RU2430376C1 - Method of obtaining pseudotuberculosis antigen polymer diagnosticum - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: claimed is method of obtaining antigen diagnosticum with application of species-specific membrane proteins of Y. Pseudotuberculosis, sensibilised on acrolein polymer spheres, for detection of antibodies to causative agent of pseudotuberculosis of the most epidemically essunsial serovars, spread among people. As sensitin used is pool from 10 protein antigens with molecular weight within 28-80 kDa and total protein concentration 0.9-1.3 mg/ml, isolated from coarse protein membranes. As sensitin carrier used are spherical polymer particles with diameter 1.5 mcm, which contain 1.3 mM/g of aldehide groups. ^ EFFECT: method improvement. ^ 5 cl, 6 ex, 1 tbl

Description

The present invention relates to medicine, namely to clinical laboratory diagnosis of dangerous infections in the detection of antibodies to the causative agent of pseudotuberculosis of the most epidemiologically significant serovars, common among people, using species-specific membrane proteins Yersinia pseudotuberculosis sensitized on acrolein polymer spheres.

Despite the large arsenal of tools in laboratory practice, the problem of diagnosing pseudotuberculosis (PT) currently remains relevant.

The most preferred method in the clinic is the determination of antibodies to the pathogen antigens in blood serum, while the peculiarity of pseudotuberculosis is the variety of its clinical manifestations caused by Y. pseudotuberculosis serovars, namely O: 1a, O: 1b, O: 2b, O: 2c, O : 3, O: 4a, O: 4b, O: 5a, O: 5b, which complicates the diagnosis and treatment, since there are no test systems that reliably and quickly identify antibodies to the pseudotuberculosis microbe.

A known diagnosticum is pseudotuberculous erythrocyte antigenic (see Guidelines MU 3.1.1.2438-09 "Epidemiological surveillance and prevention of pseudotuberculosis and intestinal yersiniosis", Moscow, 2009, p. 61), which is a lyophilized suspension of sheep’s formalin erythrocytes, or sensib protein polysaccharide complex of Y. pseudotuberculosis serovar O: 1.

However, the official diagnosticum does not allow the detection of antibodies in the blood of patients if the disease is caused by another serovar of the causative agent of pseudotuberculosis.

As a result, due to the biological lability of the carrier, poor reproducibility of the drug series is observed, and the use of a polysaccharide as sensitin gives a large number of cross-reactions with other representatives of the genus Yersinia, as well as with intestinal pathogens, thereby complicating the differentiation of antibodies to the pseudotuberculosis pathogen.

The closest in technological essence is a method for the diagnosis of pseudotuberculosis (see patent RU No. 2153172, class G01N33 / 53, published July 20, 2000), which is a test system based on a highly purified thermostable toxin with mol. mass of 45 kDa, where a species-specific porin protein from the outer membrane of Y. pseudotuberculosis is used as an antigen, a dilution of 1: 400 and 1: 800 serum is taken as a diagnostic titer, a 0.1% solution of medical gelatin is used as a reagent to prevent nonspecific binding . The result is considered positive when the ratio of the optical density of a specific serum to the optical density of normal serum is greater than or equal to 2.1.

The disadvantage of the prototype can be considered that the toxin used as antigen is produced by 82.6% of the Y. pseudotuberculosis strains studied by the developers, regardless of the serovar of the causative agent of pseudotuberculosis, which makes it possible to obtain false negative results.

The results of the study of blood serum of patients (patent No. 215372) showed that only at 3-4 weeks of illness the percentage of positive reactions in ELISA using a porin-based test system was 98.9% (average optical density 1.34). In addition, in view of the multi-stage, complexity and duration of the study, this test system cannot be referred to as drugs for the rapid diagnosis of pseudotuberculosis, which makes it impossible to use it in a mass study, as well as epidemiological threats.

The technical task of the invention consisted in the development of a drug for the rapid diagnosis of pseudotuberculosis with high species specificity and sensitivity.

