RU2731521C1 - Method for diagnosing radiation damage of organism and method for preparing antiradiation antibody bentonite preparation for diagnosing radiation injuries of organism - Google Patents

Abstract

FIELD: radiation biology.

SUBSTANCE: group of inventions relates to radiation biology, in particular to production of preparations for diagnosing radiation injuries of an organism, and discloses a method for diagnosing radiation injuries of an organism, as well as a method of producing an antitracarontal anti-radiation bentonite preparation (ARBP). Diagnostics method involves serological analysis of blood serum of irradiated animals in bentonite flocculation reaction (BFR) and detection of radiotoxins. Serological analysis of blood serum in BFR is carried out using an anti-radiation antibody bentonite preparation (ARBP), in which the antibody carrier is an anti-radial antibodies coated with antibacterial antiserum serum, bentonite particles, and on phenomenon of flocculation of sensitized bentonite nanoparticles, presence of radio-induced antigen - radiotoxin in examined serums is stated and diagnosed for radiation injuries.

EFFECT: invention enables diagnosing radiation injuries of an organism in the early period after irradiation.

2 cl, 1 tbl, 3 ex

Description

The invention relates to radiation biology, the production of drugs intended for the diagnosis of radiation damage to the body.

A known method for the diagnosis of radiation damage to the body by hematological analysis of peripheral blood and the determination of the neutrophilic index (see the book. "Veterinary anti-radiation protection". - Ed. VA Kirshin and VA Budarkov. - M .: Agropromizdat, 1990 . - P. 111).

The disadvantage of this method is its limited application, since it is designed only for the diagnosis of radiation sickness in sheep, and it is not suitable for other mammals.

A known method for the diagnosis of radiation injuries in humans by detecting autoantibodies in the blood serum by the method of indirect immunofluorescence (NRIF) on cryostatic thymic media in a 14-day culture of thymic epithelial cells of newborn mice (see collection of abstracts "Human immune status and radiation". - M ., 1991. - S. 31).

However, the known method is complex, expensive, time-consuming, and most importantly, the assessment of research results is subjective.

A known method for diagnosing radiation damage to the body by serological analysis of blood serum, irradiated animals in the reaction of indirect hemagglutination (RIGA) using antiradiation antibody erythrocyte diagnosticum (ATED) and upon detection of a radiotoxin in the blood serum, diagnose radiation sickness (see RF Patent No. 2145712, publ. 20.02.2000, bull. No. 5; RF Patent No. 2240137. - publ. 20.11.2014, bull. No. 32).

The disadvantage of this method is the use of sheep erythrocytes as an immunosorbent, which are characterized by high sensitivity to fluctuations in environmental factors (pH, the presence of chemical agents), leading to inactivation of the erythrocyte diagnosticum.

A known method for the diagnosis of radiation damage to the body by staging a reaction of bentonite flocculation (RBF) using bentonite particulates (see the abstract of the candidate of thesis. P.P. Gainullin "Development of bentonite diagnosticum for the indication of radio-induced toxic compounds." - Kazan, 2009. - 23 p.).

The disadvantage of this method is the complex technology of purification of bentonite and low sensitivity of the diagnosticum, since bentonite microparticles with a size of 60-90 microns are used as an immunosorbent, which have insufficient sorption activity and, due to their low dispersity, quickly precipitate, changing the course of the reaction and reducing the sensitivity of the RBF test. systems for immunochemical analysis of the test material.

Meanwhile, it is known from the field of nanotechnology that the transition of matter into a nanoscale state is accompanied by a change in the fundamental properties of matter, which have a gigantic scientific, technical and socio-economic potential (see the article by A.S. Radilov and V.R. Rembovsky "Nanotechnology and nanotoxicology" / / Toxicological Bulletin. - 2007. - No. 6. - P. 4-8).

Therefore, one of the promising areas for the use of minerals is the creation on their basis of nanosized medicinal and diagnostic preparations with more pronounced positive properties and, in this regard, there is a constant need for biological testing of nanosized particles obtained from mineral raw materials.

