RU2240822C2 - Method for obtaining antitularemic serum and method for obtaining a dry erythrocytic tularemic immunoglobulin test kit - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: the present innovation deals with subcutaneous immunization in rabbits with alive F.Tularensis culture of 15 NIIEG vaccine strain, blood sampling and serumal separation. Additionally, one should prepare corpuscular antigen out of the mixture of inactivated F.tularensis culture of Schu virulent strain, F.Tularensis 503 virulent strain and F.Tularensis 543 virulent strain. Immunization in rabbits should be performed by the following scheme: one should twice immunize them due to subcutaneous injection of the mixture of corpuscular antigen with incomplete Freund's adjuvant, then - thrice - due to intravenous injection of corpuscular antigen without an adjuvant, then rabbits should be twice intravenously injected with alive F.Tularensis culture of NIIEG 15 vaccine strain. The method to obtain a dry erythrocytic tularemic immunoglobulin test kit deals with isolating immunoglobulins out of antitularemic hyperimmune serum, preparation of ram's erythrocytes and their sensitization with isolated immunoglobulins and, finally, lyophilic drying of obtained erythrocytic test kit. Moreover, one should isolate immunoglobulins G out of antitularemic hyperimmune serum obtained by the first variant to sensitize ram's erythrocytes with them. Lyophilic drying of obtained test kit should be carried out in medium containing the following components (weight%): saccharose 10, bovine serumal albumin 1, sodium tetraborate 1, succinic acid 0.3, sodium azide 0.1, distilled water - the rest. Serum and test kit obtained are of high activity and specificity, serum is safe and a test kit is distinguished by its high stability.

EFFECT: higher efficiency.

2 cl, 8 tbl

Description

The invention relates to the field of medical and veterinary microbiology, in particular, to methods for the preparation of immune sera and can be used to prepare, on the basis of the obtained serum, preparations for immunodiagnosis of tularemia, in particular, a dry erythrocyte tularemia immunoglobulin diagnosticum, which allows to identify tularemia pathogen in cultures and detect tularemia antigen in materials obtained from sick people and animals, cadaveric material and environmental objects Using the indirect hemagglutination reaction (RNGA).

A known method of preparing hyperimmune tularemia horse serum (see FS 42-3492-98, RP No. 55-94 “Diagnostic tularemia dry serum for agglutination reaction (PA)”).

A known method for the production of tularemia serum based on hyperimmunization of mice-producers with tularemia microbes or antigens extracted from them (Cherepakhina I.Ya., Kozlovsky V.N., Efanova EA, etc. Dynamics of formation of antibodies to the vaccine strain F. tularensis with different immunization schemes // Microbiological journal. - 1990. - T.52, No. 2. - S.89-93).

In common with the claimed method is the sequence of stages of preparation of antigenic material, immunization of animal producers, obtaining hyperimmune serum.

Closest to the claimed solution is a method based on subcutaneous immunization of rabbits with a live culture of F. tularensis vaccine strain 15 NIIEG (see Khlebnikov BC, Golovlev I.R., Kulevatsky D.P. and others. Study of biochemical, antigenic and protective properties of the external membranes of the causative agent of tularemia // Molecular Genetics, Microbiology and Virology. - 1991. No. 7. - P.15-20).

The method includes the following steps:

growing a culture of F. tularensis;

preparation of corpuscular antigen;

immunization of rabbits with a prepared antigenic preparation.

The disadvantage of this method is the use of mycobacterium tuberculosis complete Freund's adjuvant, which leads to a decrease in the specificity of immunodiagnostic preparations based on this serum. In addition, the use of one live culture of the vaccine strain 15 NIIEG as an antigenic preparation casts doubt on the possibility of detecting virulent F. tularensis strains with a richer antigenic spectrum by these immunodiagnostic preparations.