This object is achieved by the fact that in the known method for producing a pseudotuberculosis antigenic polymer diagnosticum, including the production of sensitin using the outer membrane proteins of Y. pseudotuberculosis, followed by their use in a serological reaction, a pool of 10 protein antigens with molecular weights in the range of 28- 80 kDa, with a concentration of total protein of 0.9-1.3 mg / ml, which is dissolved in 1 ml of 0.1 M bicarbonate buffer solution (pH 9.2) and immobilized on a carrier, representing suspension of microspheres, at the rate of 1 mg of protein per 100 mg of carrier, while incubation is carried out for 2 hours with constant stirring on a mixer, after which the suspension is left for 16-18 hours at 4-6 ° C, then free aldehyde groups are washed three times with buffered saline (pH 7.2) and centrifuged at 3000 rpm for 10 min at room temperature, after which they are suspended in 3% gelatin-sucrose drying medium in a ratio of 1: 1 and poured into insulin vials 1 ml, after which lyophilization is carried out tion.

In this case, the protein pool is isolated from coarse cell membranes by adding to them a 33% aqueous solution of N-lauryl-sarcosine-Na to a final concentration of 2% in suspension and incubation for 16 hours at room temperature, then the solution is centrifuged at 50,000 rpm on an ultracentrifuge for 1 hour, the resulting precipitate was washed with distilled water and the protein spectrum was studied by SDS-disk electrophoresis in SDS page according to the Laemmly method.

In addition, spherical polymer particles with a diameter of 1.5 μm containing 1.3 mmol / g of aldehyde groups are used as a sensitin carrier.

Moreover, coarse cell membranes are obtained from previously isolated bacterial mass with a density of 80-100 billion cells. / ml, which is centrifuged at 6000 rpm for 10 min, and then resuspended in 10 mM Hepes buffer containing 20% (w / v) sucrose (pH 7.2), after which five cycles of ultrasonic disintegration lasting 45 sec with a minute interval, at a wavelength of 585 μm, after lysed cells are treated with DNAase and RNAse at the rate of 10 μg of the enzyme per 1 ml of microbial suspension for 10 minutes, the undamaged cells are removed by centrifugation at 50,000 rpm in an ultracentrifuge for 30 min and the obtained scernatant was diluted with two volumes of Hepes-buffer and followed by centrifugation at 50,000 rev / min in the ultracentrifuge during 1 hour a precipitate was obtained, which is washed with distilled water and then resuspended to obtain a preparation of outer membrane proteins.

At the same time, free aldehyde groups are blocked by adding 2 ml of a 0.5% gelatose solution in buffered saline solution (pH 7.2) to a suspension of a carrier with sensitin and leaving the mixture under constant stirring for 2 hours at room temperature.

The method is as follows.

- At the first stage, a bacterial mass is obtained. For this, the non-plasmid strain H 141/84 Y. pseudotuberculosis is seeded on a Petri dish with 2% Hottinger agar and cultured for 2 days at room temperature. Then the two-day culture is subcultured into the Hottinger broth (pH 7.2). After two days of cultivation at a temperature of 24 ° C, the bacterial suspension is subcultured on Petri dishes with 2% Hottinger agar. Then, after 2 days, the bacterial mass of Y. pseudotuberculosis in S-form is washed off with buffered saline (pH 7.2) and inactivated for 30 minutes in a water bath at 80 ° C. Then, the resulting bacterial mass is checked for specific sterility.

- The second step of the method is to obtain coarse cell membranes, which are obtained from the bacterial mass with a density of 80-100 billion mcl / ml. For this, the bacterial mass is centrifuged at 6000 rpm for 10 minutes, and then resuspended in 10 mM Hepes buffer containing 20% (w / v) sucrose (pH 7.0). Next, five cycles of ultrasonic disintegration are carried out for a duration of 45 seconds with a minute interval, at a wavelength of 585 microns. Then the lysed cells are treated with DNAase and RNAse at the rate of 10 μg of the enzyme per 1 ml of microbial suspension for 10 minutes. After that, the undamaged cells are removed by centrifugation at 10 thousand rpm. within 30 minutes. The resulting supernatant was diluted with two volumes of Hepes buffer and centrifuged at 50,000 rpm in an ultracentrifuge for 1 hour. The resulting precipitate is washed with distilled water to remove the Hepes buffer, resuspended in distilled water and used to produce the outer membrane protein preparation.

