RU2730532C1 - Method of producing antitoxic hyperimmune serum for the treatment of t-2 animal toxicity and a method of treating t-2 animal toxicity - Google Patents

Abstract

FIELD: veterinary science.SUBSTANCE: group of inventions refers to veterinary science and represents a method for producing antitoxic hyperimmune serum for treating T-2 animal toxicity, providing for three-time immunization of rabbits with a conjugate of T-2 toxin with polylysine in physiologic saline, during the first immunization with Freund's complete adjuvant, then with 3-week interval twice – with Freund's incomplete adjuvant, introduction of the obtained emulsions is performed with preliminary heating to temperature of 37 °C in dose of 2 ml in a single intracutaneous manner along the back of rabbits, in which in 2 weeks after the third immunization blood is taken from ear veins, blood serum is obtained from each immunized animal, collecting blood and holding for 30 minutes at temperature of 37 °C, then the blood clot is separated from the walls of the vessel and placed for 18 hours in a refrigerator at temperature of 4 °C, in 18 hours antitoxic serum is separated, tested in indirect ELA competitive variant for presence of antibodies, bringing titer to 1:6400 and a method of treating T-2 animal toxicity, comprising administering into the body antitoxic hyperimmune serum obtained by the disclosed method, which is administered three times intramuscularly every 10 days in dose of 0.3 mg per kg of live weight.EFFECT: declared group of inventions enables to obtain hyperimmune serum for treating T-2 animal toxicity, the use of which in the declared method of treatment provides neutralization of T-2 toxin action, normalization of homeostasis values, improved general condition, decreased mortality and body weight.3 cl, 5 tbl, 2 ex

Description

The invention relates to veterinary medicine and can be used to treat T-2 toxicosis in animals.

T-2 toxin is one of the most common [1] and toxic [2] mycotoxins of the trichothecene group.

T-2 toxin inhibits the functional activity of organs (bone marrow, spleen, thymus, lymphoid tissue), suppressing the immune response of animals and birds [3]. Thus, there is evidence that T-2 toxin inhibits the central nervous system [4, 5, 6, 7, 8].

T-2 mycotoxicosis manifests itself in the form of vomiting, hemorrhagic syndrome, erythrocytopenia, leukopenia, thrombocytopenia, anemia. In case of poisoning with T-2 toxin, animals often refuse to feed, and there is a gradual destruction of hematopoietic and immunocompetent organs [9, 10, 11, 12].

The strategy for the prevention of T-2 mycotoxicosis in animals is primarily aimed at preventing the formation of mycotoxin, eliminating contaminated feed from the diet, as well as decontaminating and detoxifying feed.

In practice, despite the complex of preventive measures, it has not yet been possible to exclude animals from eating affected feed, which requires treatment of mycotoxicosis.

Known methods of treatment for poisoning with T-2 toxin, including the use of sorbents, immunomodulators, antioxidants, detoxifying agents, and probiotics [13, 14].

Thus, the use of a number of antioxidants for the prevention and treatment of T-2 toxicosis is known: the preparation IFO-6ET [15], selenium and vitamin E [16], vitamin C, vitamin E, selenium [17, 18].

The disadvantage of this method is that the detoxifying effect of antioxidants is based on the elimination of the initiation of free radical oxidation of lipids - one of the possible mechanisms of action of trichothecene mycotoxins, and not on the elimination of the toxin itself.

One of the effective methods of treating mycotoxicosis in animals is the use of various adsorbents: zeolites [19, 20], bentonites [11, 21], activated carbon [22], shungite [23, 24], vermiculite [25], dietary fiber [26] , polysaccharides [27], glucomannans [28, 29], lignins [30, 31], white sludge [32], which can significantly prevent the absorption of mycotoxins and their metabolites in the digestive tract and their spread throughout the body.

However, these methods have a number of disadvantages, in particular, the disadvantages of sorbents based on aluminosilicates include: lack of selectivity of sorption to vitamins and nutrients, a high rate of input into the diet.

