RU2240807C2 - Method for obtaining substances out of mammalian tissues and organs being of autoimmune activity both in vitro and in vivo - Google Patents

Abstract

FIELD: medicine, clinical immunology.

SUBSTANCE: material should be typified, homogenized with quartzitic sand and 0.9%-sodium chloride solution at 1:5 ratio, extracted by Grasse, at a 5-fold cycle frozen at -17 C and defrosted at +37 C, homogenate should be centrifuged to isolate supernatant and standardize there protein due to spectrometric technique 20-22 mg/ml and lyophilically dried, moreover, additionally, during the 4^th cycle of freezing it is necessary to conduct quantum treatment of a substance with gamma-quantums at 0.37 kJ energy at integral dose being 1.8 x 10⁴ Gr for 20-25 min. As a result, substances keep initial organic tissue specificity and are free of individual properties of isoantigenic form of donor's initial material. Combination of these features provides high accuracy of tissue identification in reactions in vitro and manifests its activity in vivo, as well. Substances have high purity of the substance as it is.

EFFECT: higher efficiency.

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Description

The invention relates to medicine, in particular to clinical immunology, and is a method for producing substances (autoantigens of tissues) having immune (autoimmune) activity against tissues and organs of mammalian organisms in vitro and in vivo. These substances can be used to quantify in vitro tissue autoantibodies circulating in the blood. This is realized by setting up the indirect hemagglutination reaction (RIGA) in a microtest system with one of these substances, and the results of this reaction can be used to assess the state of the studied tissue, and hence the organ. The sequential formulation of RIGA with a set of obtained substances allows us to determine the autoimmune state of the body at a given point in time (research) and monitor autoimmunity in normal and pathological conditions.

And also these substances can cause damage and lysis of tissue homologous to them, accompanied by shockogenic, hyperergic reactions of the body, up to the death with the introduction of in vivo.

Known methods for producing immunologically active substances from tissues and organs of mammals [1], [2], [3].

The closest is the method [3], where for the preparation and preparation of an immunoactive substance (antigens of the tissues of the human eye), human tissues of group I (0) of the Rh (-) group who died from accidental injury were used. The organs were freed of fat, washed 7-10 times with chilled NaCl 0.89% pH 7.2 and crushed with sterile silica sand and NaCl 0.89% in a mortar in a ratio of 1: 5. The crushed homogeneous tissue was placed in a box at a temperature of (-20 ± 1) ° C for 18 hours, then it was transferred for 6 hours at a temperature of (+ 37 ± 1) ° C and again moved to the refrigerator. This procedure was repeated 4-5 times, ensuring the transfer of soluble antigens (AG) to the solution. Then the suspension was centrifuged at 22300 q for 30 minutes. The obtained supernatants containing soluble AH of the corresponding organ were aspirated, adjusted to a protein content of 20-25 mg / ml and freeze-dried on a KS-6 apparatus. With this method, tissue AH remained active for 1-2.5 years.

This method provides a sufficiently high purification and preparation of AH and substances like them, but when RNGA is set to titer up to 1:16, cross-reactions with antibodies of other organs occur, i.e. there is immune activity (homologous antigen - homologous antibody and homologous antigen - heterologous autoantibody). In this situation, one cannot talk about the high specificity of the substance and define it as a direct autoantigen.

This method does not provide the necessary purity and specificity of the reaction and, as a consequence, accurate diagnosis of the state of the tissue, apparently due to impurities of additional ingredients (insufficient purity of the substance, the content of direct antigens, autoantigens and substances like them), as well as because complex structure of these substances.

We first developed a method for obtaining substances from tissues and organs of mammals with autoimmune activity in vitro and in vivo. Also, these substances retain the initial organ specificity of the tissue and are deprived of the individual properties of the isoantigenic form of the source material of the donor (organism). The combination of these properties is able to provide greater accuracy of tissue identification in in vitro reactions and to manifest its activity in vivo. Substances have a great purity of the substance itself.

The method we have proposed is characterized in that the starting material in the frozen state (the fourth cycle of cryogenic treatment) is additionally irradiated with γ-rays with an energy of 0.37 kJ, an integrated dose of 1.8 · 10 ⁴ Gy, and a U-10 high-voltage linear accelerator for 20-25 minutes . The parameters and time of irradiation are selected so that the processed substance is capable of energy storage. With a longer exposure to the substance, its biological structure is damaged.

