RU2410699C1 - Method of obtaining erythrocytal anthrax antigenic diagnosticum for detection of antibodies to protective antigen - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medical microbiology, can be used for detection of antibodies to protective antigen in serum of humans and animals, sick with anthrax, in serum of infected, as well as immunised with protective antigen animals. Method of obtaining diagnosticum in accordance with invention includes processing of formalinised sheep erythrocytes with conjugating preparation, sensitisation with anthrax antigen, obtained by cultivation of toxin-producing strain of Bacillus anthracis STI in nutritional medium, intended for separation of anthrax toxin. After that obtained cell-free culture filtrate is condensed on ultrafilters which exclude proteins smaller than 10 kDa, and separate on superfine cephacryl S-300 in column. Then fractions are separated at output of 160-180 ml of eluate, concentrated on ultrafilter PM 10 until content of protein is not less than 600 mcg/ml and belonging of protein to protective antigen is determined.

EFFECT: method allows increasing sensitivity and specific activity of erythrocytal anthrax antigen diagnosticum for detection of antibodies to protective antigen.

2 cl, 6 tbl, 5 ex

Description

The invention relates to medical microbiology, can be used to detect antibodies to a protective antigen in the sera of people and animals suffering from anthrax, as well as in the sera of infected and immunized animals.

To detect antibodies in the sera of anthrax patients in humans and animals, erythrocyte diagnostics were used, prepared using the antigens of vegetative cells, which turned out to be low-sensitive and not specific enough.

Upon sensitization of formalized tanized erythrocytes with an antigen isolated from ultrasonic disintegrate of vegetative cells, a diagnosticum was obtained that detected antibodies in anthrax globulin in a titer of 1: 80000-1: 160000, in serum of vaccinated sheep in a titer of 1: 160-1: 640 (5) . However, anthrax globulin of hyperimmune serum is known to contain antibodies to the protective antigen and to cell wall antigens (6). Apparently, in RNGA with anthrax globulin, the positive result is mainly due to antibodies not to PA, but to antigens of cell surface structures, since PA is an extracellular antigen and is not detected in cell extracts (7).

To increase the sensitivity and simplify the technique of preparing erythrocyte diagnosticums, various conjugating preparations were tested (tannin, glutaraldehyde, diamond green). A diagnosticum prepared using a 0.10-0.12% brilliant green aqueous solution turned out to be more sensitive and made it possible to detect antibodies in precipitating serum in a titer of 1: 6400-1: 12800 (4).

Protective antigen (PA) is one of the main components of anthrax toxin, which plays a leading role in the pathogenesis of the disease, which led to the development of diagnostic drugs using its components (1).

The erythrocyte diagnosticum based on PA, which was isolated from the culture filtrate by acid deposition, turned out to be insufficiently sensitive and specific (2).

The erythrocyte diagnosticum, obtained by sensitization of formalized tanized PA erythrocytes, isolated from the culture filtrate by ion-exchange gel chromatography on DEAE cellulose, followed by purification on Sephadex G-50, allowed us to determine antibodies in the sera of 93% of patients and 98% of the sera of vaccinated people in titers 1: 8-1: 2056 (3).

Currently, there are no registered certified diagnosticums of erythrocyte anthrax antigenic for the detection of antibodies to a protective antigen.

The closest analogue of the proposed method is an enzyme-linked immunosorbent assay for detecting antibodies to a protective antigen, which, according to the authors, is similar to RNGA in sensitivity and specificity, is faster, more accurate and cheaper (8). Other authors consider the enzyme immunoassay method the most sensitive, but more time-consuming, requiring expensive equipment for formulation and accounting.

The purpose of the invention is to increase the sensitivity and specific activity of the diagnosticum of an erythrocyte anthrax antigenic for the detection of antibodies to a protective antigen.

This goal is achieved in that in order to preserve the native properties of the antigenic drug, after culturing the toxin-producing strain of B.anthracis STI in a nutrient medium designed to isolate anthrax toxin, the cell-free culture filtrate is concentrated on ultrafilters excluding proteins of less than 10 kDa, and they are separated on ultrafine sephacryl S- 300 in a 2.5 × 75 cm column. A fraction is taken at the exit of 160-180 ml of eluate, concentrated on a PM10 ultrafilter to a protein content of at least 600 μg / ml, protein affiliation is determined protective antigen and subsequently used to produce the antigenic and antisera diagnosticum.

