SU533376A1 - The method of obtaining Toxoplasma antigen - Google Patents

Description

one

The invention relates to the field of immunochemistry, in particular, to methods for producing biological diagnostic agents, namely, an antigen for the serological diagnosis of toxoplasmosis.

Known methods for producing antigens for the diagnosis of toxoplasmosis from an invasive material by extracting it with ether n alcohol-ether-phenol mixture.

However, this method is laborious and time consuming, while the antigen obtained by a known method does not give clear positive serological reactions with blood sera of animals that are spontaneously or experimentally infected with toxoplasma of agricultural and wild animals.

The closest in technical essence and the achieved effect of the present invention is a method including removing and washing Toxoplasma from the cellular elements of the raw material, disintegrating the Toxoplasma cells with ultrasound and isolating the obtained anti-ultrasound lysate with an antigen fraction by ion-ion fractionation.

However, this method also turned out to be unacceptable for obtaining antigen from Toxoplasma, since the resulting antigenic preparations show insufficient serification in serological reactions:

they give positive results not only with homologous toxoplasma blood sera, but also with antiserum to the native peritoneal exudate of healthy white mice, which indicates the presence in the antigens of ballast substances that reduce their activity and specificity.

The purpose of the invention is to develop an express method for obtaining a standard homogeneous antigen with highly active and specific properties, suitable for use in several immunological reactions: in the reaction of complement fixation (CSC), the reaction of indirect hemagglutination (RIGA) and the reaction of microdiffusion in an agar gel ( RP-gel) for the serological diagnosis of toxoplasmosis.

The goal is achieved by the fact that in a known method, including removing and washing Toxoplasma from the cellular elements of a raw material, disintegrating Toxoplasma by sonication and isolating the antigenic fraction by ionic fractionation, ultrasonic disintegration of the Toxoplasma cells is carried out 2-3 times with separation of the toxoplasm by 2-3 times to isolate the separation of the toxoplasm by 2-3 times with the separation of the toxoplasm by separation of the toxoplasm by means of separation of the toxoplasm by separation of the toxoplasm by means of ionization fractionation, ultrasonic disintegration of the toxoplasm by 3–2 times and 3 isolating the antigenic fraction from it with isonon fractionation with an additional effect on the lysate by ultrasound followed by purification of the antigenic fraction from

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non-specific ballast substances by adding a serum to it.

The method for producing the antigen is as follows.

The following reagents and solutions are necessary for carrying out the process: sodium chloride (h. H.), Caustic soda (h. H.), Sulfuric or hydrochloric acid (h. H.), 0.25, 0.5 and 1 n. caustic soda solutions, 0,25, 0,5 "1 n. solutions of sulfuric or hydrochloric acid; a 0.85% solution of sodium chloride (saline).

Getting the raw materials. The material for the production of the Toxoplasmic antigen is the non-ritoneal exudate of the white mice infected with the Toxoplasma Reference Label or one of the virulent colonies isolated from farm animals. To do this, white mice weighing 16–18 g in batches of 200–250 heads are infected with peritoneal exudate from the original toxoplasmic mice. The exudate for infection was diluted with sterile saline so that 1-3 toxoplasmas were visible in the microscope field (magnification 15X40). The dose of infection with mice is 0.2 ml. At 4-5 days after infection, mice are anesthetized with ether and peritoneal exudate is removed from the abdominal cavity with a sterile syringe. The breeding cavity of each mouse is additionally washed with 0.5 ml of saline. The collected material is checked for the presence of toxoplasma and the absence of extraneous microflora by microscopic smears. After testing, the exudate is centrifuged in sterile tubes for 30-45 minutes at 3-4 thousand rpm. The supernatant was removed. The pellet contains Toxoplasma and an admixture of leukocytes and histiocytes of mice peritoneal exudate. To get rid of these cellular ballast components, the pellet is resuspended in 5-10 ml of saline and then repeatedly passed through a thin needle of the syringe to destroy the cellular elements of the exudate. To the resulting suspension add 10-15 ml of physiological solution, again pass through the needle of the syringe and then centrifuge at 2-300 rpm for 45-60 minutes. The supernatant is discarded, and the pellet is repeated 2-3 times more, after which the washed Toxoplasma pellet is suitable for preparing the antigen.