The objective of the present invention is to develop a highly sensitive method for the diagnosis of radiation injuries of the body through the use of anti-radiation antibody bentonite diagnosticum, which increases the formulation of a lifetime express - diagnosis of radiation injuries in the early stages after lethal exposure.

The problem is solved by using the immunological analysis of the blood serum of irradiated animals in the reaction of bentonite flocculation (RBF), which is carried out using antiradiation antibody bentonite diagnosticum (ATBD), in which bentonite nanoparticles sensitized with antiradiation serum are used as a carrier of specific antibodies. Then, by the phenomenon of flocculation of sensitized nanoparticles of bentonite, the presence of a radio-induced antigen-radiotoxin in sera is judged, by the degree of flocculation of sensitized nanoparticles and by the titer of radiotoxin in the RBF test system, the diagnosis is made for radiation damage. For the diagnostic titer of radiotoxins in RBF, a positive reaction is taken in dilutions of the studied sera of 1: 8 and higher with a degree of flocculation of nanoparticles of 3-4 crosses, and as an assessment criterion for the severity of lethal exposure (the degree of acute radiation sickness of animals) - an 8-10-fold increase diagnostic titer of radiotoxin in blood serum in RBF (1: 64-128).

In the present invention, an antiradiation antibody bentonite preparation (ATBD) for the diagnosis of radiation injuries of the body is obtained by extracting a potato-radiotoxin from irradiated at a dose of 350-400 Gy, hyperimmunizing rabbits with it by intramuscular 4-fold injection of antigen at a dose of 1.0 with an interval of 14 days -2 cm ³ , conjugated with incomplete Freud's adjuvant in a 1: 1 ratio, obtaining diagnostic sera, sensitization of the latter to bentonite nanoparticles. Dispersed bentonite powder (thermally activated bentonite clay) with a particle size of 75-85 nm is used as bentonite nanoparticles, which is obtained on an ultrasonic unit "UZU-0.25" at a frequency of 18.5 kHz, an output power of 80 W and a waveguide vibration amplitude of 5 μm , and sensitization of bentonite nanoparticles is carried out by first mixing bentonite nanoparticles with distilled water to obtain a 0.19-0.20% aqueous suspension, and then mixing it with hyperimmune serum in a ratio of 3-7, respectively, followed by thermostating the mixture at a temperature of 30 ° C for 30 min, then the mixture is centrifuged at 3000 rpm for 7 min, the resulting precipitate is washed twice with distilled water at 3000 rpm for 10 min, the supernatant is decanted, and the precipitate is resuspended in distilled water to a concentration of 0.19 -0.20%, then the indicator component is introduced into the resulting immunological complex - 0.1% methylene blue at the rate of 0.7 cm ³ per 100 cm ^{3 of the} suspension obtained by the anti-radiation antibody bentonite diagnosticum (ATBD) is stored in a refrigerator at a temperature of 2-6 ° C, used as specific antibodies in the bentonite flocculation reaction (RBP) to detect radiotoxins in the studied sera.

A significant difference between the proposed method for the diagnosis of radiation injuries of the body and the method of obtaining anti-radiation antibody bentonite preparation for the diagnosis of radiation injuries of the body is that a highly specific and sensitive test system is introduced in the irradiated body - the reaction of bentonite flocculation (RBF) to detect a specific radio-induced antigen - a radiotoxin. ) using bentonite nanoparticles with sizes of 75-85 nm, obtained by dispersing bentonite powder in an ultrasonic device "UZU-0.25" at a frequency of 18.5 kHz, an output power of 80 W and a waveguide vibration amplitude of 5 μm, and taking into account the timing the appearance of radio-induced antigens and the degree of their concentration from the dose of lethal radiation.