- The third stage is the allocation of a pool of proteins of the outer membrane of Y. pseudotuberculosis. To one suspension of coarse cell membranes add a 33% aqueous solution of N-lauryl-sarcosine-Na to a final concentration of 2% in suspension and incubate for 16 hours at room temperature. After that, the solution is centrifuged at 50,000 rpm in an ultracentrifuge for 1 hour. The resulting precipitate is washed with distilled water, and then resuspended in distilled water.

The total protein content of the resulting protein outer membrane preparation was determined by the Lowry method. The protein concentration is 0.9-1.3 mg / ml.

The study of the protein spectrum of the obtained preparation is carried out by SDS-disk electrophoresis in SDS page according to the Laemmly method. The results of the study confirm the presence in the preparation of a block of 10 protein antigens with a molecular weight of 28-80 kDa.

Thus, the resulting sensitin is a pool of proteins encoded by chromosomal genes, porins of the outer membrane, as well as a thermostable toxin. These proteins have pronounced species-specific properties and are even present in strains of Y. pseudotuberculosis lacking the plasmid virulence pYV.

Assessment of immunochemical specificity was carried out according to the Western blot method.

- The fourth step is to immobilize sensitin to the carrier. As a carrier, spherical polymer particles with a diameter of 1.5 μm containing 1.3 mmol / g of aldehyde groups are used. The carrier was obtained by anionic polymerization of acrolein in an aqueous alkaline medium. Particle staining is done by adding dye to the polymerization mixture during the synthesis process.

A suspension of microspheres in an amount of 100 mg, which corresponds to 2 ml of a suspension of microspheres, is washed twice with 0.1 M bicarbonate buffer solution in a volume of 5 ml (pH 9.2) by centrifugation at 3000 rpm for 10 minutes at a temperature of 20 ° C. The washed carrier is suspended in 2 ml of bicarbonate buffer, and with constant stirring on a magnetic stirrer, it is combined with sensitin at the rate of 1 mg protein / 100 mg of the carrier, leaving it to contact for 2 hours at room temperature. Further, immobilization is carried out at 4-6 ° C for 16-18 hours.

To block free aldehyde groups, 2 ml of a 0.5% gelatose solution in buffered saline (pH 7.2) are added to a suspension of a carrier with sensitin and left under constant stirring on a magnetic stirrer for 2 hours at room temperature.

After that, the diagnosticum is washed three times with buffered saline (pH 7.2) and centrifuged at 3000 rpm for 10 minutes at room temperature. The final precipitate is suspended in 8 ml of a 0.1% gelatose solution in buffered saline (pH 7.2). After that, determine the activity and specificity of the liquid antigenic diagnosticum.

The finished product is suspended in 3% gelatin-sucrose drying medium in a ratio of 1: 1 and poured into 1 ml insulin vials. Lyophilization is carried out for 15 hours.

The activity and specificity of the diagnosticum is determined in the volumetric agglomeration (RAO) reaction. For its use, a control pseudotuberculosis rabbit serum against the outer membrane proteins, rabbit serum against Y. pestis, rabbit serum against Y. enterocolitica and hyperimmune serum against pathogens associated with the group of intestinal infections: escherichiosis, salmonella, shigellosis.

To set up a bulk agglomeration reaction, 50 μl of normal rabbit serum at a 1:50 dilution are added to each well of a 96-well immunological reaction plate. To determine the specific activity, 50 μl of control pseudotuberculosis serum at a dilution of 1:10 was pipetted into the first well with a pipette and titrated twice to the end of the row, to 1: 2560. From the last well, 50 μl is removed.

