Method for preparing allergenic preparation for diagnosis radiation injury in body and method for diagnosis of body radiation injury

FIELD: medicine, radiation biology.

SUBSTANCE: invention proposes a method for preparing allergenic preparation used for diagnosis of body radiation injures. Method involves irradiation of potato tubers by the dose 350-400 Gr, the following extraction of quinoid radiotoxin by extraction with ethanol followed by removing extractant in rotary evaporator and additional extraction with ethyl acetate. The final prepared fraction is subjected for chromatography and separated in the system: (a) 2% acetic acid, and (b) mixture benzene - acetic acid - water in the ratio = 2:4:1, respectively. The prepared allergenic fraction is eluted with 2% acetic acid solution and standardized by dry matter as measured for 1 mg/cm3. Also, invention proposes a method for diagnosis of body radiation injures involving intracutaneous administration of specific allergen and detection of autosensitization symptom. Diagnosis of radiation diseases is proved by the allergy index. Proposed methods provide preparing the specific radiation allergen for detection of radiosensitization of body and to carry out diagnosis of radiation disease. Invention can be used in radiation biology.

EFFECT: improved preparing method, improved method for diagnosis.

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The invention relates to radiation biology, in particular the production and use of products intended for the diagnosis of radiation injuries of the body.

It is known that among the phenomena that develop after ionizing radiation, play a significant role allergic reactions, leading to the development in the irradiated organism one of the main symptoms of radiation damage - radiosensibility. Therefore, timely detection of radiation allergies (radiosensibility) is the key to successful treatment of radiation sickness. Therefore, development of effective methods and means of detecting radiation allergies is an actual problem of veterinary medicine. One of the conventional methods for detecting the allergic condition is intradermal introduction of a relevant allergen to obtain local giperergicakie reaction. However, despite the potential of this method, allergic diagnosis of radiation injuries is still not developed as veterinary and medical practice does not have specific allergenic drug.

There is a method to detect the condition of autocannibalism irradiated organism by intralobular the introduction of white mice as allergens sterile milk, sterile horse serum, mist day is offered by the agar culture of Escherichia coli, as well as extracts of homologous tissues of intact and irradiated animals - small intestine, liver, spleen, bone marrow, and evaluate the response on a 4-point system (see Lamparska NN. and other Local samples as a method for detecting the state of autocannibalism irradiated organism. - Bull. the experimental. Biol. and the honey. - 1958. No. 12. -Pp.28-32).

The disadvantage is the very high soreness of vnutrigornoj (intraluminal) samples for animals to run at large animals is problematic, and evaluation of the results of labial reaction is subjective, not amenable to instrumental measurement. Used to execute the specified method of detecting radiation sensitization allergens are nonspecific, because many of them are not true allergens.

The closest to natural allergens from the time-tested means are extracts from homologous tissues of intact and irradiated animals - small intestine, spleen and bone marrow (see ibid., p.30). However, it is estimated that, of these antigens only extracts of the small intestine revealed the state of autocannibalism in irradiated mice substances tissues of the same species (homosociability), but this antigen may not be used as a beam of allergen because, firstly, for vyyavleny the radiation sensitization according to this method, it is necessary to apply homologous allergens for each species, i.e. for white mice, the extract of the intestine of mice, rats extract intestine of rats, Guinea-pigs - extract intestine of Guinea pigs, etc. that represents a technically impossible task, because you need to prepare many kinds of allergens, as there are species of animals and, secondly, tissue extracts, being a complete protein antigens when injected into the body to induce the formation of protivougonnykh, protivotankovyh antibodies, which violates one of the requirements for allergens - they are introduced into the body must not form antibodies against this drug. Therefore, tissue antigens (extracts from authorities) do not respond to the main requirement of Allergy - allergens should not have antigenicity, and therefore cannot be used as allergens with particular pathologies.

The task of the invention to provide specific radiation antigen to detect the state of radiosensibility organism and emergency measures to desensitizing therapy radiation sickness.

The problem is solved in that a method of producing drug for the diagnosis of radiation injuries of the body, including the extracting of the irradiated objects allergenic material, as allergen using chromatographic clean fra is of quinoid radiotoxins, isolated from an ethanol extract of irradiated dose of 350-400 G of potato tubers, which is injected animals intradermally into the region of the inner thigh (small cattle), from the outside base of the ear (the pig), the upper third of the neck (cattle, horses) in an amount of 0.1-0.2 cm3(1.5 to 2 μg per animal) and the index of allergies (IA)equal to or in excess of 1.12 (IA greater than or equal to 1.12), is diagnosed with radiation sickness.

