SU1069783A1 - Method of producing tuberculosis diagnosticum - Google Patents

Abstract

THE METHOD OF CONSOLIDATION. TUBERCULAR DAAGNOSTICUMA, including the cultivation of Mycobacterium tuberculosis, the introduction and purification of the phosphatide antigen from the microbial mass with acetone and methanol, followed by a sensitization of the sorbent, and with the aim of increasing the activity of the target product, cultivate the product and cultivate it. / St and Valu, after muthanol purification, the phosphatide antigen is evaporated to a sediment, treated with chloroform and red blood cells are sensitized at the rate of 0.4 1, 0 mg of antigen per 1 ml of 10% suspension of erythrocytes.

Description

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The invention relates to medicine and can be used to obtain an antigenic erythrocyte tuberculous diagnosticum.

A known method for producing a tuberculosis diagnosticum, including the cultivation of mycobacterium tuberculosis, the isolation and purification of phosphatide antigen from the microbial mass with acetone and methanol, with subsequent sensitization of kaolin by them to SP.

However, the known method does not allow obtaining a high activity diagnosticum and, moreover, is difficult to perform.

The purpose of the invention is to increase the activity of diagnosticum.

This goal is achieved by the fact that according to the method of obtaining a tuberculosis diagnosticum, including cultivation of mycobacteria of tuberculosis, isolation and purification of phosphatide antigen from a fine tartar with acetone and methanol with subsequent sorbent sensitization, mycobacterium Dt / St and Valu are cultured after a methane filter. sediment, are treated with chloroform and red blood cells are sensitized at the rate of 0.4-1.0 mg of antigen per 1 ml of 10% suspension of erythrocytes. ,

The method is carried out as follows.

G cobacterium tuberculosis - Dt / St and Valu are grown in separate matrices filled with Linnikova and Mogilevsky medium (, medium composition: distilled water 200 l, glycocol 1.7 kg, ammonium citrate monobasic 1.0 kg; anhydrous sodium carbonate 0.6 kg , sodium chloride 0.4 kg, magnesium sulfate 0.2 kg, iron citrate oxide 0.01 kg; glycerin 14 kg, orthophosphoric acid 60-90 ml; potassium phosphate disubstituted 0.6 kg), for 6-8 weeks at 38 ° C. After that, sterilization of the cultures is carried out by autoclaving with flowing steam for 1 hour. The microbial cultures are then mixed in equal volumes, after which the culture fluid is removed by filtration through asbestos plates to obtain microbial bodies.

The obtained microbial cells are dried by treatment with acetone.

Microbial cells dried with acetone are poured with acetone at the rate of two volumes of acetone per volume of microbial mass and placed in a thermostat at 38 ° C for 3 h, after which. acetone is drained. This operation is performed twice.

The acetone treated cells are poured with methanol from a calculation of 0.5 liters of methanol per 50 g of cells and shaken for 5 hours at 37 ° C, after which the microbial cells are separated by centrifugation. This operation is also performed twice.

The ketanol extracts of the first and second extracts (in the above operation) are combined and placed in a vacuum-drying oven, in which the methanol extract is evaporated until a phosphatide antigen precipitates. Evaporation should be carried out at a vacuum of 0.8-0.6 kgf / cm and a temperature of 48-52 s. The end of the evaporation operation is established either visually by precipitation (for which the vacuum drying cabinet must have a transparent window for observation) or by a time pre-set depending on the equipment used and the specific mode of evaporation on the basis of evaporation to 1/3 of the volume. Evaporation to 1/3 of the initial volume of methanol extract is sufficient to precipitate a phosphatide antigen without coprecipitating impurities.

The resulting phosphatide antigen precipitate is separated by centrifugation and dissolved in chloroform. Two volumes of acetone are added to this solution to precipitate the phosphatide antigen. The precipitated antigen is washed with acetone and dried in a vacuum desiccator.