To determine the specificity, 50 μl of heterologous sera at a dilution of 1:10 are added to the wells of each row after normal rabbit serum is added and two-fold dilutions are sequentially made up to 1: 2560. 50 μl is removed from the last well.

Then, 25 μl of diagnosticum in working dilution are pipetted into all wells with a pipette-dispenser. To test the diagnosticum for spontaneous agglutination, 50 μl of normal rabbit serum and 25 μl of diluted diagnosticum are added to any two wells. After adding a diagnosticum, the contents of the wells are mixed by shaking the tablet for 1 minute and left at room temperature for 2-2.5 hours.

A colored pink agglomerate, lining the entire bottom of the well evenly, or agglomerate, lining the bottom of the well with clearly defined edges, was taken as a positive result. In negative cases and in the control, a compact ring or “dot” was formed in the center of the well.

To determine the sensitivity of the diagnosticum, a series of rabbit immune serum (control) was used in the volumetric agglomeration reaction (RAO) with titers of 1: 2560.

After freeze drying, the activity of the diagnosticum did not change.

The use of the obtained pseudotuberculosis antigenic polymer diagnosticum is confirmed by examples (see table).

A series of studies of blood serum of patients with intestinal infections in the reaction of agglomeration of volumetric (RAO) using antigenic pseudotuberculosis diagnosticum. The diagnostic titer of the drug is 1:80.

For the production of radioactive waste, the studied sera are prepared. The test human serum is diluted with buffered saline (pH 7.1) 1:10 before the study. The diluted serum is heated for 30 minutes at 56 ° C. After that, leave at room temperature for 10 minutes and then examined in the RAW.

Hyperimmune pseudotuberculosis rabbit serum is also diluted 1:10 with buffered saline (pH 7.2).

Example 1

Row A - examine the patient's serum (1) with suspected pseudotuberculosis. A preliminary diagnosis is made on the basis of clinical manifestations. Symptoms of the disease are observed for about 14 days. The serum titer in RA with a live culture of Y. pseudotuberculosis was 1: 320. A positive reaction (+) observed in 1-4 wells (color bright pink agglomerate) indicates the content of antibodies to the causative agent of pseudotuberculosis in a titer of 1: 160. The results confirm the preliminary diagnosis of pseudotuberculosis.

Example 2

Row B - patient's blood serum (2) with a clinical diagnosis of pseudotuberculosis. Serum titer in RA 1: 1280. A positive reaction (+) in 1-5 holes corresponds to a titer of 1: 640, which is consistent with the clinical diagnosis.

Example 3

Row C - patient's blood serum (3) with a diagnosis of intestinal yersiniosis. Caption in RA with live culture Y.enterocolitica 1: 640. A negative reaction (-) observed in all wells indicates the absence of antibodies to the causative agent of pseudotuberculosis.

Example 4

Row D - patient serum (4) with a diagnosis of salmonellosis. Title in RNGA with commercial salmonella diagnosticum 1: 640. In RAO with pseudotuberculosis antigenic diagnosticum, a negative reaction (-) is observed in all wells.

Example 5

Series E - rabbit hyperimmune serum to the plague pathogen. Working titer of serum 1: 1280. In a reaction with a pseudotuberculosis antigenic diagnosticum, a negative result (-) is observed in all wells of the series.

Example 6

Series F - rabbit hyperimmune pseudotuberculous serum (positive control). In 8 holes a positive reaction (+) is observed, the titer is 1: 2560, which indicates the high activity and specificity of the proposed diagnosticum.

Series G - control of the diagnosticum for spontaneous agglutination. In the two wells used, the result is negative (-).

Therefore, the developed diagnostic drug in the RAO allows to detect antibodies to the causative agent of pseudotuberculosis, to differentiate this disease from plague, intestinal yersiniosis and other intestinal infections. The results obtained are consistent with additional studies and a clinical diagnosis. Thus, the proposed diagnosticum is promising for use in clinical laboratory diagnostics, including early stages of the disease, as well as conducting monitoring in the field and research work.