The use of nanosized bentonite up to 100 nm (75-85 nm) in the bentonite flocculation reaction promoted an increase in its sensitivity by 9-10 times compared to the microanalogue (the maximum titers of the standard antigen-radiotoxin in RBF with a known diagnosticum were 1: 220-1: 250, and when using the proposed (nanodiagnosticum) - 1: 2250-1: 2500.

The essence of the proposed method is based on the fact that under the influence of an ionizing reaction in the body, radiolysis of water occurs with the appearance of highly active radicals, which, by attaching to adducts of proteins, lipids, amino acids, form toxic products of molecular modification - quinoid and lipid radiotoxins with antigenic properties, entering into immunochemical reaction with homogeneous antisera in specific test systems (RNGA, RBF). The timing of the appearance of radio-induced antigens (radiotoxins) in the body of irradiated animals falls on the first hours and days after radiation injury and is recorded during the entire period of the peak of acute radiation sickness (the first 10-14 days after exposure).

The use of the proposed antibody bentonite diagnosticum (ATBD) in the immunochemical test system (RBP) allows detecting radiation antigens-radiotoxins (RT) in the blood serum of an irradiated organism in 1.5-2 hours, which appear in the first hours and days after irradiation in concentrations, corresponding to the severity of radiation damage.

This technology leads to a significant simplification of the method, increased speed, labor productivity, reliability of analysis results and, most importantly, the ability to predict the course and outcome of acute radiation sickness (ARS).

A method for diagnosing radiation injuries to an organism and a method for producing an anti-radiation antibody bentonite preparation (ATBD) for diagnosing radiation injuries in an organism is carried out as follows.

At the first stage, an immunosorbent of bentonite nanoparticles is obtained by dispersing bentonite of any origin (thermostated bentonite clay) in an ultrasonic unit UZU-0.25 at a frequency of 18.5 kHz, an output power of 80 W and an amplitude of vibration of an ultrasonic waveguide of 5 μm (see the abstract by Motina T .Yu., "Pharmacotoxicological evaluation of nanosized bentonite" Kazan. 2014. - 23 p.).

The specified modes of ultrasonic treatment of bentonite provide nanoparticles with sizes of 75-85 nm. The obtained nanosized bentpowder is suspended at the rate of 190-200 mg / 100 cm ^{3 of} distilled water and a 0.19-0.20% aqueous suspension of bentonite is obtained.

At the second stage, sensitin, a specific antiradiotoxic serum (ARTS), is obtained by hyperimmunizing rabbits with a conjugated antigen containing a specific radiomimetic - radiotoxin, which is used as an ethanol extract of irradiated plant tissue. To obtain a radiotoxin, potato tubers are used, which are irradiated with gamma rays at a dose of 350-400 Gy at an exposure dose rate of 2500 Gy / h. Irradiated tubers are kept at a temperature of 18-22 ° C for 24 hours. Further processing of the irradiated object in order to isolate the antigenic material - radiotoxin is carried out according to the method of S.K. Melnikova and V.A. Kopylov (see the book. Radiotoxins, their nature and role. Ed. By A.M. Kuzin. - M .: Atomizdat, 1966. - S. 86-91). The top layer 3-4 mm thick is removed from the potato tubers and the remaining tissue is homogenized in a threefold volume of 96% ethanol. The extraction is carried out for 1 hour with constant stirring, the stroma is separated and the alcohol is removed under vacuum at a temperature of 28-33 ° C.

In the resulting ethanol extract, protein is determined according to Lowry, quinones - according to A.M. Kuzin and V.A. Kopylov (Radiotoxins. - M .: Nauka, 1983, - S. 27-28), the dry matter content - by the generally accepted method of drying, biological activity - in the test of 50% hemolysis of erythrocytes by the method of acid erythrograms (I.A. Terskov, Gitelzon IN. - Biophysics, 1957, No. 2, P. 259.), antigenic activity - in the reaction of diffusion precipitation - RDP according to E.N. Shlyakhov (Antigens of macroorganisms and responses. - Kishinev, 1962, - S. 123-126).