Known methods for the preparation of erythrocyte diagnostic kits designed to identify the causative agents of many infectious diseases (see the Guide to vaccine and serum / Edited by PN Burgasov. - M., 1978; Karalnik MP Erythrocyte protein diagnostics. - M. , 1983).

In common with the claimed method is the sequence of steps for obtaining formalized red blood cells, isolation of specific immunoglobulins from hyperimmune sera, and sensitization of red blood cells by them.

Closest to the claimed solution is a method for producing a diagnosticum of erythrocyte tularemia immunoglobulin dry, developed by the Central Asian NIPCH in 1990 (see the Schedule for the production of a diagnosticum of erythrocyte tularemia immunoglobulin dry for RNGA and RNAg / RP No. 230-90, Alma-A in 1990 in Alma-A) with the requirements of FS 42-308 AC - 90.

The method includes the following steps:

obtaining immunoglobulin from tularemia agglutinating serum;

preparation of sheep erythrocytes to an immunoglobulin load;

sensitization of formalized red blood cells with immunoglobulin;

freeze drying of the erythrocyte diagnosticum.

The main disadvantage of this method is that the diagnosticum is prepared on the basis of the gamma-globulin fraction, which includes all types of serum immunoglobulins, most of which cross-react with heterologous microorganisms, or create steric barriers to the interaction of tularemia antigens and antibodies. As a result, the diagnosticum obtained by this method gives positive results in RNGA not only with tularemia microbe, but also with cultures of pathogens of brucellosis, glanders, melioidosis, plague, cholera and escherichiosis. The extremely low specificity negates the practical value of the diagnosticum, which, apparently, was the reason for the impossibility of reapproving the normative and technical documentation for the preparation and use of this drug. In addition, the diagnostic prototype is not very stable, since it retains its activity only for 6 ... 8 months.

The technical result of the method for producing anti-tularemia hyperimmune rabbit serum is an increase, in comparison with the prototype, of its specific activity and specificity in the absence of fatal outcomes in rabbits, as well as a three-fold increase in the volume of the resulting serum due to the use of vaccine strain 15 tulipensis NIIEG for immunization as a live culture , as well as mixtures of inactivated cultures of virulent strains - typical representatives of three geographic rastularemia microbe (Schu, 503, 543), introduced internally Riven in the later stages of immunization.

A new method for producing anti-tularemia hyperimmune rabbit serum is that a mixture of inactivated cultures (particle antigens) F. tularensis of virulent Schu strains 503, 543 and live culture of vaccine strain 15 NIIEG is used to immunize rabbits, which enhances the animal's immune response and enriches the spectrum of animals specific antibodies in the resulting sera.

The use of incomplete Freund's adjuvant, which contains no Mycobacterium tuberculosis, is used to immunize rabbits, which increases the specificity of the drug.

New is that corpuscular antigens obtained from inactivated cultures of tularemia microbe and live culture of F. tularensis vaccine strain 15 NIIEG are administered to rabbits intravenously in the late stages of immunization, which does not cause specific injuries and death from anaphylactic shock.

The technical result of the proposed method for preparing a diagnosticum of erythrocyte tularemia immunoglobulin dry is to increase the sensitivity and specificity of the drug, reduce the time to obtain the final result, as well as increase the persistence of the diagnosticum due to the use of class G immunoglobulins as a sensitin, as well as stabilization of class G immunoglobulins diagnosticum in the original drying medium containing 10% ca Arosa, 1% bovine serum albumin (BSA) and sodium borax, 0.3% succinic acid and 0.1% sodium azide.

New in achieving the technical result is the use of highly active anti-tularemia hyperimmune rabbit serum with a wide spectrum of antibodies to all F. tularensis races as a raw material for the preparation of the preparation, which increases the sensitivity of the prepared diagnosticum.

The use of not all gamma-globulin fraction as a sensitin, but of class G immunoglobulins isolated from it is also new, which increases the specificity of the prepared diagnosticum.