Known methods of detoxification in mycotoxicosis using probiotics based on various bacterial strains: Bacillus subtilis-93 [33], the drug Sakhabactisubtil - contains bacterial strains Bacillus subtilis-TNP-3 and Bacillus subtilis-THn-5 [34]. However, the effectiveness of these drugs is based on the ability of the microorganisms that make up their composition for vitamin-forming, acid-forming, antagonistic and immunostimulating activities.

Known complex preparations, which may contain organic and mineral components, cultures of probiotics and prebiotics, inhibitors, enzymes, biologically active substances (vitamins, minerals, immunostimulants): "Fungistat" [35]; a mixture of natural zeolites and enzyme preparations [36]; a mixture of sorption material, acids, etc. [37]; a feed additive containing bee podmore, bentonite, grass meal, biomass and culture liquid of M. Gisevii Lindau kombucha, bifidumbacterin, antidiarin [38].

The disadvantage of these combined preparations is that the simultaneous presence of components with different physicochemical properties can lead to mutual compensation (absorption) of useful properties.

Despite the growing list of pharmacological drugs for the treatment of T-2 toxicosis, the choice of effective means of protection against mycotoxins is still extremely insignificant, since specific prevention has not yet been developed. There are also no antitoxic sera in medical and veterinary practice, this is primarily due to the fact that mycotoxins are low molecular weight compounds that do not have the ability to form antibodies.

The objective of the present invention is to create an antitoxic hyperimmune serum for the treatment of T-2 toxicosis in animals, which neutralizes the action of T-2 toxin, normalizes homeostasis indicators, improves the general condition, decreases mortality and increases body weight.

The task is solved by the fact that the method of obtaining antitoxic hyperimmune serum for the treatment of T-2 toxicosis of animals provides for three-fold immunization of rabbits with a conjugate of T-2 toxin with polylysine in saline, during the first immunization - with complete Freund's adjuvant, then with an interval of 3 weeks twice - with incomplete Freund's adjuvant. The introduction of the obtained emulsions is carried out with preliminary heating to 37 ° C, in a dose of 2 ml intradermally, once, along the back of rabbits, from which, 2 weeks after the third immunization, blood is taken from the veins of the ear and kept for 30 minutes at a temperature of 37 ° C, then the clot is separated from the walls of the vessel and placed for 18 hours in a refrigerator at + 4 ° C, after 18 hours, the antitoxic serum is separated, tested in an indirect competitive ELISA for the presence of antibodies, the titer is adjusted to 1: 6400. The antitoxic serum obtained in this way is administered to sick animals intramuscularly three times with an interval of 10 days at a dose of 0.3 ml per kg of live weight. Store the serum in the refrigerator at + 4 ° C. And the conjugate of T-2 toxin with polylysine is obtained on the basis of hemisuccinate T-2 (GS-T-2), to which is first added a mixture of acetonitrile with water in a ratio of 6: 1, and then EDK (1-ethyl-3- (3- dimethylaminopropyl) carbodiimide), incubate the mixture for 10 minutes at a temperature of 200 ° C and pH 4.5, then portionwise add a solution of polylysine in physiological saline in a ratio of 1: 3, conjugate for 2 hours at pH 6.5, then dialyze in within 18 hours and used to obtain antitoxic serum. The use of the obtained antitoxic hyperimmune serum allows for effective treatment of animals with T-2 toxicosis by neutralizing the action of the T-2 toxin, normalizing homeostasis indicators, improving the general condition of animals and reducing mortality and increasing the body weight of animals.

The method for the treatment of T-2 toxicosis in animals provides for three times administration of antitoxic hyperimmune serum intramuscularly with an interval of 10 days at a dose of 0.3 mg per kg of live weight.

The method of obtaining antitoxic hyperimmune serum for the treatment of T-2 toxicosis in animals was carried out as follows.

To obtain blood serum, male rabbits of the "Gray Giant" breed of dark or gray color, not older than 2 years, with a body weight of 2.0-2.9 kg were used. A conjugate of T-2 toxin with polylysine was used as an antigen for hyperimmunization of rabbits.