Using the present invention, due to the unique sensitin, presented in the form of a pool of membrane proteins encoded by chromosomal genes, porins of the outer membrane, as well as thermostable toxin, antibodies to the causative agent of pseudotuberculosis can be detected regardless of serovar Y. pseudotuberculosis, while not giving cross-reactions with other representatives of the genus Yersinia, as well as intestinal pathogens.

The advantage of the diagnosticum is the possibility of conducting a study in one stage, followed by visual consideration of the color reaction. Obtaining the result of the study within 2 hours allows us to attribute the proposed diagnosticum to the means of express diagnostics, and the absence of the need to use special equipment and training personnel makes it possible to use it in the field.

Table Row Dilution of test sera in a tablet one 2 3 four 5 6 7 8 9 10 eleven 1:20 1:40 1:80 1: 160 1: 320 1: 640 1: 1280 1: 2550 1: 5120 BUT + + + + - - - - - AT + + + + + + - - - FROM - - - - - - - - - D - - - - - - - - - E - - - - - - - - - F + + + + + + + + - G - -

Claims (5)

1. A method of obtaining a pseudotuberculosis antigenic polymer diagnosticum, including the production of sensitin using proteins of the outer membrane of Y. pseudotuberculosis, followed by their use in a serological reaction, characterized in that a pool of 10 protein antigens with molecular weights in the range of 28-80 kDa is used as sensitin , the concentration of the total protein of 0.9-1.3 mg / ml, which is dissolved in 1 ml of 0.1 M bicarbonate buffer solution (pH 9.2) and immobilized on a carrier, which is a suspension of microspheres, and calculation of 1 mg of protein per 100 mg of carrier, while incubation is carried out for 2 hours with constant stirring on a mixer, after which the suspension is left for 16-18 hours at 4-6 ° C, then free aldehyde groups are blocked, washed three times with buffered saline (pH 7.2) and centrifuged at 3000 rpm for 10 min at room temperature, then suspended in a 3% gelatin-sucrose drying medium in a ratio of 1: 1 and poured into 1 ml insulin vials, after which lyophilization is carried out. 2. The method according to claim 1, characterized in that the pool of proteins is isolated from coarse cell membranes by adding to them a 33% aqueous solution of N-lauryl-sarcosine-Na to a final concentration of 2% in suspension and incubation for 16 hours at room temperature, then the solution is centrifuged at 50,000 rpm in an ultracentrifuge for 1 h, the resulting precipitate is washed with distilled water and the protein spectrum is studied by SDS-disk electrophoresis in SDS page using the Laemmly method. 3. The method according to claim 1, characterized in that spherical polymer particles with a diameter of 1.5 μm containing 1.3 mmol / g of aldehyde groups are used as a sensitin carrier. 4. The method according to claim 2, characterized in that the coarse cell membranes are obtained from pre-isolated bacterial mass with a density of 80-100 billion cells / ml, which is centrifuged at 6000 rpm for 10 minutes and then resuspended in 10 mm Hepes- a buffer containing 20% (w / v) sucrose (pH 7.2), after which five cycles of ultrasonic disintegration are carried out for a duration of 45 s with a minute interval, at a wavelength of 585 μm, after lysed cells are treated with DNAase and RNAse from calculating 10 μg of the enzyme per 1 ml of microbial suspension for 10 min, remove indestructible The cells were centrifuged at 50,000 rpm in an ultracentrifuge for 30 minutes and the resulting supernatant was diluted with two volumes of Hepes buffer, followed by centrifugation at 50,000 rpm in an ultracentrifuge for 1 h, a precipitate was obtained, which was washed with distilled water, and then resuspended obtaining the preparation of proteins of the outer membrane. 5. The method according to claim 3, characterized in that the blocking of free aldehyde groups is carried out by adding to the suspension of the carrier with sensitin 2 ml of a 0.5% gelatose solution in buffered saline (pH 7.2) and left with constant stirring on a stirrer in for 2 hours at room temperature.