A significant difference of the method of producing drug for allergic diagnosis of radiation injuries of the body is that as a specific allergen use the main etiopathogenetic factor radiation damage is a natural radiomimetic - radiotoxic responsible for the development in the irradiated organism radio-sensitizing effect. Following exposure to a lethal dose of damaged cells radiolysis products of the tissues enter the blood and lymph, which are autoimmunization of the body, lead to a gradual formation of the it process anaphylactoid type, characterised by hypersensitivity of the delayed type neck surgery to toxic products, i.e. developing autoallergy. When intradermal toxic component radiotoxins irradiated animal develops from is to maintain the body's response in the form of giperergicakie allergic skin reactions with different intensity, showing (identifying) the fact of radiosensibility in the tested animal.

The use of intradermal allergen in a sample of natural radiomimetic - plant radiotoxins - can unify diagnostic drug and greatly improve its specificity, because, firstly, he is free from abdykanovich (whole group) antigens contained in tissue extracts, and, secondly, the drug has no antigenicity, as it is the protein content is minimal (1.5-1.8 per cent) and, mainly, it contains low-molecular glikopeptid - product integration quinoid "prosthetic groups of residues of amino acid and peptide.

The use of intradermal allergen in the sample allows the initial time after irradiation (the first 5-6 days) to identify development in the body condition of radiosensibility and an emergency desensitization of the body, thus preventing radiation death of animals.

The way to obtain allergic drug is as follows.

The tubers subjected to a single external gamma irradiation at a dose of 350-400 G when the exposure dose of 250 Gy/h Irradiated tubers incubated at a temperature of 18-22°within 24 hours the Further processing of the irradiated object with the purpose of isolating allergene what about the material - quinoid radiotoxins is carried out according to the standard technique (see kN. Radiotoxic, their nature and role. Edited Ammesia M: Atomizdat, 1966. - Pp.86-91). To do this with potatoes off the top layer with a thickness of 3-4 mm and the remaining tissue is homogenized in three volumes of 96%ethanol. The extraction can be performed during 1-1,5 h with constant stirring, stroma separated by centrifugation and the alcohol removed under vacuum at a temperature of 28-33°C. the Precipitate, which is formed in the concentrate, separate and transparent, slightly yellowish solution is acidified with hydrochloric acid to a pH of 7.2 and extracted with ethyl acetate. Received an ethyl acetate layer is applied on a paper chromatogram (20×20 cm) and scatter in two solvents: 2%acetic acid in a mixture of benzene - acetic acid - water (2:4:1). The obtained chromatogram is subjected to infrared spectrophotometry according to the standard technique (Kopylov V. the Mechanism of formation and identification of toxic substances quinoid nature, formed in the irradiated organism. M: Atomizdat, 1966. - P.18-28). Faction, fluorescent blue, then passing in a bright blue color, elute with 2%solution of acetic acid, unite in a common fraction is neutralized to a pH of 7.2. The resulting eluate determine protein by Lowry, quinones - Amosine and V.A.Kopylov (Radiotek the ins. M.: Nauka, 1983. - P.27-28), the dry matter content is a common method of drying to constant weight, biological activity in the test 50%hemolysis of erythrocytes by the method of acid urethrogram (IAASB, Iasiello. - Biophysics, 1957. No. 2. - S).

Obtained by the above method radiotoxic dried to constant weight and standardize by diluting it with distilled water with a pH of 7.2 to a concentration of 1.0 mg/ml, stored at a temperature of 2-6°With and use for their intended purpose - as a specific radiation allergen in the intradermal reaction to detect radiosensibility animals.

Identifying the state of radiosensibility in irradiated animals is as follows. In Guinea pigs on the side of the back surface, in cattle and horses in the upper third of the neck wool clip out the size of 3×3 cm3at other types of animal hair (fine cattle and pigs) not clip out, the allergen is injected into the inner thigh (sheep, goats), in the outer side at the base of the ear (in pigs).

The allergen is injected strictly intradermally in a volume of 0.1-0.2 cm3(1.5 to 2.0 μg per animal) in the above region of the respective species. Reading the reactions are conducted through 24, 48, 72, 96, 120, 144 h after irradiation, registering the character is eacli on the allergen and evaluation of reactions carried out on a 4-point system: 1 point slight redness at the injection site, 2 points - bright redness, swelling, 3 credits hyperemia, edema, hemorrhage, 4 points - hemorrhage, necrosis. Processing of results calculate the average score (index of Allergy - IA). Establish that animals (non-irradiated animals) average score (index of allergies) ranges from 0.3 to 1.0, and irradiated from 1,12 and above (IA>1,12). Therefore, the diagnostic index of allergies, equal to or greater than the value of 1.12 shows the defeat of the body to ionizing radiation, and the degree of lesion correlates with the index of allergies (IA): for acute radiation sickness mild this index ranges from 1,12-2.0; for medium - from 2.1 to 3.1 and severe - between 3.1 and above.