A new method is the stabilization of the diagnosticum in the original drying medium containing 10% sucrose, 1% BSA and sodium tetraborate, 0.3% succinic acid and 0.1% sodium azide (the final concentration of each component was selected experimentally), which compared with the prototype, reduces by 7 times the time to obtain the final result in the RNGA micromethod and increases by 3 times the retention of properties of the diagnosticum.

Comparative analysis shows that the claimed technical solution is characterized in that for the preparation of hyperimmune anti-tularemia rabbit serum, combined immunization of animals with vaccine and inactivated virulent F. tularensis strains is used according to the original scheme, and the erythrocyte diagnosticum is prepared on the basis of class G immunoglobulins and stabilized in special drying medium containing 10% sucrose, 1% bovine serum albumin and sodium tetraborate, 0.3% yang ary acid and 0.1% sodium azide, that the invention meets the criterion of "novelty".

New significant features inform the claimed technical solution of the new properties, manifested in increasing the sensitivity, specificity, retention of the diagnosticum, as well as reducing the time to obtain the result of the analysis using the declared diagnosticum, which meets the criterion of "industrial applicability".

A method of obtaining protivotularemia hyperimmune rabbit serum is as follows.

1. Growing cultures of tularemia microbes

The starting material is lyophilized tularemia cultures of strains 15 NIIEG, Schu, 503, 543, stored in ampoules under vacuum and grown on FT-agar (VFS 42 / D-013 VS-94, Obolensk) in accordance with the requirements of FS 42-3951 -00.

2. Preparation of corpuscular antigens

The culture of strain 15 NIIEG formed in the matrix is washed off and diluted with a sterile 0.9% sodium chloride solution to a concentration of 1.0 billion cells in 1.0 ml according to OSO 42-28-59-86P, equivalent to 5.0 billion cells of tularemia microbe in 1.0 ml.

The grown cultures of tularemia microbes of strains Schu, 503, 543 are washed off and diluted with a sterile 0.9% sodium chloride solution to a concentration of 40.0 billion cells in 1.0 ml, after which they are inactivated by the addition of a 5% phenol solution and 1% solution of Tween-20 to final concentrations of 0.5% and 0.1%, respectively, followed by 5 minutes boiling in a water bath. The resulting suspension is mixed in equal volumes. For 5 immunization cycles, 10 rabbits are prepared with at least 100.0 ml of the mixture.

Freund's incomplete adjuvant is placed in a glass vial and sterilized by boiling in a water bath for 30 minutes. It is cooled to room temperature and mixed in a volume ratio of 1: 1 with the calculated amount of the mixture of killed cultures of F. tularensis. The resulting suspension is repeatedly (at least 20 times) drawn into a syringe and released back with force until a stable homogeneous emulsion is formed, the drops of which should not disintegrate on the surface of distilled water for 30 minutes or more.

3. Immunization of rabbits

Use at least 10 animals. With intravenous injections, immunogens are injected into the marginal vein of the ear, and with subcutaneous injection into the right and left paravertebral areas (along the spine at 4 points, 5 cm distant from each other). Drugs are administered as follows:

3.1. 0.2 ml of a mixture of inactivated cultures of F. tularensis with incomplete Freund's adjuvant subcutaneously at 4 points of the right paravertebral region, repeated after 20 days to the left paravertebral region;

3.2. after 20 days - cyclic intravenous immunization with a mixture of killed cultures of F. tularensis without adjuvant in (0.8-1.6-2.4) ml with a 5-day interval between injections; 3. after 12 days, cyclic intravenous immunization with a live culture of F. tularensis strain 15 NIIEG (0.8-1.6) ml with a 2-day interval between injections.

4. Obtaining immune serum

10 days after the last injection in rabbits, blood is drawn. Blood is collected in glass cylinders and placed in a coagulation thermostat. The resulting serum is drained, canned by adding a solution of sodium azide to a final concentration of 0.1% and purified by centrifugation.