The method is as follows: To prepare the substance from the tissues of the liver, kidneys, heart, stomach, brain, pancreas, thymus, thyroid gland, skin, typed tissue sections of animals and a healthy person I (0) of group Rh (-), who died from accidental injury. Typed tissues of the small intestine and lung were obtained from animals and a stillborn child. After release from fat, a weighed portion of organs was ground with quartz sand and 0.9% NaCl solution at the rate of 1: 5, and the substance was extracted according to Grasse with five freezing (-17 ° С °) and thawing (+ 37 ° С °). Moreover, in the fourth freezing cycle, the substance was treated with gamma rays with an energy of 0.37 kJ with an integrated dose of 1.8 · 10 ⁴ Gy. In this case, the irregularity of the irradiation did not exceed 5%, which was achieved by defocusing the electron beam. The radiation dose was controlled by an air-equivalent device (VA-K-252 bar camera), working in conjunction with the VA-J-18 device. The deceleration of high-energy electrons (up to 5 MeV) occurred on a tungsten target and its center was taken as the origin of the distribution of the gamma radiation field. Irradiation doses were calculated according to a pattern in which there was a direct dependence of the irradiation time and the inverse of the target object’s square of the distance. The reference point of radiation was at a distance of 100 cm from the exit window of the accelerator. Exposure to 20–25 minutes.

The supernatant after centrifugation (22300 q) was standardized for protein (20-22 mg / ml), lyophilized and the target substance was obtained.

Example.

The guinea pig liver tissue was sampled. The tissue is typed, freed from connective fibers, fat, homogenized. A sample was taken and ground with quartz sand and 0.9% NaCl solution at the rate of 1: 5. The substance was extracted according to Grasse with a five-fold freezing cycle (-17 ° C) and thawing (+ 37 ° C °). After the third thawing, the drug was divided into two equal parts, which were frozen. One part in the fourth freezing cycle was subjected to treatment with gamma rays of 0.37 kJ energy with an integrated dose of 1.8 · 10 ⁴ Gy. for 23 min at the U-10 installation, and the other part was not subjected to treatment. Conducted another cycle of freezing and thawing. Obtained after thawing, both parts of the homogenate were centrifuged (22300 q), the liquid part of the supernatants was separated, protein standardized (22 mg / ml in both portions), both portions of the obtained supernatant (irradiated and not irradiated) were poured into glass ampoules (5 ml), marked and subjected to freeze drying. Dry matter was obtained in both ampoules (one subjected to gamma radiation, the other not subjected to gamma radiation), after which the ampoules were hermetically sealed under sterile conditions.

A comparative assessment of the structures of the obtained substances was made.

1. A tissue substance treated with gamma rays (γ +).

2. Tissue substance without treatment with gamma quanta (γ-) The study was carried out by high performance liquid chromatography (HPLC - gel exclusion chromatography), which allows us to separate the test substances by molecular weight and ultraviolet scanning (UV), which allows us to assess the purity of the separated preparations by determining the wavelength of maximum adsorption, taking into account the spectra of each peak at a wavelength of 200 to 350 nm, with an interval of 5 nm.

HPLC was performed in isocratic mode using a finished gel filtration column NSK300SW, size 7.5 × 300 mm. For elution, a buffer prepared with deionized water containing 0.14 M NaCl and 0.02% NaN ₃ at a flow rate of 1 ml / min was used. A two-channel flow spectrophotometer with a variable wavelength tuned to 254 and 280 nm served as a detector.

During chromatography, it was found that the liver (γ-) sample has four peaks with a retention time of 4.5; 8.5; 11.2 and 12.3 minutes, which indicates the inhomogeneity of the drug and its multicomponent nature (at least 4) (Fig. 1a). UV scanning of these four peaks also showed heterogeneity of the drug with several peaks of absorption maximum. The fourth peak was the most homogenous and differed from the three absorption peaks (Fig. 1.1-1.4).

A sample of liver tissue substance (γ +) during HPLC analysis came out with only one peak with a retention time of 12.3 minutes. (Fig. 2a) and when UV scanning was found one of its maximum absorption, which indicates the homogeneity of the drug (Fig.2.1-2.3). According to the spectral characteristics and retention time, it corresponded to the fourth peak of a sample of liver substance (γ-). In other words, treatment with γ-quanta “cut off” the first three peaks of the γ- preparation and made the γ + preparation homogeneous and homogeneous.