To obtain a diagnosticum, a volume of 2.5% suspension of sheep erythrocytes stabilized with formalin is mixed with an equal volume of a solution of sodium alkyl sulfate (ASN) at a concentration of 1.56 mg / ml and incubated with periodic stirring in a water bath at 45 ° C for 15 min . To the erythrocyte sediment washed with a pH 7.2 phosphate-buffered saline (PBS), PA is added with a protein concentration of at least 300 μg / ml in a pH 5.9 phosphate-saline solution. A suspension of red blood cells is incubated with periodic stirring at 45 ° C for 30 minutes, then placed in a refrigerator at 4 ° C for 18 hours. A phosphate-buffered solution of pH 7.2 with 1% formalin for 10 minutes is added to the erythrocyte sediment formed after 18 hours. . The washed red blood cells are suspended at a concentration of 1% in the PBS with bovine serum albumin 5 mg / ml and 0.05% Tween 20 (PBS). FBTA with 1% formalin is used as a diluting liquid (RG) when setting the micromethod of the indirect hemagglutination reaction (RNGA) and the inhibition of indirect hemagglutination (RTNGA).

The sensitivity of a diagnosticum with a positive control sample of serum obtained for PA corresponds to a titer of 1: 128000 at 100% reproducibility of the test. A diagnostic titer of 1:20 is determined for human sera, 1:10 for guinea pig sera. The specific activity of the diagnosticum with paired sera of people suffering from cutaneous anthrax corresponded to titers of 1: 20-1: 10240 and depended on the duration of the disease. In the sera of immunized and non-immunized guinea pigs that survived after infection of 50-2000 spores of the virulent strain B.anthracis 81/1, antibodies to PA were found in titers 1: 10-1: 81920. The specificity of RNGA is confirmed by RTNGA with protective antigen.

Examples of specific performance, confirming the implementation of the proposed method.

Example 1. Isolation of a protective antigen from a culture filtrate of B. anthracis STI.

To obtain a culture filtrate (CF) of B.anthracis, STI is grown in the R medium used to obtain anthrax toxin (8). In a 500 ml flask with 200 ml of medium, B. anhracis STI is grown on blood agar for 18 h at 37 ° C to a concentration of 1.10 ³ -1.10 ⁴ μ / ml. The flasks were closed with parafilm, shunted at 75 rpm at 37 ° C for 21-22 hours. The culture medium was sterilized by filtration through a DR-045 filter, checked for sterility and concentrated on a DS-2 apparatus with an HLX-50 ultrafilter, then PM-10 (Amicon) to a protein content of 20 mg / ml.

Separation of the components of CF is carried out on a superfine Sephacryl S-300 with a volume of 2.5 × 75 cm. 2.5 ml of concentrated CF is applied to the gel, elution is carried out at a rate of 25 ml / h, the sample volume in a test tube is 1 ml. The protective antigen is contained in 160-180 test tubes and is the dominant protein in CF (9).

The selected fraction is concentrated on a PM-10 ultrafilter to a protein content of 600-1000 μg / ml, PA membership is determined and used to obtain serum according to the rabbit immunization scheme (9), as well as an antigenic erythrocyte diagnosticum.

The belonging of the fraction proteins to the protective antigen is determined by the following criteria:

- the protective antigen is dominant in the culture filtrate of B.anthracis STI - a single-plasmid toxin-producing strain;

- Twice administration to guinea pigs of 50 and 100 μg of protective antigen with incomplete Freund's adjuvant determines the survival of the animals upon infection with 50 LD ₅₀ spores of B.anthracis 81/1;

- serum to a protective antigen in immunoblotting reveals proteins of molecular weight 84 kDa, characteristic for PA.

Example 2. The preparation of the diluting liquid and the method of setting RNGA and RTNGA

To 100 ml of phosphate-buffered saline pH 7.2 ± 0.2 (PBS), add 0.05 ml of Tween-20 Serva detergent (PBS). Bovine serum albumin (BSA) (TU 644-2-278-79) 2 g is dissolved in 20 ml of FBI (100 mg / ml). BSA (100 mg / ml) was added to the FBRT at a ratio of 1:20 diluting liquid (RH). Preparation of an erythrocyte diagnosticum - 10% of an erythrocytic diagnosticum is diluted to 1% concentration in the RG with 1% formalin.

RNGA and RTNGA set in a volume of 100 μl. When setting RNGA serum in a volume of 50 μl is titrated in an equal volume of dilution fluid to well 11. Well 12 of each row is a control. 25 μl of dilution liquid and 25 μl of a 1% erythrocyte diagnosticum are added to all wells of the plate, incubated in an incubator at 37 ° C for 40 min, then at room temperature. The result of RNGA take into account for 1.5 hours

When staging RTNGA, serum is titrated as in RNGA, 25 μl of diluent with PA at a concentration of 10 μg / ml is added to all wells. The plate is incubated in an incubator at 37 ° C for 40 min, then 25 μl of a 1% erythrocyte diagnosticum is added to all wells, re-incubated in an incubator at 37 ° C for 40 min, then at room temperature. The result of RTNGA is taken into account within 1.5 hours. The final result after 24 hours.