The control of the activity of the obtained serum is determined in the RDP with cultures of F. tularensis strains 15 NIIEG, Schu, 503, 543. The antibody titer in the immune serum must be at least 1: 128.

To compare the prototype and the proposed method, rabbits were immunized and the sera obtained from them were studied (tables 1-3).

Observations showed that after the second cycle of immunization according to the prototype, animals often had necrosis of the skin of the inguinal region, and after the third cycle, most rabbits fell from anaphylactic shock, which, in our opinion, is associated with repeated use of the most sensitizing organism subcutaneous injection of immunogens.

The general condition of rabbits immunized by the claimed method, throughout the experiment remained satisfactory, which contributed to a sufficiently high yield of the final product (serum). Based on one rabbit taken into work, 3 times more serum was obtained than in the control (table 1). And the serum itself was characterized by the absence of nonspecific and higher content of specific antibodies than the serum obtained by the prototype (tables 2 and 3).

The presence of a causal relationship between the set of essential features of the proposed method and the achieved technical results are shown in table 4.

The proposed method for producing anti-tularemia hyperimmune rabbit serum has the following advantages:

does not cause death of rabbits from specific lesions and anaphylactic shock, which contributes to obtaining, in terms of one animal, about 3 times more serum than in control;

provides immune serums with a higher titer of specific antibodies.

The high content of specific antibodies in the obtained sera made it possible to propose these preparations for the preparation of a diagnosticum of erythrocyte tularemia immunoglobulin dry on their basis, the preparation method of which is carried out as follows.

1. Preparation of immunoglobulin

An equal volume of a saturated solution of ammonium sulfate is added to tularemia agglutinating serum with constant stirring for 5 minutes. After daily incubation at 4 ° C, the suspension is centrifuged for 15 min at 5500 g. After removing the supernatant, the immunoglobulin precipitate is dissolved in a 0.05 M solution of phosphate buffer (FBI) pH 7.2 in a volume equal to the initial volume of the taken serum.

The deposition operation is repeated. After centrifugation, the precipitate was dissolved in 0.05 M PBS, pH 7.2 in a volume equal to half the initial volume of the taken serum.

The solution of immunoglobulins is dialyzed for two days against 1 liter of 0.05 M PBS, pH 7.2, cooled to 4 ° C with a buffer change after 2, 8 and 24 hours. It is clarified by centrifugation at 5500 g for 15 minutes. The supernatant is carefully poured into a clean bottle and the protein concentration is determined spectrophotometrically.

The dialyzed immunoglobulin solution is injected by means of a peristaltic pump with a downward flow rate of (5.0 ± 0.5) ml · h / cm ² into a chromatographic column (size 2.6 × 40 cm) with DEAE-sephacelum based on not more than 10 mg of protein per 1 ml of gel. The sample was eluted with 0.05 M PBS, pH 7.2, controlling the extinction protein yield at a wavelength of 280 nm using a flow absorber and a potentiometric recorder. Immunoglobulin G elutes with one broad peak. With the beginning of the release of protein from the column, the eluate is collected in tubes (5.0 ± 0.5) ml. Fractions of the first peak containing protein in a concentration of at least 5.0 mg / ml are combined. The immunoglobulin G solution is preserved with sodium azide added to a final concentration of 0.1%.

2. Preparation of formalized sheep erythrocytes

Blood from intact rams is taken 500 ml into glass flasks, with sterile beads placed in them. After blood collection, the flasks were continuously stirred for 20 minutes to adhere fibrin to the beads. Defibrinated blood is filtered through nylon tissue; erythrocytes are washed from serum proteins by 20-minute centrifugation at 1100 g and 4 ° C with 0.9% sodium chloride solution (pH 6.8), cooled to the same temperature. The supernatant is removed; the operation of washing red blood cells is repeated 3-5 times.