The obtained substances from the tissues of the organs of γ + and similar γ- were used to obtain antiorgan erythrocyte antigenic diagnostics (PED), which make it possible to identify homologous autoantibodies in serum in the RNGA microtest system according to classical methods. In this case, hyperimmune rabbit anti-hepatic serum was obtained (similarly, all other organ-specific hyperimmune rabbit serums were obtained). This was accomplished by subcutaneous administration of 1 ml of a substance obtained from liver tissue diluted in 2 ml of distilled water, together with 5 ml of heated (45 ± 1) ° C of Freund's complete adjuvant. Every 7 days, the rabbit was injected intramuscularly three times with 1 ml of a substance obtained from liver tissue, followed by bleeding 10 days after the last injection. For every 100 ml of serum, 0.5 ml of a 10% aqueous quinosole solution (preservative) was added and poured into ampoules.

The sensitivity control and specific activity of the anti-organ erythrocyte diagnosticums PED (γ +) compared to PED (γ-) were studied in RIGA using the C. Takatsy micromethod (1952) according to the “Instructions for the use of serological diagnostic methods for epizootological examination of natural plague foci” (Saratov, 1974 )

The specificity of the positive result of RNGA (negative control - the absence of passive hemagglutination) was checked in the inhibition of indirect hemagglutination (RTNGA) when a corresponding tissue substance with antigen properties was added to the serum, which is accompanied by the formation of complexes like antibody + antigen and erythrocyte agglutination.

It was found that when using PED (γ +) for liver tissue material with the corresponding hyperimmune antihepatic rabbit serum, the absolute titers in RNGA were (1: 512-1: 1024), and with PED (γ-), respectively (1: 64-1: 128), that is, the sensitivity of the method increased by 3-4 times when using PED (γ +).

In addition, the use of PED (γ +) made it possible to increase the specificity of RNGA by eliminating cross-reacting tissue substances with antigen activity in the substance. For example, a liver tissue antigen (according to the prototype) in a small titer (1: 8) in some cases gives a positive reaction with rabbit hyperimmune serum to kidney tissues. In other words, strictly “antihepatic” autoantibodies are identified, since the substance after treatment with γ-quanta has become strictly “hepatic”.

Thus, treatment of tissue substrates with γ-quanta is accompanied by a change in their isoantigenic structure, “architectonics” and contributes to a more specific structure of the obtained substance, which, when used as sensitins for RNGA, is accompanied by an increase in the sensitivity of the reaction by a factor of 3-4 and its specificity.

The resulting substance can be considered as a specific marker of tissue, which is able to register homologous autoantibodies of the studied tissues in blood serum (in vitro).

When this substance is introduced into the recipient's body (we examined the reaction of the organism and liver tissue of the guinea pig to the administration of the substance we obtained), a number of hyperergic type disorders occur, in the form of sequential chocogenic reactions that trigger general body thanatogenesis due to lysis of hepatocytes and rejection of damaged liver tissue. Control by electron microscopy confirms the lysis of hepatocytes and the preservation of tissue indifferent in their isoantigenic structures, which once again confirms the high specificity of this substance and its activity (in vivo).

EFFECT: obtaining a substance possessing immune (autoimmune) activity in vitro and in vivo, increasing its activity and tissue specificity, which is manifested in increasing the reliability of diagnostic results, greater specificity of the reaction, stability and repeatability of the results.

Literature

1. Kargina I. B., Sysoeva O. B., Arion V. Ya., Lopukhin Yu.M. A method for producing immunologically active substances from neurosecretory structures of the mammalian brain epithalamus. 1999 year

2. Shanina L.N. Autoimmune reactions in plague and cholera vaccine and infectious processes and methods for their correction. Dis. MD, Saratov, 1985

3. Gulida O.L. Obtaining an erythrocyte diagnosticum for eye antigen tissues (lens, vitreous body) 1996

Claims (1)

A method of obtaining substances from tissues and organs of mammals with autoimmune activity in vitro and in vivo, characterized in that the material is typed, homogenized with quartz sand and 0.9% sodium chloride solution in a ratio of 1: 5, extracted with Grasse five times cycle, freeze at -17 ° C and thaw at + 37 ° C, homogenate is centrifuged and the supernatant is isolated, the protein in it is standardized with a 20-22 mg / ml spectrometric method, lyophilized, and an additional gamma quantum is carried out in the fourth freezing cycle th processing agents γ quanta energy of 0.37 kJ, integrated dose of 1.8 × 10 ⁴ Gy, for 20-25 minutes.

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