The obtained concentrated extract containing quinoid radiotoxins is standardized by dilution with sterile distilled water at pH 7.2 to a concentration of 1.0 mg / ml. The resulting preparation, consisting mainly of quinones, is defective in antigenic terms, because due to the insignificant (1-1.8%) amount of protein, it has a low molecular weight (5-6 kDa), which characterizes it as a hapten and, in view of this cannot cause an immune response in the immunized organism. Therefore, to convert the hapten into a complete protein antigen, the obtained ethanol extract (0.1% solution of plant radiotoxin) is mixed in a 1: 1 ratio with Freund's incomplete adjuvant (NAF). The conjugation of the antigen and the immunomodulator is carried out on a shaker at an intensity of 110-120 swings per minute for 15-17 minutes. A conjugated antigen containing a specific radiation substance, a radiotoxin, is used as an immunizing agent for hyperimmunization of laboratory (rabbits) or farm (sheep) animals.

Hyperimmunization of rabbits with a live weight of 2.0-2.5 kg is carried out according to the following scheme. The conjugated antigen prepared according to the above method in the form of an emulsion is heated in a water bath to 37 ° C before use and injected into rabbits 4 times, intramuscularly in the area of the inner surface of the thigh with an interval of 14 days between injections of 1.0-2.0 cm for each introduction. On the eighth day after the last injection of antigen (AH), a blood sample is taken from the ear vein in rabbits to determine the level of antiradiotoxic antibodies, which is studied in the indirect hemagglutination reaction (RIGA) according to K. Malborg (Immunological methods M .: Medicine, 1987. - P. 211-218). Plant radiotoxin (RT) is used as a positive antigen, and AG from unirradiated potato tubers is used as a negative antigen. The production of antiradiotoxic serum is carried out according to RF Patent No. 2240137 dated 20.11.2004 Bull. No. 32.

At the next stage, nanoparticles are sensitized (loaded with specific antitoxic antibodies) by mixing a 0.19-0.20% aqueous suspension of bentonite with hyperimmune antiradiotoxic serum in a ratio of 3: 7 (30 cm of bentonite suspension + 70 cm of hyperimmune serum). The resulting mixture is thoroughly mixed and placed in a thermostat at 30 ° C for 30 minutes, then the mixture is centrifuged at 3000 rpm for 7 minutes, the resulting precipitate is washed twice in 100 cm ³ distilled water at 3000 rpm for 10 minutes, the supernatant decanted. The resulting precipitate (centrifugate) is resuspended in distilled water to a concentration of 0.19-0.20%.

For visual indication of the antigen (radiotoxin) - antibody complex, in the RBF test system the resulting complex is labeled with a special indicator - methylene blue at the rate of 0.7 cm ³ per 100 cm ^{3 of the} diagnosticum.

The antibody bentonite diagnosticum (ATBD) obtained by the above method is used as a source of antiradiotoxic antibodies in the reaction of bentonite flocculation (RBP) to detect radiotoxins in the blood serum of animals and is stored in a refrigerator at a temperature of 2-6 ° C.

The diagnosis of radiation damage to the body is carried out as follows. Peripheral blood is taken from the examined animals and the serum is obtained from it by a conventional method.

The resulting serum is used as an antigen-containing component in the reaction of bentonite flocculation (RBF), the statement and evaluation of the results of which is carried out in accordance with the methodological recommendations of Z.V. Pruchkina and others (USSR AS No. 952260 A61K 39/00. Publ. 08/23/1982. Bull. No. 31).

As a source of specific antibodies in RBP, the ATBD obtained by the above-described method is used.

The reaction is accompanied by appropriate controls: 1) serum of intact animals (negative control); 2) serum of irradiated animals (positive control); 3) standard radiotoxin (positive control); 4) serum of irradiated animals (heterologous control).

For the "positive" reaction of bentonite flocculation, the sedimentation of bentonite nanoparticles (AGBD) sensitized with antiradiotoxic antibodies at the bottom of the wells of immunological plates with an estimate of 3-4 crosses in a dilution of the tested sera of 1: 8 and higher in a negative reaction with heterologous and negative controls is taken.