To obtain an 8% suspension, a dense precipitate of washed red blood cells is diluted 12.5 times with 0.9% sodium chloride solution, heated to 37 ° C. In formalin solution (GOST 1625-75), the percentage of formaldehyde is determined in accordance with MUK 4.1 / 4.2.588-96, pp. 100-102. The formalin solution volume required for formalization of red blood cells is calculated by the formula: X = 3V / C, where X is the desired volume, V is the volume of the prepared 8% suspension of red blood cells, C is the percentage of formaldehyde in the formalin solution used.

To obtain a 3% formaldehyde solution, (V-X) ml of a 0.9% sodium chloride solution is added to X ml of formalin solution.

A 3% formaldehyde solution, heated to 37 ° C, is poured into the prepared 8% suspension of red blood cells with constant stirring. The bottle with the mixture is placed in the original red blood cell formalinization apparatus, which automatically maintains the temperature of the water in the water bath and ensures continuous mixing of the water in the bath and red blood cells inside the bottle. Formalization is carried out at 37 ° C for 18 hours. Then, formalin erythrocytes are washed four times in a 10-fold volume of a 0.9% sodium chloride solution by centrifugation at 1100 g for 15 minutes. A dense precipitate is diluted 10 times with 0.9% sodium chloride solution. A 10% suspension of formalin erythrocytes is preserved by adding formaldehyde to a final concentration of 0.8%. Store at 4 ° C for 3-5 years. For the preparation of an erythrocyte diagnosticum, red blood cells are optimal after storage for a year.

Before use, red blood cells are monitored for homogeneity and lack of tendency to spontaneous bonding. To control homogeneity, red blood cells are diluted with 0.9% sodium chloride solution and one drop of a 0.5% suspension of red blood cells is applied to a glass slide, covered with a coverslip and examined at a small magnification of the microscope. Red blood cells must lie in isolation, 2-3 clusters are allowed, containing no more than 10 red blood cells in 10 fields of view. No tendency to spontaneous bonding: to 0.4 ml of a 0.9% solution of sodium chloride in a polystyrene plate add 0.05 ml of a 2.5% suspension of red blood cells, homogenize and leave at room temperature for 2-3 hours. Red blood cells should precipitate in the form of a “button” or a small ringlet with a flat edge. In the event of spontaneous agglutination, a series of red blood cells are rejected.

3. Preparation of erythrocyte tularemia immunoglobulin diagnosticum

The required amount of 10% formalin erythrocytes is washed from formalin with 0.9% sodium chloride solution (pH 7.2) by 4-fold centrifugation at 1100 g for 4 minutes. Then the red blood cells are diluted with a 0.9% sodium chloride solution (pH 7.2) heated to 37 ° C to a concentration of 5%. To the resulting suspension with constant stirring, add an equal volume of a solution of tannin at a dilution of 1: 20,000. Tanization is carried out for 15 min at a suspension temperature of 37 ° C.

The suspension is washed with a 0.9% sodium chloride solution (pH 6.4) by centrifugation at 3 × 20 minutes at 1100 g. Then the red blood cells are diluted with 0.9% sodium chloride solution (pH 6.4) to a concentration of 5%.

Before preparing the main batch of diagnosticum for this series of formalized red blood cells and this series of immunoglobulin, the optimal sensitizing dose of the latter is determined. For this, 16 ml of the obtained red blood cells are poured into 4 ml in 4 tubes. An aliquot of the prepared immunoglobulin is diluted with 0.9% sodium chloride solution (pH 6.4) to 10, 20, 40, 80 μg / ml and 4 ml of each concentration are added to test tubes with red blood cells (one concentration of immunoglobulin per tube).

After a 20-hour incubation at 4 ° C, sensitin is fixed for 2 hours by adding formaldehyde to a final concentration of 0.4%. Tubes with sensitized red blood cells are washed 4 times with 0.9% sodium chloride solution (pH 7.2) by centrifugation at 1100 g for 10 minutes.