The obtained semi-finished product (phosphatide antigen) is stored in ampoules sealed under nitrogen before the operation. Sensitization of erythrocytes is dissolved in methanol. This solution is added dropwise to saline, followed by evaporation of the methanol at 50 ° C on a magnetic stirrer to the original volume of saline. In this way, a working solution of a phosphatide antigen is obtained to sensitize red blood cells, since the phosphatide antigen does not dissolve directly in the saline solution. The amount of saline in the preparation of phosphatide emulsion depends on the desired concentration of phosphatide antigen, which provides a high titer of anti-phosphatide antibodies in the corresponding immune serums and the economical expenditure of phosphatide antigen. It is advisable to take such an amount of saline so that the weight concentration of phosphatide antigen in it is 0.4 1, 0 mU / ml. For sensitization, a 10% suspension of formalized erythrocytes in a volume equal to the volume used for

preparation of phosphatide emulsion of saline. Erythrocyte sensitization is provided most of all with a ratio of ingredients: 0.41, 0 mg of phosphatide antigen per 1 ml of 10% suspension of erythrocytes.

The sensitization operation is carried out by combining equal volumes of phosphatide emulsion and 10% formalized red blood cells, followed by holding the mixture for 14 hours at 31 ° C with constant stirring. Then centrifugation and washing of sediment with saline are performed. From the sediment washed

Antibody titer in rabbit immune serum in RIGA with tuberculosis diagnostics, obtained by sensitization of erythrocytes in various doses. phospha1: the one anshigeny

sensitized erythrocytes prepare a 10% suspension by adding a protective environment. A 5% sucrose solution, a sucrose-gelatin stabilizer, 5% peptone or peptone broth (MPB) with sucrose and succinic buffer can be used as a protective medium. In the selected protective environment, the target product is subjected to lyophilization.

Data on the preference of the mode of sensitization of erythrocytes and the choice of protective environment are given in Table. 1 and 2

Table 1

0.1

ABOUT

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0.2

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0.3

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0.4

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0.6

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Antibody titer in rabbit immune serum in RIGA with erythrocyte-diagnosticum, lyophilized in various protective media

Sucrose

Saccharose gelatin stabilizer

+++

+++

+++

+++

++ rf +++

+++ +++

table 2

1: 128 o

1: 128 1:32 1: 4

1: 641: 32 1: 8

Peptone

25

BCH.

Sucrose Ostal Barant Amber 1: 128 Buffer

The data table. 1 illustrates the operation of sensitization of sheep erythrocytes with various doses of phosphatide antigen. As can be seen from the table. 1, the optimal sensitizing vine is 0.4-0.6 mg / ml, since a lower dose does not provide sufficient detection of antibodies, a-large doses result in wasteful antigen consumption.

As can be seen from the table. 2, the most preferred for lyophilization and storage of the preparation is a protective environment of the following composition:

M septonny broth,

vol.% 25

Sucrose, wt.% 5

Borate-amber.

buffer

The drug, lyophilized in this medium, can be stored for 3 years, whereas when using other protective media, the shelf life is no more than 6 months.

The strains used in the proposed method are new only in relation to the selected prototype. They are used in this technology for producing tuberculin. In this case, the Dt / St strain is a human type strain. It was obtained from the USA (G. Washington, commission of agriculture) and is stored in the collection of them. L.A. Tarasebic. The Valu strain is a bovine strain. It is obtained from the Central Research Institute of Tuberculosis and is stored in the GISC collection named after L.A. Tarasevich. Photocopies of the passport strains are attached.

Replacement of the strains was carried out in conjunction with the fact that the strains, especially the APU, used in the prototype method led to the detection of antibodies characterizing mainly post-vaccination immunity, which reduces the specificity of this serology. Continuation of the table. 2

dough. In addition, the achieved use of the same strains both for the preparation of tuberculin and the proposed tuberculosis diagnosticum makes it possible to carry out waste-free production of both at the same time (drugs: with this, in the technological scheme of their preparation, the common stage will be cultivation tuberculin, and from the microbial mass (which was previously a waste product / release of phosphatide antigen for the proposed obtaining of tuberculosis stikum.