To obtain a conjugate of T-2 toxin with polylysine, the synthesis of hemisuccinate T-2 (GS-T-2) was first performed. The introduction of the carboxyl group into the hapten molecule was carried out according to the generally accepted procedure [39]. This reaction is widely used in immunochemistry.

We took 25 mg of mycotoxin T-2, dissolved in 5 ml of pyridine freshly distilled over NaOH and placed in a 25 ml flask equipped with a reflux condenser with a calcium chloride tube. To the solution was added 0.54 g of succinic anhydride (100-fold excess). The reaction mixture was boiled at 120 ° C for 4 hours. After that, 100 μL of the solution was taken from the reaction mass into a 10 ml conical flask to determine the degree of reaction, then 1 ml of chloroform was added to the pyridine solution, washed with distilled water 4 × 1 ml and 0.1 N HCl 3 × 1 ml to remove traces of pyridine and succinic anhydride, washed chloroform containing the reaction products, was investigated by analytical thin layer chromatography on a glass plate 5 × 10 cm (kieselgel 60 (Mezek)) in the solvent system KLF: methanol (9: 1). Detector: 1% solution of H ₂ SO ₄ in methanol, heated to 180 ° C for 2 minutes Rf (mycotoxin T-2) = 0.72; Rf (hemisuccinate T-2) = 0.35. The reaction is stopped when no traces of the original T-2 remain on the plate. Pyridine was evaporated on a rotary evaporator, the resulting oily precipitate was dissolved in 50 ml of HLF, then the solution was transferred to a separatory funnel and washed first with distilled water (4 × 150 ml), then, to remove traces of pyridine, it was washed with 0.3 N HCl (6 × 150 ml). The chloroform layer was dried over activated carbon and calcium chloride, filtered, and evaporated to dryness on a rotary evaporator. The resulting precipitate is a glassy yellowish mass - hemisuccinic - T-2 (GS-T-2). The product yield is 24 mg (75%).

The synthesis of GS-T-2 conjugates with polylysine (PL) was carried out using the activated ester method [40], which is widely used in the creation of immunoreagents [41].

The (GS-T-2) -PL conjugate was synthesized according to a typical procedure. We took 2.6 mg of GS-T-2 and dissolved it in a mixture of acetonitrile - water in a 6: 1 ratio and added 1.8 mg of EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide), the mixture was kept for 10 minutes at 200 ° C and pH 4.5, then a solution of polylysine in physiological saline was added in portions (in a ratio of 1: 3), then conjugated for 2 hours at pH 6.5 and dialyzed against saline for 18 hours. The resulting conjugate T-2 polylysine was used to obtain antitoxic hyperimmune serum.

The method of obtaining antitoxic hyperimmune serum was carried out as follows.

The day before immunization, the hair was shaved on the back of the animal and wiped with a 7% solution of ethyl alcohol. The antigen solution was prepared immediately before immunization: 300 μg of the T-2 toxin - polylysine conjugate was dissolved in 1 ml of sterile saline, then thoroughly mixed with 1 ml of Freund's complete adjuvant for the first injection. Then, with an interval of 3 weeks, 300 μg of the T-2 conjugate toxin-polylysine was dissolved in 1 ml of sterile saline and mixed with 1 ml of Freund's incomplete adjuvant for the 2nd injection and after 3 weeks - in the same way for the 3rd injection. The resulting emulsions were preheated to 37 ° C and in a dose of 2 ml was injected with a sterile syringe once intradermally along the shaved back of rabbits 10-15 points, and the remainder of the emulsion (about 0.2 ml) was injected intramuscularly into the thigh. On the 7th, 28th and 49th days after the third immunization, blood samples were taken from the veins of the rabbits' ear, they were examined in an indirect ELISA method for the specificity of antibodies, the maximum accumulation of antibodies occurred after 3 weeks. Then blood was collected from each immunized animal, kept for 30 minutes at 37 ° C, then the blood clot was separated from the walls, placed in a refrigerator at 4 ° C for 18 hours, and the resulting antitoxic hyperimmune serum was separated after 18 hours. The ELISA was tested in an indirect competitive variant for the presence of antibodies, the titer was adjusted to 1: 6400 and used to treat sick animals with T-2 toxicosis and stored in a refrigerator at 4 ° C.