A positive reaction of bentonite flocculation (RBP) with the tested sera at a dilution of 1: 8 and higher (flocculation degree 3-4 of the cross) indicate the damage to the body by ionizing radiation, causing acute radiation sickness (ARS) of mild, moderate and severe severity.

At the same time, the differential diagnostic criteria for the degree of ARS are the titers of the radiotoxin in the RBF: an 8-10-fold excess of the diagnostic titer (1: 64-1: 128) indicates that the body is damaged by lethal (fatal), and the titers are within 1: 16-1: 64 - about the defeat by sublethal (non-lethal) radiation doses.

The method for diagnosing radiation injuries has been tested on laboratory (guinea pigs, rabbits) and agricultural (sheep) animals. The animals were irradiated on a Puma gamma device with a ¹³⁷ Cs radiation source at an exposure dose rate of 5.68 R / min. The development of radiation sickness was assessed in terms of clinical and hematological parameters, dynamics of death, pathological changes in organs characteristic of radiation sickness and animal survival.

Example 1. The effectiveness of the method for the diagnosis of radiation injuries of animals using the known (microanalogue) and the proposed diagnosticum. For this purpose, 15 guinea pigs were subjected to lethal gamma irradiation at the Puma installation at a dose of 6.0 Gy; 10 animals were not irradiated and used as biological control. In control and irradiated animals in dynamics after 1; 1.5; 2; 4; 8; 10 daily for the first 7 days, and then after 10; fourteen; 18; 22; On days 26 and 28 after irradiation, blood samples were taken for immunological analysis in the RBF test system. The results of the studies are presented in table 1.

From the data in the table it can be seen that the proposed diagnosticum is much more sensitive (4-8 times) than the known analogue (microparticles of bentonite), which, within 1 hour after irradiation, reveals radiotoxins in a diagnostic titer (1: 8), while a microanalogue detects them In 1.5 hours. At the same time, by the beginning of the ARS height (day 8), the titers of radiotoxin in RBF with nanodiagnosticum were 1: 256 versus 1:48 with microdiagnosticum. The maximum radiotoxinemia (1: 128 with the known diagnosticum and 1: 1024 with the proposed one) of the blood serum of lethally irradiated guinea pigs coincided with the period of maximum radiation death of the irradiated animals, i.e. 10-20 days after irradiation.

Example 2. Checking the specificity of the nanodiagnosticum. For this, the experiments were carried out on 15 rabbits, divided into 3 groups of 3 animals each. Rabbits of the 1st group were subjected to gamma-irradiation at a lethal dose (11.0 Gy), the 2nd group - thermal burns by applying a metal plate heated to 100 ° C to the lateral surface of the body. The animals of the 3rd group were not irradiated and were not twitched with thermal effects - they served as biological control. In control, irradiated and burnt animals in dynamics (daily for 10 days after thermal and radiation exposure), blood samples were taken for immunodiagnostics for the detection of radiotoxin and thermotoxin.

The results of the immunochemical analysis of blood sera in dynamics showed that in the irradiated rabbits, already 1 hour after irradiation, radiotoxins in the diagnostic titer (1: 8) were found in the blood serum, which gradually increased and after 72 hours they reached values of 1:64 with the maximum value the titer of radiotoxin by the 10th day (1: 512). In the blood serum of non-irradiated and thermally exposed animals, the desired radiotoxins were not found at all times of the study, which indicates the specificity of the proposed diagnosticum.

Example 3. The effectiveness of the studied method for the diagnosis of radiation injuries of animals and the possibility of differentiating lethal from sublethal (non-lethal) irradiation was carried out on farm animals, which were 18 adult sheep with an average live weight of 35-38 kg, using 6 animals for each variant of the experiment. The sheep of the 1st group were irradiated with gamma rays at a lethal dose (6.0 Gy), the 2nd - with a half-lethal (4.0 Gy), and the 3rd - non-lethal (2.0-2.5 Gy). Blood samples were taken from all sheep in dynamics on days 7, 10, 14, 18, 22 and 26 after irradiation and subjected to their immunochemical analysis in RBF with the proposed nanodiagnosticum (ATBD).