The use of only one of these strains in the proposed method, as well as in the production of tuberculin, is not sufficient for the complete detection of the corresponding antibodies in tuberculosis patients.

Treatment of phosphatide antigen with chloroform is introduced to dissolve the target product, which allows it to be released from the chloroform-insoluble ballast substances produced by the tuberculosis mycobacterial strains used. In this case, the evaporation of the methanol extract is not complete, but only until sedimentation occurs (in which the phosphatide antigen is contained). It was established experimentally that otherwise (i.e., with complete evaporation of the methanol extract to obtain a dry precipitate), the precipitate becomes dark, which indicates co-precipitation of ballast pigmented substances and cannot be dissolved in chloroform. It is possible that this requirement is due to the use of these strains. , 1: 128 1: 12v 1: 128 1:64 1:64 Use as an erythrocyte sorbent makes it possible to obtain a drug in the form of erythrocyte tuberculosis diagnosticum, since it is used not in PKA, but in the reaction of indirect hemagglutination (PHTAJ Except In addition, the erythrocyte diagnosticum is subject to lyophilization, which increases its shelf life. Example: Mattresses, previously sterilized by temperature treatment, with a capacity of 1.5 liters, are sterile filled with a synthetic nutrient medium Linnikova and Mogilevsky at the rate of 600 ml per 1 mattress, the pH is 6, U-7.1. Od One half of the mattresses are seeded with the Dt / s strain and the other with the Valu strain. Mycobacterium tuberculosis is grown in these mattresses at 38 ° C for 6 weeks. During this period, the crops are examined daily for the detection of mattresses with growth of extraneous flora which reject and disinfect. Mattresses with 6-8 weeks of well-grown tuberculosis; cultures are placed in galvanized crates in which they are disinfected by autoclaving with flowing steam for 1 hour at 120 ° C. The disinfected cultures are mixed by merging them into 20 l bottles in equal volumes for each strain a). For the preparation of phosphatide antigen, 1012 liters of mixed culture of both MOB strains are taken. The mixed culture is filtered through asbestos plates to obtain microbial cells, which are then washed with 7 liters of distilled water and 3 liters of acetone. It then extracts the acetone-soluble components by treating one volume of microbial mass with two volumes of acetone for 3 hours, after which the acetone is drained. This operation is performed twice. The cells treated with acetone are poured, with methanol at the rate of 0.5 l of methanol per 50 g of cells, and placed on a shooter in a thermostat at 37 ° G, shaking the reaction mixture for b h. The precipitate is separated by centrifugation at separation factor F 1500 for 20 minutes, after which they are re-extracted with methanol under the same conditions. The methanol extracts of the first and second extracts are combined and evaporated in a vacuum oven with 0.7 kgf / cm and 50 ° C for -. 5 hours. During this time, the solution is evaporated to 1/3 of the initial volume, which is sufficient for precipitation of the phosphatide antigen without co-precipitation of impurities. The phosphatide antigen precipitate is separated by centrifugation at the indicated mode, then washed with acetone at, again centrifuged and dissolved in chloroform based on one volume of methanol used and 0.1 volume of chloroform. A double volume of pre-cooled acetone is added to the chloroform solution of the phosphatide antigen. Dropped out The phosphatide antigen is separated by centrifugation under the same conditions, washed 3 times with acetone and dried in a vacuum desiccator at room temperature for 48 hours. The yield of dry phosphatide antigen is 2-2.5 g from 50 g of microbial cells. Phosphatid antigen Packed in ampoules of 10 mg and sealed last under nitrogen. It has been established that in this form the phosphatidic antigen can be stored for 5 years. To sensitize formalized sheep erythrocytes, a weighed portion of phosphatide antigen b, O mg was taken from the ampule in 3, O ml of methanol. This solution is added dropwise to 12 ml of a 0.85% isotonic solution of sodium chloride, after which the methanol is evaporated on a magnetic stirrer at 50 ° C to the original volume of saline (i.e. up to 12 MP. Obtained 12 ml phosphatide emulsions are combined with 12 ml of 10% formalized erythrocytes with constant stirring. The concentration of phosphatide antigen is 0.5 mg per 1 ml of 10% formalinized erythrocytes (ram) .The mixture is kept for 1 hour in water bath at 37 ° C and with constant stirring The suspension is then centrifuged at separation factor F 2000, after which the precipitate is washed twice from excess antigen with saline pH 7.0-7.2. A 10% suspension is prepared from the precipitate of washed sensitized erythrocytes by adding 12 ml of protective medium of the following composition: M Sapepton broth: vol.% Is Sucrose, weight, 5 Borate-amber buffer Other The resulting liquid preparation of erythrocyte tuberculosis diagnosticum is poured into 1.0 ml in ampoules and frozen to -70 C, after which the preparation is lyophilized. ootnoshenii ingredients prepared 12 dry ampoules erythrocytic diagnosticum with tuberculous titrs l RIGA immune rabbit serum 1: 128. For the first time in world practice, erythrocyte tuberculosis diagnosis has been obtained. Since, at present, in clinical practice, the results of serological main groups are