Graduated Toxoplasma Disintegration. Disintegration is carried out in zero ultrasonic waves of a low-frequency disperser (USDN) graduated in three steps: the first two treatments are carried out in a physiological solution, the last — the main process — in distilled water.

1-2 g of washed Toxoplasma are added to a glass vessel with water cooling, 50-100 ml of physiological saline are poured and ultrasonic vibrations with a frequency of 22-35 kHz and an acoustic power of 20-50 W / cm are created in a contact medium for 5-10 min The suspension of partially destroyed Toxoplasma is centrifuged at B-10 thousand.

rpm for 15 minutes The supernatant (lysate I) is drained, and 50-100 ml of saline is added to the precipitate and the treatment of toxoplasma is repeated at a frequency of 22-35 kHz, but with a power of 50-75 w / cm

and already within 30-40 minutes A suspension of disrupted Toxoplasmas is centrifuged at 8-10 thousand rpm for 15 minutes. The supernatant (lysate II), which is the salt-soluble nrotoplasmic part

Toxoplasma is separated from the sediment of the shells of Toxoplasma, which is washed in distilled water by 2-3-fold centrifugation at the aforementioned mode. The washed precipitate is resuspended in 20-30 ml of distilled water, the medium is averaged 0.25-0.5 n. caustic alkali solution in the range of pH 8.0 - 8.2, transferred into the vessel for sound and processed by ultrasound with a frequency of 22-35 KHz, with a capacity of 40-60 W / cm 5-10 minutes.

A small amount of the undissolved precipitate is separated from the liquid portion by centrifugation.

The resulting supernatant is an aqueous solution of Toxoplasma membranes (lysate

Iii) - is a material for obtaining a specific Toxoplasma antigen.

Ultrasonic isoionic focusing and the release of a specific antigen fraction. Fractionation of ultrasound lysate III begins with focusing of ballast fractions, which may be contained in the lysate with insufficient pretreatment of toxoplasm and insufficiently careful graduated separation of toxoplasm with ultrasound into lysate III and non-specific protoplasmic lysates I and II. For this, lysate III is transferred to a 50-100 ml beaker and subjected to a 2-3-minute exposure to an ultrasonic frequency of 22-35 kHz and a power of 50-75 w / cm (without the use of a cooling system), which leads to rapid heating of the treated medium. to an optimum temperature for subsequent fractionation, of 35-40 ° C. Electrometric titration using a pH meter and 0.5-1.0 n. caustic soda solution lysate III is alkalinized to a pH of 10.5-11.5, within which a small amount of sediment is focused in the form of small flakes - the antigen fraction "A. For complete precipitation, the beaker with lysate III is additionally kept in a thermostat at 37 ° C for 20-30 minutes. The precipitate formed is separated

15-20-minute centrifugation at 8-10 thousand rpm.

The supernatant (lysate IV) is again heated by ultrasound to the optimum temperature of 35-40 ° C, slow neutralization of 0.5-1.0 n. sulfuric acid solution

pH is adjusted to 9.0-9.5 and the antigen fraction is focused: B :. Lysate IV incubated in a thermostat for 20-30 min, and then centrifuged. A third precipitate is precipitated from the supernatant liquid (lysate V) in a similar manner at pH 6.8-7.2 and the “C” antigenic fraction is obtained and a basic, cleansing lysate VI is obtained, from which a pH of 3.5-5.5 specific antigenic fraction "/). After centrifugation, the specific " D fraction with ultrasound is dissolved in 20-30 ml of distilled water at pH 8.0-8.2 and is subsequently purified.

Immunochemical purification of the specific antigen fraction "/) is carried out with antisera to the native peritoneal exudate of the white mice (on the ballast substances). It is obtained by immunizing rabbits with positive peritoneal exudate of healthy white mice. 0.2 ml of saturated sodium chloride solution is administered intraerythially to the muscles. After 30 minutes, the saturated solution is reintroduced at a dose of 0.4-0.5 ml. After one hour from the moment of the first injection of a saturated solution, the mice are anesthetized, opened and then the exudate is removed from the abdominal cavity with a syringe. The collected material is centrifuged at 3 thousand revolutions per mission for 30 minutes. The resulting material was administered 4 times to rabbits in increasing doses with an interval of 2-3 weeks between the first and second injections and one week between the next (I immunization cycle). At the first injection, the material is injected in a mixture with Freund's supplement under the skin of the side surface of the rabbit's body. The remaining 2-3 injections of the material are made intramuscularly without Freund's supplement. Reimmunization (and immunization cycle) is carried out in 30-40 days since the last introduction of the material. It consists of one or two injections of material without additional muscle in the thigh with an interval of 4-5 days between injections. On days 7, 14 and 21 after the last administration of the material, the animals take blood and check the serum titer in the RAC or RP in the agar gel. A full cycle of immunization is 4-5 months, and then the antiserum is ready for purification of the specific fraction “D.