It was found that in all irradiated animals in the blood serum of radiotoxins, the concentration of which depended on the dose and the period of ARS development after irradiation. At the same time, it was found that the titers of radiotoxin (RT) in RBF in sheep irradiated with a lethal dose (6.0 Gy) ranged from 1: 256-1: 1024 and all of them died 10-15 days after exposure with a severe ARS clinic ... Animals, in whose sera RT was recorded in titers 1: 64-1: 128, had ARS of moderate severity, and some of them (50%) died on the 18-25th day after irradiation. In sheep, whose sera were found to have PT in titers of 1: 16-1: 64 in the RBF test system, they had mild ARS and deaths among them were not recorded.

Thus, the proposed diagnostic method and a method for producing an anti-radiation antibody bentonite preparation for the diagnosis of radiation injuries to the body makes it possible to urgently diagnose radiation sickness, determine its degree, and predict its outcome, and can be applied both in veterinary and medical practice in radiation damage to the body.

Claims (2)

1. A method of obtaining antiradiation antibody bentonite diagnosticum (ATBD) for the diagnosis of radiation injuries to the body, including the extraction of radiotoxin from irradiated potato tubers at a dose of 350-400 Gy, hyperimmunization of rabbits by intramuscular 4-fold injection of antigen at a dose of 1.0 with an interval of 14 days -2.0 cm ³ , conjugated with incomplete Freund's adjuvant (NAF) in a ratio of 1: 1, obtaining diagnostic sera, sensitization of the latter to bentonite nanoparticles, characterized in that dispersed bentonite powder (thermoactivated bentonite clay) with a particle size of 75 is used as bentonite nanoparticles -85 nm, which is obtained on an ultrasonic installation at a frequency of 18.5 kHz, an output power of 80 W and an ultrasonic waveguide vibration amplitude of 5 μm, and sensitization with bentonite nanoparticles is carried out by first mixing bentonite nanoparticles with distilled water to obtain 0.19-0, 20% aqueous suspension, and then mixing it with hyperimmune serum in a ratio of 3: 7, respectively, followed by thermostating the mixture at a temperature of 30 ° C for 30 minutes, then the mixture is centrifuged at 3000 rpm for 7 minutes, the resulting precipitate is washed twice with distilled water at 3000 rpm in for 10 minutes, the supernatant is decanted, and the sediment is resuspended in distilled water to a concentration of 0.19-0.20%, followed by the addition of an indicator component - 0.1% methylene blue to the immunological complex at the rate of 0.7 cm ³ per 100 cm ³ bentonite suspensions and the resulting product - antiradiation antibody bentonite diagnosticum (ATBD) are stored in a refrigerator at 2-6 ° C. 2. A method for diagnosing radiation injuries to the body, including serological analysis of the serum of irradiated animals in the reaction of bentonite flocculation (RBF) and the detection of radiotoxins in it, characterized in that serological analysis of blood serum in RBF is carried out using antiradiation bentonite diagnosticum (ATBD) obtained according to the method according to claim 1, in which nanosized particles of bentonite sensitized by antiradiation serum are used as a carrier of specific antibodies, and according to their phenomenon of flocculation of sensitized nanoparticles of bentonite, the presence of a radio-induced antigen - radiotoxin in the studied sera is judged; lesions, a positive reaction in dilutions of the studied sera of 1: 8 and higher with a degree of flocculation of nanoparticles of 3-4 crosses is taken for the diagnostic titer of radiotoxins in RBF, and as a differential criterion for lethal irradiation, the abdomen They take an 8-10-fold increase in the diagnostic titer of radiotoxin in blood serum in RBF (1: 64-1: 128).