211

68.72

Patients with tucked out tuberculosis As can be seen from the table. 3, in total, 386 people were examined using the preparation obtained by the proposed method, and with tuberculin, 1106 people. At the same time, RIGA was diagnosed with tuberculosis using the Boyden method. Among the examined patients with pulmonary tuberculosis, the most difficult to diagnose clinical form 1 / s prevailed: disseminated, infiltrative and focal tuberculosis. Among non-tuberculosis patients, patients with pneumonnio and prevailed, which have a differential value in the diagnosis of pulmonary tuberculosis. Mathematical data processing of the table was made on the ECVM Minsk658

54,795L, 66 62,45 Tuberculosis agnostics are used, as a rule, tuberculin, the most basic is to compare the effectiveness of the use of the obtained drug with tuberculin. Data for comparative analysis are given in Table. 3. Table 3 reactions for ani 22. 22. Significance of differences in the compared groups of the examined individuals is not lower than the 5% significance level. At the same time, the detection of antiphosphatid antibodies (which is possible with the use of a new drug), respectively, is mainly active. tuberculous process, which is the advantage of the resulting diagnosticum over tuberculin. The erythrocyte tuberculosis diagnosticum is used in VEGA, while the preparation obtained by a known method is used in the kaolin agglutination reaction. Corresponding data are given in table. four.

Diagnosis of tuberculosis staged by RIGA with erythrocyte, diagnosticum and RCA according to the Takahashi method

Patients aKTHBHfcJM pulmonary tuberculosis

Sick

As can be seen from the table. 4, with a similar probability of diagnosing tuberculosis infection, the resulting drug is more than 2 times less likely to give a positive reaction when examining patients with non-tuberculosis diseases.

In tab. 5 presents information about the activity obtained by the proposed method of intermediate products.

Antigen content,.%

Table 4

68.7

70.2

that - phosphatide antigen. The activity of β-phosphatide antigen can be judged by the phosphorus content in the preparation of the selected antigen. The phosphorus content of the resulting antigen was determined by the Chen method.

. In tabl; 5 shows the phosphorus content in preparations of phosphatide antigen, obtained by known and proposed methods.

TableB

Claims (1)

METHOD FOR PRODUCING. TUBERCULAR DIAGNOSTICUM, including the cultivation of mycobacterium tuberculosis, isolation and purification of the phosphatide antigen from the microbial mass with acetone and methanol, followed by sensitization of the sorbent with it, characterized in that, in order to increase the activity of the target product, mycobacteria Dt / St and Valu phosphate are cultured after methane the antigen is evaporated until a precipitate appears, it is treated with chloroform and the red blood cells are sensitized based on U, 4 1.0 mg of antigen per 1 ml of 10% suspension of red blood cells. with QO ss>