Results recording allergic research in the dynamics shows that the initial reaction to the allergen in the irradiated animals begin to show 48-72 h after irradiation and reaches a maximum at 4-6 days. With the introduction of the allergen on the 5th day after irradiation, the reaction to the allergen appears after 24 hours (6 days), with the introduction of the allergen on the 10th day after irradiation, the reaction appears in the same day (4-6 hours), with the introduction of the allergen on the 15th day the reaction to the allergen is manifested through 24-48 hours, and in less pronounced form, i.e. the index of allergies even when severe ARS the leaves in the range of 2.0-2.5.

Therefore, the timing of the emergence of allergic reactions to intradermal injection of the drug in irradiated animals allow us to determine the stage of the disease: the emergence of a reaction to an allergen in 4-6 days - on the initial period of radiation sickness (I stage), the emergence of giperergicakie reaction to an allergen in the 24 h of the stage of clinical signs of disease (stage II), the emergence of a reaction to an allergen in the same day about the introduction of the disease in stage height (stage III), and the emergence of a reaction to an allergen 24-48 hours after the drug is on the stage of convalescence (stage IV).

The way to obtain allergic drug testing on irradiated animals are illustrated by the following examples.

Example 1. Receiving radiation allergen.

To isolate specific allergenic substance, the tubers were subjected to gamma irradiation at doses of 200, 250, 300, 350, 400, 450, 500 Gr, kept them at a temperature of 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25°C for 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 h, removed the top layer thickness 1, 2, 3, 4, 5, 6, cm, homogenized in a 1-, 2-, 3-, 4-, 5 - and 6-fold volume of 80-, 85-, 90-, 91-, 92-, 93-, 94-, 95-, 96-, 97-, 98%ethanol, were extracted for 0,5, 10, 1,5, 2, 2,5, 3 h, kept the alcohol when 20, 22, 24, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37°allocated I, II, III, IV, V, VI fractions by the method of paper chromatography. Have established that the optimal output allergenic mater is Ala (on dry matter) is achieved by irradiation of potato tubers at a dose of 350-400 G, keeping 23-24 h at room temperature (18-22°C)removing the top layer of tubers thickness of 3-4 cm, extraction 1-1,5 h, removal of alcohol on a vacuum evaporator at a temperature of 28-33°and the allocation fraction VI faction by paper chromatography. Any changes to the settings for natural radiomimetic quinoid radiotoxins (change the dose, time of extraction, the ratio of the extractant, and temperature distillation, fractional changes in the composition and so on) did not provide the active series of allergen.

Example 2. Check the activity and specificity of the radial allergen in skin reactions in Guinea pigs.

To test the activity and specificity of the obtained allergen in the experiments were used 30 Guinea pigs, divided into 10 groups of 3 animals each. The 1st and 2nd group of animals was senzibilizirani bacterial antigen was subcutaneously injected anthrax vaccine STI-1 in the optimal immunizing dose; 3-th and 4-th-were immunized jehovam antigen from burnt animal; 5 and 6 were irradiated with gamma rays at a dose of 5.5 Gy (LD90); 7-th and 8-th group of animals was immunized and not irradiated (biological control). Through 10 days after exposure and immunization the animals of the 1st, 3rd, 5th, 7th groups intradermally into the region of the side surface of the back (pre-wybrew plot p is ewedu 3x3 cm) was injected beam allergen - vegetable radiotoxic 1 dermal dose of 1.5 μg/ animal), and animals 2-nd, 4-th, 6-th and 8-th groups, respectively cloth allergen from thin intestine obtained from irradiated mice. Reading reactions were performed 24-48 h after injection of allergens. It is established that the tested allergen - radiotoxic positively with index allergies 2,5-3,2 reacted only irradiated at a dose of 5.5 Gy Guinea pigs. The reaction to the allergen in the intact (non-irradiated), inoculated with the anthrax vaccine, and grafted burns antigen animals in all cases was negative. In contrast, we offer, is known (tissue) allergen from the intestine of irradiated mice (heterologous allergen) gave false positive reactions in all variants of experiments with index allergies from 1.1 to 2.1 as in irradiated and in vaccinated and control animals. Therefore, the proposed allergen surpassed known not only activity, but also on the main criterion of specificity.