The serum was separated by a conventional method [42].

Example 1. Experimental data on the effectiveness of specific treatment of T-2 toxicosis with hyperimmune serum were obtained in experiments on 72 male white rats weighing 160-190 g, divided according to the analogy principle into 4 groups. Before the experiment, the animals were kept on a diet free of mycotoxins for two weeks. The first group of animals served as a biological control and for 30 days of the experiment received "pure" autoclaved feed. Group 2 rats were given “toxic food” contaminated with T-2-toxin (at a dose of 0.4 mg / kg of food, which is 4 times higher than the maximum permissible concentration in the Russian Federation); the third group received "toxic food" and treatment in the form of injections of hyperimmune serum at a dose of 0.3 ml per kg of body weight at the beginning of the experiment and again on days 10 and 20 of the experiment; the rats of the fourth group received "toxic food" and injections of intact serum according to the same scheme as the rats of the third group.

The experiment lasted 30 days. During the experiment, the survival rate, the dynamics of changes in body weight were recorded. At the end of the experiment, the animals were euthanized with blood sampling for laboratory studies.

For experimental studies, crystalline T-2 toxin was used, obtained in the laboratory of mycotoxins of the Federal State Budgetary Scientific Institution "FCTRB-VNIVI", with a mycotoxin purity of 99.8%. The toxin was introduced by inclusion in the diet by sequential and stepwise thorough mixing. The feed was also preliminary tested for the content of mycotoxins regulated in Russia by enzyme immunoassay [43] and indicators of biological safety - the feed corresponded to the quality certificate for safety.

Hematological studies, including the determination of the number of erythrocytes, leukocytes, hemoglobin, were carried out by conventional methods, the total protein content was established by refractometric analysis, and the quantitative ratio of protein fractions was determined by the nephelometric method using KFK-2 with a red filter [44, 45].

The digital material was processed by the method of variation statistics using the Student's t-test.

In rats with prolonged intake of a toxic diet, a decrease in food and water consumption was noted, as well as ruffled skin. The animals that received specific treatment did not show clinical signs. The data and indicators of changes in body weight are presented in table 1.

Table 1 shows that the survival rate of the rats receiving the toxin without treatment was 66.7% with 100% safety in the biological control and treatment groups. In the second group, pronounced clinical signs of toxicosis appeared already on the 10th day of the experiment in the form of a decrease in appetite, depression, anorexia and diarrhea, while in the third group, the clinical picture was in 93% of animals only at the end of the experiment.

In rats receiving toxic food without treatment, a decrease in live weight was observed, so the average live weight of white rats by the end of the experiment was 23.1% lower than the initial indicator, and in the group of animals receiving treatment, a positive increase was observed, amounting to 0 , 6% versus 7.41% in the biological control group. In the fourth group, where the rats received a course of injections of intact serum, a decrease in live weight was observed, amounting to 26.3%.

From the results of hematological studies of the blood of white rats presented in Table 2, it can be seen that by the 30th day of the experiment in the second group there was a regular pronounced decrease in the number of erythrocytes by 13.3% (p <0.01), leukocytes by 47.8% (p <0.01), hemoglobin - by 19% (p <0.001), in the fourth group the decrease in similar indicators was 5.88%, 49.6% (p <0.001 and 15.23% (p <0.0001), While against the background of specific treatment, these indicators were on the same level with the control group.

Progressive erythrocyto-leukopenia and anemia, with a simultaneous increase in ESR values, are one of the characteristic symptoms of mycotoxicosis and indicate serious destructive changes in the hematopoietic and immunocompetent organs, which are known to be the target organs of T-2 toxin [46, 47].

