The method of preparation of red blood cells to stimulate the immunological reactivity in laboratory animals

 

(57) Abstract:

The invention relates to medicine and biology and can be used to stimulate an immune response in laboratory animals. The method provides a more expressive stimulation of immunological reactivity in laboratory animals by introducing a solid mass of syngeneic, or autologous red blood cells, or their heavy fraction is subjected to electromagnetic waves of millimeter range of extremely high frequency. The method is as follows: the experimental animals before immunization intravenously twice in the marginal vein of the tail enter the erythrocytes in the form of a 5% suspension in medium 199 at a dose of 1 ml containing 8 x 108cells/ml, pre-exposed to electromagnetic radiation of millimeter range, which was performed using a millimeter-wave device "Yav-1", when the oscillation frequency 42,19 GHz, the wavelength of 7.1 mm, when the flux density of radiation power up to 10 mW/cm2. The exposure time was 1, 2, 5 and 10 minutes, the Dose of microwave radiation was 2.4 to 6.0 j/, see table 5., 1 Il.

The invention relates to medicine and biology and can be used for stimulation in animals by injection of erythrocytes, treated in vitro with trypsin (Author's certificate N 1037453, A 61 K 39/395 "Method of preparation of red blood cells to stimulate animal - producers immune serum". Authors: L. G. Prokopenko, A. I. Hemp, G. I. Krylov). Syngeneic erythrocytes, treated with a solution of trypsin in phosphate buffer, pH of 7.6 to 8.0 at a concentration of 6-810-3mg/ml for 2.5-3.5 hours at 36-38oC and washed from trypsin, resuspended in physiological solution of sodium chloride, form a 5% solution trypsinization erythrocytes, which are used for five intravenous experimental animals before immunization dose of 4108cells per 1 kg of body weight every 24 h to stimulate a humoral immune response (GMOs). The level of development of GMOs determine the titers of antibodies (Antibodies) in the serum reaction indirect agglutination: the higher titer of antibodies to 5-th and 8-th day compared to control.

The disadvantages of the described method of stimulation is a long treatment of erythrocytes with a solution of trypsin in vitro (for 2.5-3.5 hours). It is possible that the intake, along with the modulated red blood cells, alien agent - trypsin, resulting in the possible development of adverse side effects in imunological reactivity in laboratory animals by introducing a solid mass of syngeneic or autologous erythrocytes, or heavy fraction is subjected to electromagnetic waves of millimeter range of extremely high frequency (EM IIM EHF).

This is achieved by two intravenous experimental animals (Wistar rats) in the marginal vein of the tail of erythrocytes in the form of a 5% suspension in medium 199 at a dose of 1 ml containing 8108cells/ml, pre-exposed to EHF. The impact of electromagnetic waves of millimeter range of extremely high frequency conducted with the use of EHF-apparatus "Yav-1", when the oscillation frequency 42,19 GHz, the wavelength of 7.1 mm, when the flux density of the radiation energy is not more than 10 mW/cm2. The exposure time was 1, 2, 5 and 10 minutes, the Dose of microwave radiation was 2.4 to 6.0 j/cm2.

The method is illustrated by examples.

Example 1. The red blood cells of intact rats were subjected to EM IIM EHF for 1, 2, 5 or 10 minutes With this purpose, 15 ml of a 15% suspension was placed in a cuvette, and the top had the radiator, most bringing it to the surface of the erythrocyte suspension. Irradiated erythrocytes in the form of a 5% suspension in medium 199 at a dose of 0.5 ml containing 8108cells/ml, was injected intravenously in the marginal vein of the tail intact recipient of the Ana (EB), which was injected once intraperitoneally at a dose of 2108cells per 100 g of body weight. After 5 days, rats were killed and determined in the spleen, the number of antibody productive (KLA) and rosette (ROCK) cells in the spleen of animals. The degree of stimulation of the immune response to be judged by the performance of the KLA and the ROCK in the group of control animals, which were introduced only EB.

A single injection of erythrocytes exposed to EM IIM kWh for 10 min, stimulates the formation of humoral immune response (GMOs) on DL. The number of the KLA and the ROCK in the spleen of experimental animals were respectively 1.6 and 2.9 times higher compared to the control animals (introduction of intact erythrocytes of rats) (table. 1).

The experimental results show that the red blood cells of animals exposed to GM IIM EHF in vitro, acquire the ability to stimulate GMOs on DL when administered to healthy allogeneic recipients.