The obtained erythrocytes sensitized by various concentrations of immunoglobulin are tested in RNGA with killed cultures of F. tularensis strains 15 NIIEG, Schu, 503, 543 for sensitivity, as well as with inactivated cultures of Brucella, Yersinia, Vibrio, Salmonella, Escherihia. RNGA is carried out according to the instructions for use of the drug. The optimal sensitizing dose of immunoglobulin is considered to be the one at which a diagnosticum is obtained that has the highest sensitivity and does not detect cultures of the above heterologous microorganisms at a concentration of 1 · 10 ⁸ m.k. / ml.

To prepare the main batch of diagnosticum, an equal volume containing 1.5 optimal doses of immunoglobulin in 0.9% sodium chloride solution (pH 6.4) is added to a 5% suspension of tanized erythrocytes with constant stirring. After a 20-hour incubation at 4 ° C, sensitin is fixed for 2 hours by adding formaldehyde to a final concentration of 0.4%. The drug is washed 4 times with 0.9% sodium chloride solution (pH 7.2) by centrifugation at 1100 g for 20 minutes and adjusted to a 10% concentration with the same solution. Preservative - sodium azide 1: 5000.

4. Lyophilic drying of the prepared diagnosticum

The prepared preparation is precipitated by 10-minute centrifugation at 1100 g, the precipitate is resuspended in a 10% sucrose solution, centrifuged for 30 minutes at 1100 g.

To prepare the drying medium, 650.0 ml of distilled water are dissolved in 100.0 g of sucrose, 10.0 g of BSA, 10.0 g of sodium tetraborate and 1.0 g of sodium azide. To the resulting solution, under pH control and with constant stirring, (100.0 ± 20.0) ml of a 3% solution of succinic acid are poured to obtain a pH value (7.0 ± 0.1). The volume was adjusted with distilled water to 900.0 ml, the solution was thoroughly mixed and stored at 4 ° C for no more than one year.

The precipitate washed with 10% sucrose erythrocytes is diluted with a drying medium to a 10% concentration and poured into ampoules in (1.0 ± 0.5) ml.

The ampoules with diagnosticum installed in the cassettes are placed on the shelves of the MASS-5 freeze-drying chamber previously cooled to a temperature of minus (30 ± 2) ° C. The chamber is hermetically sealed. The diagnosticum is frozen for (2.5 ± 0.5) hours until the material temperature reaches no higher than minus 25 ° С, then a vacuum is created in the chamber, maintained during the whole process of freeze drying at the level of 1.1 ^-1 Torr (13.33 n / m ² ). After (2 ± 0.5) h, all shelves are heated. After 1 h, the shelves warm up to a temperature of (28 ± 1) ° C, maintained until the end of freeze drying, while the temperature in the ampoule with diagnosticum after (13 ± 1) hours also reaches (28 ± 1) ° C, after which freeze drying is continued within (6 ± 0.5) hours. The duration of freeze drying of the diagnosticum is (21 ± 1) hours. At the end of the process, the ampoules are sealed and the quality of the dry diagnosticum is controlled according to the following parameters:

4.1. Solubility - when 4.0 ml of 0.9% sodium chloride solution is added to the ampoule, the drug should dissolve within 1 min.

4.2. PH - should be within (7.0 ± 0.2).

4.3. The loss in mass upon drying is not more than 3%.

4.4. The homogeneity of the resuspended drug - under a small magnification of the microscope - no more than 3 clusters of 10 or less red blood cells in 10 fields of view.

4.5. Lack of spontaneous hemagglutination - when adding 0.4 ml of a 0.9% solution of sodium chloride, 0.05 ml of a 2.5% suspension of red blood cells to a plate placed in a hole for plates of RNGA by a macro method, after 3 hours a button or a narrow ring should form .