The erythrocyte sedimentation rate increased in the second and fourth groups of rats on the 30th day of the experiment by 146.1% (p <0.01) and 138.26% (p <0.001), respectively, while in the group with treatment - by 26, 1% (p <0.05).

According to the data presented in Table 2, it can be seen that the most pronounced violation of protein metabolism occurred in rats receiving toxic food without treatment, so in the second and fourth groups, the content of total protein in the blood serum compared with the control data decreased by 30 days by 26.0 % (p <0.01) and 27.6% (p <0.001), respectively. The introduction of specific serum prevented protein imbalance, so in the third group there was a decrease in total protein on day 30 by 12.6% (p <0.05).

Thus, the studies carried out have established that in case of T-2 toxicosis in white rats, the use of specific treatment reduced the negative effect of the ecotoxicant, contributed to an increase in the safety of animals and the normalization of hemato-biochemical parameters.

Example 2. It is known that fur animals are more sensitive to mycotoxins than laboratory and farm animals [48, 49]. Therefore, to confirm the data on the effectiveness of specific treatment of T-2 toxicosis with hyperimmune serum in white rats, a similar experiment was performed on rabbits of the "Gray Giant" breed. For this purpose, 4 groups of rabbits were formed, 3 animals each, divided according to the principle of analogues. The first group served as a biological control, received normal food without toxins; the second - with the T-2 diet toxin at a dose of 300 μg / kg feed; the third - with the T-2 diet toxin at a dose of 300 μg / kg of feed and treatment in the form of injections of hyperimmune serum at a dose of 0.3 ml per kg of body weight at the beginning of the experiment and again on days 10 and 20 of the experiment. The fourth group received with the T-2 diet toxin at a dose of 300 μg / kg of feed "toxic feed" and injections of intact serum according to the same scheme as the animals of the third group.

Before the experiment, the animals were kept on a diet free of mycotoxins for two weeks. The experiment lasted for 30 days, every 10 days hematological and biochemical blood tests were performed.

For experimental studies, crystalline T-2 toxin was used, obtained in the laboratory of mycotoxins of the Federal State Budgetary Scientific Institution "FCTRB-VNIVI", with a mycotoxin purity of 99.8%. The toxin was introduced into the feed by sequential and stepwise thorough mixing. The feed was also preliminary tested for the content of mycotoxins regulated in Russia by enzyme immunoassay [43] and indicators of biological safety - the feed corresponded to the quality certificate for safety.

Clinical signs, body weight dynamics, hematological and biochemical parameters were used as the parameters of the effectiveness of the use of antitoxic serum in T-2 toxicosis.

The number of erythrocytes, leukocytes, the hemoglobin content in the peripheral blood was determined according to generally accepted methods, the total protein was determined by refractometry [50, 45]. The biochemical parameters of blood serum (glucose, urea, uric acid, ACT, ALT, total calcium, inorganic phosphorus) were determined using an Express Plus analyzer.

The digital material was processed by the method of variation statistics using the Student's t-test.

Clinical signs of intoxication were recorded only in animals that received toxic food, which manifested itself as apathy, decreased appetite and response to external stimuli, short-term upset of the gastrointestinal tract on the 25th day of the experiment.

The dynamics of the live body weight of rabbits with T-2 toxicosis against the background of the use of antitoxic serum are presented in table 3, from which it can be seen that in almost all experimental groups, with the exception of the biological control group, there was a decrease in live weight.

As can be seen from the table, in the second and fourth groups, by day 20, body weight decreased by 10.8 and 10.4%, by day 30 - by 15.5 and 15.0%, respectively. In the first and third groups, relative to the initial data, an increase in body weight was noted by 7.97% and 7.68%, respectively.

The results of a blood test of rabbits with T-2 toxicosis against the background of specific treatment are presented in Table 4, from which it can be seen that the decrease in hematological parameters in the group of rats receiving "toxic food" without the use of antitoxic serum, for the entire study period, was lower than the initial values. ...