The specific antigenic fraction "D" is checked for purity by precipitation with antiserum for native peritoneal exudate of white mice (for ballast substances). To test tubes with a progressively decreasing amount of antigen fraction (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 ml), add 0.5 ml of whole or diluted (in depending on the titer) antisera on ballast vendestva. The mixture is thoroughly mixed and incubated at 37 ° C for 1.5-2 hours, and then placed 18-24 hours in a refrigerator at 4 ° C. Controls are

antiserum and antigein fractions with physiological solution. The reaction of the ballast is considered positive if a precipitate (precipitate) is noted in one or several samples. If the pre-titat is absent in all microtubes (negative reaction), the antigen fraction is immediately used as a specific Toxoplasma antigen. In the case of a positive reaction (precipitate), a tube with the maximum amount of precipitate is used to calculate the required amount of antisera to precipitate ballast and add it to the whole volume of the antigen fraction. The mixture is also incubated for 1.5-2 hours in a thermostat and incubated for 18-24 hours in the refrigerator. The precipitate formed is separated by centrifuging for 20-30 minutes at 8-10 thousand rpm. A purified specific antigen fraction was isolated from the supernatant by ultrasonic iso-ionic focusing at pH 3.5-5.5.

Preparation and standardization of antioxidants. The sediment of the antigenic fraction “purified from ballast“ D and washed in distilled water by 2-3-fold ultrasonic dissolution at 0.8–8.2 and reprecipitation with a pH of 3.5–5.5. The washed precipitate is dissolved in 20-30 lb. of distilled water at a pH of 8.0-8.2. This solution of the antigen fraction is a specific toxoasel antigen.

Standardization of the antigen is carried out by serological activity by titration with hyperimmune toxoplasma serums, the optimal working titer of antigea in RSK and RIGA 1: 20 - 1: 160, and for RP in agar gel - the original dilution. The sensitivity of the Toxoplasma antigen to the detection of specific tukloplaz: ennyx antibodies in serum of hyperimmune, experimentally infected and spontaneously sick animals is characterized in RSK in dilution of serum 1: 5 - i: 640 and in PIU.4 1; 40 - 1: 2560, and in RP in agar gel - one line of precipitation.

To stabilize, the antigen is lyophilized in a working titer with medium. In dried form, in ampoules, it is a white-melted pill which readily dissolves in distilled water and physiological saline. The shelf life of the lyophilized antigen is 2-3 years.

The Toxoplasma antigen prepared according to the described method was tested by a co. Mission, tested in VGIKI and in a production experiment and showed high specificity in the serological diagnosis of toxoplasmosis.

Claims (1)

Invention Formula The method of obtaining Toxoplasma antigen for serological diagnostics, including the removal and washing of Toxoplasma antibodies from cellular elements of the raw material, the disintegration of Toxoplasma by sonication and the isolation of the antigen fraction by anti-anionic fractionation, 5 different in that, in order to increase the specific activity of anti-antigens by anti-anesthetic fractionation, 5, in order to increase the specific activity of anti-antigens, in order to increase the specific activity of anti-angens, in order to increase the specific activity of anti-antigens, which are 5 in order to increase the specific activity of anti-antigens by anti-angens, in order to increase the specific activity of anti-antigens, in order to increase the specific activity of anti-antigens, in order to increase the specific activity of anti-antigens, in order to increase the specific activity of anti-antigens, in order to increase the specific activity of anti-antigens, in order to increase the specific activity of anti-antigens, in order to increase the specific activity of anti-antigens, in order to increase the specific activity of the anti-antigens, in order to increase the specific activity of the anti-antigens, in order to enhance the specific activity of t 2-38 times with the separation of the lysate of the toxoplasma membranes and the release of the antigenic fraction from it by isoionin fractionation with additional tive impact on sonicated lysate, followed by purification antigenic fractions from nonspecific ballast not societies addition thereto antiserum.