4.6. The number of red blood cells is in 1.0 ml of a 2.5% diagnosticum (5.4 ± 0.6) · 10 ⁵ m.k. / ml.

4.7. Sensitivity - F. tularensis should be detected at a concentration of 3.12 · 10 ⁶ m.k. / ml by the macromethod and 6.25 · 10 ⁶ mc / ml by the micromethod in RNGA.

4.8. Specificity - should not be detected in RNGA cultures of Brucella, Yersinia, Vibrio, Salmonella, Escherihia at a concentration of 1.0 · 10 ⁸ MK./ ml, inactivated by 20 minutes boiling and adding formalin to a final concentration of 2%.

4.9. The quality of sealing of ampoules is in accordance with MUK 4.1 / 4.2.588-96, p. 63-65.

The guaranteed shelf life of the diagnosticum at a temperature of 2 to 10 ° C is 2 years.

A comparative assessment of the specificity and sensitivity in RNGA of tularemia immunoglobulin erythrocyte diagnosticums prepared by the proposed method and prototype was carried out with cultures of F. tularensis (strains 15 NIIEG, Schu, 503, 543), Br. abortus (strain 19-BA), Y. pestis (strain EV), V. cholerae (strain 569-B), S. typhimurium (strain 1), E. coli (strain HB-101). RNGA staging by micro- and macro-methods was carried out according to the instructions for use of the drug with the following features: to assess the sensitivity, F. tularensis strains were titrated with a double step from the 1st to the 9th hole, inclusive, starting from a concentration of 5.0 · 10 ⁷ mk ./ml, and strains of heterocultures were used for verification of specificity at concentrations of 1.0 · 10 ⁹ , 2.0 · 10 ⁸ and 4.0 · 10 ⁷ MK / ml each.

The results of the specificity study, presented in table 5, indicate that the claimed sample under these conditions did not give positive reactions in RNGA with any of the studied heterologous microorganisms. In contrast, the diagnostic prototype cross-reacted with all strains of heterocultures. For S. typhimurium, the minimum detectable concentration was 1.0 · 10 ⁹ m.k. / ml, and with strains of brucellosis and plague microbes, a positive reaction was observed even at their concentration of 4.0 · 10 ⁷ m.k. / ml. With regard to sensitivity, as follows from the data given in table 6, the prototype showed sensitivity corresponding to the NTD sensitivity only with respect to the Schu strain of tularemia microbe, while the claimed sample with respect to all the studied strains.

Analysis of the obtained data allows us to conclude that the diagnosticum prepared according to the prototype, inferior in sensitivity to the claimed sample, has significantly worse specific properties. Such a low specificity is a significant disadvantage of the prototype.

The diagnostic value of the obtained drug was studied during state tests. In accordance with the Test program for the diagnosticum of erythrocyte tularemia immunoglobulin dry series 1, 2, 3, F. tularensis cultures (strains 15 NIIEG, Schu, 503, A-108 from the collection of GISK named after L.A. Tarasevich; 15 NIIEG from collections of the NIIM MO RF, included in the delivery set of the diagnosticum), as well as Br. abortus (strain 19-BA), V. cholerae (strain 569-B), E. coli (strain 18), S. typhi enteritidis (strain WHO), Y. enterocolitica (strain 237 serovar 09) from the collection of GISK im. L.A. Tarasevich.