Example 3. The effectiveness of the proposed allergens for the diagnosis of radiation injuries of animals and the possibility of differentiation lethal exposure to sublethal (non-fatal) were tested for farm animals, which are used adult sheep with average live weight 35-38 kg, with 3 animals in each variant of the op is the one. While sheep of the 1st group were irradiated with gamma rays in lethal (Gr 5-6), the 2nd group - in sublethal (2.5 to 3 Gy), 3rd group were immunized bacterial (anthrax) vaccine, the 4th group was subjected to a heat treatment to burn, animals, the 5th group was not subjected to any impact (biological control). 24 hours after physical (radiation burns) and biological (anthrax immunization vaccine) effects of experimental and control animals intradermally into the region of the inner thigh has introduced the proposed plant allergen - quinoid radiotoxic 1 dermal dose of 1.5 μg/animal) in the amount of 0.1 cm3. Reading reactions were carried out through 24, 48, 72, 96, 120, 144 h after injection of the allergen. Found that the first reaction used the allergen was shown for 3-4 days in irradiated fatal (5-6 Gy) dose sheep, in the initial period with the intensity of 2.1 and 2.7, for 5-6 day - 3,1-3,5. Intradermal reaction to the allergen in animals exposed to sublethal (2.5 to 3 Gy) dose was less intense (1,12-1,17) and maximum response (1,19-2,21) have evolved to 5-6 days after exposure. In contrast to irradiated animals reaction to the tested allergen in sheep vaccinated anthrax vaccine and subjected to a heat treatment (burn)in all study periods was negative.

Comparative characteristics of methods p the radiation beam allergens and their use for allergic diagnosis of radiation injuries of the body presented in the table.

As can be seen from the table, the proposed method for obtaining antigen for the diagnosis of radiation injuries of the body allows to exclude from the process the use of expensive laboratory or C. farm animals, their slaughter, the removal of organs and obtaining tissue antigens, and replace them with cheap and easy process raw potatoes, increase the specificity of the drug by overcoming tissue barrier, i.e. exceptions to the allergen main factor non-specificity - a whole group of tissue antigens, to reduce the time of manufacture of the drug and to expand the range of its application, i.e. the resulting preparation is optimized biochemical composition, uniform and suitable for research of all kinds of animals.

1. The way to obtain allergic drug for the diagnosis of radiation injuries of the body, including extraction from irradiated at a dose of 350-400 G of potato tubers quinoid radiotoxins by ethanol extraction, removal of solvent on a rotary evaporator, the sludge acidification with hydrochloric acid to a pH of 7.2 and an additional extraction with ethyl acetate, characterized in that received an ethyl acetate fraction is subjected to chromatographic purification by applying the above fraction to chromatographies the second paper (20× 20 cm) and dispersed in the system: a) 2%acetic acid; b) a mixture of benzene - acetic acid - water in the ratio 2:4:1, respectively; the resulting specific allergenic fraction of the chromatogram elute 2%solution of acetic acid, and the final product will standardize on the dry substance at the rate of 1 mg/cm3.

2. A method for the diagnosis of radiation injuries of the body, including intradermal introduction of specific allergen and identification of autocannibalism, characterized in that as a specific allergen animals injected purified fraction of plant quinoid radiotoxins in the amount of 0.1-0.2 cm3(1.5 to 2.0 μg per animal) into the region of the inner thigh (sheep and goats), in the upper third of the back (cattle and horses) and the rear side at the base of the ear (pigs), and detection of autocannibalism set by the reaction of the body (skin) at the site of injection of the allergen through 24, 48, 72, 96, 120, 144 h after irradiation and the index of allergies (IA)equal to or in excess of 1.12 (IA≥1,12), establish the diagnosis of radiation sickness.



 

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FIELD: medicine, radiation biology.

SUBSTANCE: invention proposes a method for preparing allergenic preparation used for diagnosis of body radiation injures. Method involves irradiation of potato tubers by the dose 350-400 Gr, the following extraction of quinoid radiotoxin by extraction with ethanol followed by removing extractant in rotary evaporator and additional extraction with ethyl acetate. The final prepared fraction is subjected for chromatography and separated in the system: (a) 2% acetic acid, and (b) mixture benzene - acetic acid - water in the ratio = 2:4:1, respectively. The prepared allergenic fraction is eluted with 2% acetic acid solution and standardized by dry matter as measured for 1 mg/cm3. Also, invention proposes a method for diagnosis of body radiation injures involving intracutaneous administration of specific allergen and detection of autosensitization symptom. Diagnosis of radiation diseases is proved by the allergy index. Proposed methods provide preparing the specific radiation allergen for detection of radiosensitization of body and to carry out diagnosis of radiation disease. Invention can be used in radiation biology.

EFFECT: improved preparing method, improved method for diagnosis.

1 tbl, 3 ex

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