According to table 4, it can be seen that in the second group of rabbits, the content of erythrocytes in the blood compared with the initial data decreased on the 10th day of the experiment by 5.81%, on the 20th day - by 17.1%, on the 30th day - by 21.15 %. In the third group, on day 10, the number of erythrocytes increased by 2.8%, then the number of erythrocytes decreased relative to the initial data on days 20 and 30 of the study by 2.3 and 6.85%, respectively. In the fourth group, the decrease in erythrocytes in the same period was 6.37% in the same period; 14.35 and 21.1%, respectively.

The hemoglobin content in the blood of rabbits of the second group compared with the initial data decreased by 5.73% on the 10th day of the experiment, by 10.40% on the 20th day, and by 16.06% on the 30th day. In the third group, on day 10, the hemoglobin content increased by 3.01%, then the hemoglobin content decreased relative to the initial data on days 20 and 30 of the study by 1.97 and 6.45%, respectively. The hemoglobin content in the blood of the fourth group of rabbits in comparison with the initial data on days 10, 20 and 30 of the experiment decreased by 4.2%, 12.68% and 15.51%, respectively.

ESR in the blood of the second group of rabbits in comparison with the initial data increased on the 10th day of the experiment by 20.71%, on the 20th day - by 63.31%, on the 30th day - by 100%. In the third group, on day 10, ESR decreased by 4.32%, then increased relative to the initial data on days 20 and 30 of the study by 7.56 and 16.75%, respectively. ESR in the blood of rabbits of the fourth group increased on days 10, 20 and 30 of the experiment by 19.28%, 66.27 and 95.18%, respectively.

As can be seen from table No. 4 in the second group of rabbits, the content of leukocytes in the blood compared with the initial data increased on the 10th day of the experiment by 13.7%, on the 20th and 30th days there was a decrease in the number of leukocytes by 18.55 and 33.1%, respectively ... In the third group, on the 10th day, the number of leukocytes increased by 3.45%, subsequently on the 20th and 30th days of the study, the number of leukocytes decreased relative to the initial data by 1.93% and 6.83%, respectively. The content of leukocytes in the blood of rabbits of the fourth experimental group decreased on the 20th and 30th days of the experiment by 17.95 and 30.91%, respectively.

The total protein content in the second group of rabbits in comparison with the initial data by the 10th day of the experiment decreased by 6.06%, by the 20th day - by 12.12%, by the 30th day - by 24.24%. In the third group of animals, by the 10th day of the experiment, the total protein content was higher than the initial data by 2.8%, by the 20th day, this indicator had decreased by 6.7%, by the 30th day - by 9.3%. In the fourth group, on days 10, 20 and 30 of the experiment, the decrease in total protein was 6.1; 12.6 and 23.5%, respectively.

In the course of the experiment, we studied some biochemical parameters of the blood of rabbits with T-2 toxicosis against the background of specific treatment. The data are presented in Table 5.

From the data in Table 5 it follows that in the second group, the glucose content on days 10, 20 and 30 decreased by 4.45; 17.3 and 27%, respectively. In the third group of rabbits, the glucose content on days 10 and 20 of the study, relative to the initial data, increased by 5.7 and 1.8%, respectively. When studying the blood of rabbits on the 30th day, a decrease in glucose concentration by 7.7% was noted. In the fourth group, the blood glucose content of rabbits on days 10, 20 and 30, relative to the initial data, decreased by 1.83; 18.3 and 28.3%, respectively.

In the second and third groups, the content of inorganic phosphorus on days 10, 20 and 30, relative to the initial data, increased by 6.9; 24.5; 40.1% and 2.4; 0.9; 6.7% respectively. In the fourth group, on the contrary, a decrease in this indicator was observed in the same period by 7.3; 25.8 and 39.1%, respectively.