The results showed that the test drug has significant advantages over the prototype in the following indicators:

specificity: diagnosticum prepared by the present method does not react RNHA with cultures of heterologous microorganisms (Brucella, Yersinia, Vibrio, Salmonella, Escherihia) at concentrations of 2.0 · 10 ⁸ m.k. / ml and 1.0 · 10 ⁹ m .k / ml, while the diagnostic prototype cross-reacts at both concentrations with all the heterocultures studied except S. typhimurium;

sensitivity: the claimed sample allows identification of F. tularensis strains 15 NIIEG, Schu, 503, 543 in the concentration of 3.12 · 10 ⁶ mk / ml by the macromethod and 6.25 · 10 ⁶ mk / ml by the micromethod , while the prototype reveals in this concentration only F. tularensis strain Schu;

according to the time of obtaining the results: the results of RNGA with the test drug were taken into account after 3 hours for the micromethod finally, for the macromethod - preliminary with the final count after 20 hours (preliminary and final results coincided); with the diagnostic prototype, the final recording of the results was possible no earlier than after 20 hours

The obtained results confirmed the correspondence of the sensitivity and specificity of the proposed diagnosticum to the data given in tables 3 and 4, and also showed that the test drug meets the requirements of FS 42-308 BC-90 and the developed draft NTD according to the following indicators:

in appearance: amorphous porous mass of brown color;

for solubility, transparency and color: soluble for 1 min when 4.0 ml of 0.9% sodium chloride solution is added to the ampoule; with shaking, a homogeneous brown suspension;

according to the concentration of red blood cells in the preparation: in all three series, within 5.4 · 10 ⁵ ± 0.6 · 10 ⁵ cells in 1 ml.

The results of the state tests made it possible to recommend the introduction of a erythrocyte tularemia immunoglobulin dry diagnosticum manufactured in accordance with the claimed method into health care practice and to draw up all the necessary regulatory and technical documentation ("Rules for the production of erythrocyte tularemia immunoglobulin dry diagnosticum", "Pharmacopoeia article", "Instructions for . The rules for the production of erythrocyte tularemia immunoglobulin dry diagnosticum are registered under RP number 1018-00. Pharmacopoeia article on the diagnosticum erythrocyte tularemia immunoglobulin dry is registered under the number FS 42-3951-00.

The presence of a causal relationship between the set of essential features of the claimed object and the achieved technical results is shown in table 7. From its analysis it follows that the tularemia erythrocyte diagnosticum prepared by the claimed method shows results that significantly exceed the prototype in terms of specificity and stability of storage properties.

The possibility of implementing the proposed method can be demonstrated by the following example.

At the ATL, created at the Research Institute of Microbiology of the Ministry of Defense of the Russian Federation, 3 series of erythrocyte tularemia immunoglobulin diagnosticum were prepared in accordance with the approved regulatory and technical documentation. The characteristics of the series are presented in table 8.

From the data presented in table 8 it follows that all series of erythrocyte tularemia immunoglobulin diagnosticum fully met the requirements of FS 42-3951-00.

Claims (2)

1. A method of producing anti-tularemia hyperimmune serum, including subcutaneous immunization of rabbits with live culture of F. Tularensis vaccine strain 15 NIIEG, blood sampling and separation of serum, characterized in that they further prepare a corpuscular antigen from a mixture of inactivated cultures of F. Tularensis virulent Schu strain, F. Tularensis virulent strain 503 and F. tularensis virulent strain 543, and rabbits are immunized according to the following scheme: they are immunized twice by subcutaneous injection of a mixture of corpuscular antigen with incomplete ad Freund's juvenile, then three times by intravenous administration of a corpuscular antigen without adjuvant, after which the living culture of F. Tularensis vaccine strain 15 NIIEG was administered twice to rabbits. 2. A method of obtaining a diagnosticum of erythrocyte tularemia immunoglobulin dry, comprising isolating immunoglobulins from anti-tularemia hyperimmune serum, preparation of sheep erythrocytes, sensitization of sheep red blood cells with isolated immunoglobulins and freeze drying of the obtained erythrocyte testosterone, which is different, immunoglobulins G, which sensitize sheep erythrocytes, and freeze-drying the floor scientific diagnosticum is carried out in an environment containing the following components, wt.%: Sucrose 10 Bovine Serum Albumin 1 Sodium tetraborate acid 1 Succinic acid 0.3 Sodium Azide 0.1 Distilled Water Else