Example 2. The development of delayed-type hypersensitivity (GST) differs according to the properties of the main types of cells and secreted factors (Medunitsyn N. C. hypersensitivity of the delayed type // M.: Medicine, 1983. - 160 C.). GST caused a twofold introduction EB-Spa right foot (106cells in 0.15 M NaCl), and in the ball of the left foot - a solution of 0.15 M NaCl (control). The intensity GST judged by the difference between the masses of regional and contralateral lymph nodes, and the difference in the number of carnotite in them after 24 h after injection of the resolving dose (Chernousov, A. D., Fontain L. N., Kondratyev since the Formation of delayed-type hypersensitivity to sheep erythrocytes with separate and combined with the introduction of antigen and cyclophosphamide // bull. the experimental. biology and medicine. -1979. -N 5. -S. 449-452).

In rats twice, with an interval of 24 h, the introduction of erythrocytes exposed to EM IIM kWh for 10 min, the indicators GST were higher than in intact animals (see drawing).

The experimental results show that the red blood cells of animals exposed to GM IIM EHF in vitro, acquire the ability to stimulate GST at their introduction healthy allogeneic recipients.

Example 3. For a more directed and effective influence on the organism of animals examined together with immunomodulating capacity of different fractions of erythrocytes exposed to EM IIM EHF. To do this, before irradiation of erythrocytes was fractionally in the gradient of the density of the weft, having a density of 1,091-1,105 g/cm3and heavy ("old") erythrocytes density 1,117 g/cm3.

15 ml of 5% suspension of red blood cells in medium 199 was placed in a special quartz cuvette and subjected to EM IIM EHF within 10 minutes Irradiated erythrocytes in a dose of 0.5 ml containing 8108cells/ml, was injected intravenously in the marginal vein of the tail intact recipients twice with an interval of 24 h, with immunization in the last day of the introduction of irradiated red blood cells.

The data obtained showed that EM IIM EHF induces the appearance of immunostimulatory properties only heavy ("old") of red blood cells, as evidenced by high values of KLA and ROCK in the spleen of the experimental rats and titers of Antibodies serum compared with the control (table. 2). Intermediate density of erythrocytes and their light fraction ("young") after exposure to physical factors remained immunologically inactive (table. 2).

The results show that cells exposed to EM IIM EHF, stimulate the immune response, faster and more significant (especially the fraction of erythrocytes high density (d>1,117 g/cm3) improve immune response in healthy animals.

Example ctively in terms of secondary immunodeficiency (Lazarev Century N. The Alekhine E. K. immune Stimulants // M.: Medicine, 1985. -256 C.). To check these terms of Immunostimulants conducted a series of experiments on Wistar rats weighing 120-180 g; as models of secondary immunodeficiency States were used as follows: a single intraperitoneal injection of cyclophosphamide (CP at a dose of 150 mg/kg), infection with Staphylococcus, experimental acute pancreatitis (OP) (Bystrova N. A. abstract. dis... Kida. the honey.Sciences. Kiev, 1990, 24 S.).

The results of the experiments showed that intraperitoneal injection of the living culture of Staphylococcus aureus, cyclophosphamide or modeling of experimental acute pancreatitis inhibits the development of GMOs on the DL (see table. 3, 4, 5).

Solid IIM-erythrocytes cancel emerging immunosuppression, and the fraction of heavy IIM-erythrocyte not only cancel the immunosuppression in terms of the OP, staphylococcal infections and the introduction of cyclophosphamide, but have an immunostimulatory effect, which confirms the effectiveness of the proposed method immunostimulation not only healthy animals, but also in terms of secondary immunodeficiency.

Thus, the research results prove that the proposed method caffeinism undesirable chemical agents (trypsin), faster and to a greater extent (especially heavy fraction of red blood cells) increases the immune response in healthy rats, and also effective in terms of secondary immunodeficiency.

Description of the drawing. The influence of electromagnetic waves of millimeter range of extremely high frequency on the development of the reaction GST induced EB.

Y-axis: I - the difference is the weight (mg) regional and contralateral lymph lymph nodes;

II - the difference in the number of kariolou (million) in these lymph nodes;

On the x-axis: 1 - introduction of sheep red blood cells intact recipients;

2 - fixation; 3 - introduction IIM-red blood cells, irradiated for 1 min; 4 - introduction IIM-red blood cells, irradiated for 2 min; 5 - introduction IIM-red blood cells, irradiated for 5 min; 6 - introduction IIM-red blood cells, irradiated for 10 minutes

The method of stimulation of immunological reactivity in laboratory animals, including blood sampling, separation of red blood cells with their subsequent processing, wherein the solid mass of syngeneic autologous erythrocytes and their heavy fraction is subjected to the influence of electromagnetic waves of millimeter range of extremely high frequency within 10 m

 

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FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

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