In the second group, the total calcium content on days 10, 20 and 30 decreased by 4.5; 25.8 and 39.1%, respectively. A similar trend towards a decrease in this indicator by 4.8; 26.8% and 40.2%, respectively, were observed in the fourth group. In the third group, this level on days 10 and 20 increased by 6.4 and 4.8%, respectively; on the 30th day it decreased by 5.2%.

In the second group, ACT activity on days 10, 20 and 30 increased, relative to the initial data, by 24.2; 86.8 and 150%, respectively, in the fourth group the increase in this indicator in the same period was 23.8; 87.6 and 145%, respectively. In the third group, this indicator on the 10th day decreased by 13.1%; on days 20 and 30 increased by 4.6 and 38.8%, respectively.

ALT activity on days 10, 20 and 30 increased in the second group relative to the initial data by 35.5; 107 and 146.9%, respectively, in the fourth group the increase in this indicator in the same period was 36.7; 108 and 155%, respectively. In the third group, this indicator decreased by 3.7% by 10 days of research; on 20 and 30 days - increased by 26.7 and 78.6%, respectively. In the third group of rabbits, ALT activity decreased by 11.5% on day 10; on the 20th and 30th days - increased by 12.6 and 59.7%, respectively.

In the second, third and fourth groups, the activity of alkaline phosphatase on days 10, 20 and 30 increased by 49.1; 79.0; 125.2%; by 9.5; 22.5; 43.6% and 48.5; 78.0 and 120.0%, respectively.

Thus, the proposed method for producing antitoxic hyperimmune serum for the treatment of T-2 toxicosis in animals allows one to overcome the toxic effect caused by the presence of T-2 toxin in their diets, normalize homeostasis indicators, improve the general condition and reduce the death of animals and increase their body weight.

Thus, the use of a specific method of treating T-2 toxicosis will prevent the toxic effect in animals caused by the presence of T-2 toxin in their diets. During passive immunization with specific antitoxic hyperimmune rabbit serum obtained by multiple immunization of rabbits with T-2 polylysine conjugate, the negative effect of T-2 toxin is neutralized, homeostasis indicators are normalized, general condition improved, mortality decreased and animal body weight increased.

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Claims (3)

1. A method of obtaining antitoxic hyperimmune serum for the treatment of T-2 toxicosis in animals, providing for three-fold immunization of rabbits with a conjugate of T-2 toxin with polylysine in saline, during the first immunization - with complete Freund's adjuvant, then with an interval of 3 weeks twice - with incomplete Freund's adjuvant , the introduction of the obtained emulsions is carried out with their preheating to a temperature of 37 ° C at a dose of 2 ml intradermally once intradermally along the back of rabbits, from which, 2 weeks after the third immunization, blood is taken from the veins of the ear, blood serum from each immunized animal is obtained, blood is collected and kept 30 minutes at 37 ° C, then the blood clot is separated from the vessel walls and placed in a refrigerator at 4 ° C for 18 hours, antitoxic serum is separated after 18 hours, tested in an indirect competitive ELISA for the presence of antibodies, the titer is adjusted to 1: 6400, the obtained antitoxic serum is injected intramuscularly to sick animals but three times with an interval of 10 days at a dose of 0.3 ml per kg of live weight, store the serum in a refrigerator at 4 ° C. 2. The method according to claim 1, characterized in that the conjugate of T-2 toxin with polylysine is obtained on the basis of hemisuccinate T-2 (GS-T-2), to which is added first a mixture of acetonitrile with water in a ratio of 6: 1, and then EDK (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide), incubate the mixture for 10 minutes at a temperature of + 200 ° C and pH 4.5, then portionwise add a solution of polylysine in physiological saline in a ratio of 1: 3, conjugate to for 2 hours at pH 6.5, and then dialyzed for 18 hours and used to obtain serum. 3. A method of treating T-2 toxicosis of animals, providing for the introduction into the body of a specific antitoxic drug, characterized in that as a specific antitoxic drug, antitoxic hyperimmune serum obtained according to the method according to claim 1 is used, which is administered three times intramuscularly with an interval of 10 days in a dose 0.3 mg per kg of live weight.