proposed technical solution meets the criterion of "novelty."

An analysis of the sources of information available to the authors showed that there is no information about receiving the transfer factor from the lymph nodes of cattle.

Thus, all these distinguishing features are necessary and essential, are closely related to each other, and therefore it is difficult to distinguish the contribution of each individual attribute in achieving the goal.

Tests of the proposed method for producing a transfer factor confirm its advantages over the prototype method.

Example: a scheme for obtaining a transfer factor.

1. Obtaining an extract of lymphocytes. Regional lymph nodes (pre-scapular, patellar folds and others), selected during the slaughter of healthy cattle, are released from connective tissue and fat and crushed into small pieces, 100 g of tissue are placed in a homogenizer, distilled water is added in a ratio of 1: 1 and homogenized to homogeneous consistency at 20 thousand rpm for 2-3 minutes

2. Hydrolysis of the extract of lymphocytes and obtaining a transfer factor.

Then carry out the hydrolysis of proteins in the homogenizer.

To do this, prepare a 0.5% solution of pepsin in a 0.5% solution of hydrochloric acid. The suspension obtained after homogenization of the lymph nodes is mixed in a glass container with this solution in a ratio of 1:10. At the same time, for the depolymerization of DNA, MgSO ₄ crystals in the amount of 0.015-0.017 mg and lyophilized enzyme DNA-ase in the amount of 0.004-0.006 mg are introduced into the reagent mixture. Then the vessel is placed in a thermostat for 24-28 hours at a temperature of 37-38 ° C and the pH of the medium is periodically checked. If necessary, bring it with the initial solution of pepsin and hydrochloric acid to the optimum value of 1.75-2.0. The control of the end of hydrolysis is the lack of response to protein (biuret reaction).

The resulting target product in an amount of 1200 ml is filtered and canned with a carbolic acid solution to a final solution concentration of 0.5%.

The cattle transfer factor is a slightly opalescent liquid of a light yellow or light brown color with a slight precipitate that breaks easily upon shaking, does not contain protein, contains polypeptides and oligonucleotides, is resistant to repeated freezing and thawing, lyophilization, and is resistant to RNA -ases, DNA-bases, trypsin.

The obtained advantages of the proposed method allow us to recommend it for widespread use in veterinary practice in order to obtain a transfer factor.

Literature

1. Petrov R.V. Immunology. - M .: Medicine, 1983 - 368 p.

2. Smirnov B.C. Immunodeficiency conditions. - St. Petersburg: "Tome", 2000 - 568 p.

3. Fremel G. Immunological methods. - M .: Medicine, 1987 - 472 p.

4. Schroder I., Rovensky J. In vitro determination of effects of dialyzable leukocyte extracts containing transfer factor activity. - Allergol. et Immunopathol., 1982, 10, 171.

5. Uotila A. Studies on the chemical nature of dialyzable transfer factor. Comparison of human leukocyte dialyzate and dialysates derived from human serum and from mammalian lymphoid and non-lymphoid organs. - Immunobiol., 1979, 156, 353.

6. Wilson G. B., Fudenberg H. H., Horsmanheimo M. Effects of dialyzable leukocyte extracts with transfer factor activity on leukocyte migration in vitro. 1. Antigen-dependent inhibition and antigen-dependent inhibition and enhancement of migration. 2. Separation and partial characterization of the components in DLE producing antigen-dependent and antigen-independent effects. - J. Lab. Clin. Medicine, 1979, 93, 800.

7. Bhardwaj N., Brummer E., Foster L. G. et al. Transfer of DTN to SK-SD and tetanus toxoid in BALB / c mice by TFd prepared from purified subpopulations of murine lymphocytes. - In: Immune regulators in transfer factor / Eds. A. Khan. et al. - New York: Academic Press 1979, 285.

8. Brummer E., Foster L. G., Bhardwaj N. et al. A murine model for studying the transfer of DTH with dialyzable human transfer factor. - ibid., p. 27.

Claims (1)

A method of obtaining a transfer factor, including the preparation of an extract of lymphocytes, hydrolysis with DNA, filtration, followed by preservation of the target product, characterized in that the regional lymph nodes of cattle are used as a source of lymphocytes, which are crushed in distilled water in a ratio of 1: 1, then homogenize to a homogeneous mass at 20,000 rpm for 2-3 minutes, the homogenate is mixed with a 0.5% solution of pepsin in 0.5% hydrochloric acid in a ratio of 1:10, add 0.015-0.017 mg MgSO ₄ 0.004 -0.006 mg DNAase, term Stateira reaction mixture at 37-38 ° C 24-28 hours, the pH of 1.75-2.0, the desired product is separated by filtration and conserve carbolic acid solution.