With a positive RNGA result, red blood cells drop out on the bottom of the well with a thin layer in the form of an “umbrella”, sometimes with scalloped creeping edges. "Umbrella" can line the entire bottom of the hole (4+) or 2/3 of it (3+). With a negative result of RNGA and in the control, red blood cells fall out in the form of a “button” or a ring of various sizes. In the case of RTNGA, erythrocyte sedimentation in the form of a “button” is considered a positive result.

Example 3. Determination of the optimal conjugating dose of sodium alkyl sulfate

As the conjugating component, sodium alkyl sulfate is used (GOST 19433). 500 mg of sodium alkyl sulfate is dissolved in 40 ml of a pH 7.2 phosphate-buffered saline (PBS) at 37 ° C for 15 minutes, clarified by filtration through a paper filter. 0.5 ml of 10% red blood cells are washed by centrifugation of 4 ml of the FBI 1 time, the erythrocyte sediment is suspended in 8 ml of the FBI and divided into 4 equal parts, the red blood cells are precipitated. The erythrocyte sediment is suspended in 0.5 ml of PBS, 0.5 ml of 2.5% suspension of red blood cells is obtained.

An equal volume of an ASN solution in a dilution of 1: 2 (6.25 mg / ml), 1: 4 (3.12 mg / ml), 1: 8 (1.56 mg) is added to 0.5 ml of a 2.5% suspension of red blood cells. / ml) 1:16 (0.78 mg / ml) and incubated in a water bath with periodic stirring at 45 ° C for 10 minutes. The red blood cells are washed with 4 ml of PBS, the precipitates are suspended in 0.5 ml of PA with a protein concentration of 125 μg / ml in a phosphate-saline solution of pH 5.9, incubated in a water bath with periodic stirring at 45 ° C for 30 minutes, then placed in a refrigerator at 4 ° C for 18 hours. After 18 hours, the supernatant is removed, the red blood cells are suspended in 0.5 ml of 1% formalin, pH 7.2, and incubated for 10 minutes. After sensitization, red blood cells are washed alternately in 4 ml of PBS and RG. Red blood cells in the RH are suspended from 1% formalin to 1% concentration. A diluting liquid with 1% formalin is used in the formulation of the indirect hemagglutination reaction (RNGA) and the indirect hemagglutination inhibition reaction (RTNGA). Sensitized PA erythrocytes are checked in the RNHA micromethod with a positive control sample of serum obtained for PA.

The optimal conjugating dose for 2.5% of formalized red blood cells was a final concentration of ASN of 0.78 mg / ml. Treated ASN and PA sensitized erythrocytes were agglutinated with a control serum at a dilution of 1: 32000 and settled compactly (in the form of a button) in control wells (table 1).

Example 4. Determination of the optimal sensitizing dose of protective antigen

When determining the optimal sensitizing dose of PA, red blood cells are treated with the optimal conjugating dose of ASN. The precipitate obtained from 2.5% of the suspension of red blood cells is suspended with various concentrations of PA, which is added in a volume corresponding to 10% of the suspension of red blood cells. Conjugation with ASN and sensitization of PA red blood cells is carried out under the conditions described in example 3.

The minimum sensitizing dose determined the concentration of PA 300 μg / ml protein, which led to the highest titer of RNGA (1: 128000 and higher) with a control positive serum to PA and compact erythrocyte sedimentation in the controls (table 2).

Example 5. Criteria for the qualitative assessment and suitability of a diagnosticum for the detection of antibodies to a protective antigen in the sera of infected animals and people with anthrax. The diagnosticum was evaluated with the following sera:

- 10 paired sera from 5 patients with cutaneous anthrax, which were obtained in September 2003 and 2008;

- 32 sera (control) from individuals who applied to the clinic for examination, received in April 2005;

- 24 sera of guinea pigs surviving after infection with B.anthracis 81/1 and 10 sera of normal guinea pigs, obtained in June and October 2001

Serum of humans and animals inactivated at + 56 ° C for 30 minutes, preserved with 1: 10000 merthiolate, and stored in a household refrigerator at + 4 ° C.

The sensitivity of three series of diagnosticum in five replicates was determined in RNGA with a positive control serum to PA No. 903 and corresponded to a dilution of serum 1: 128000 with 100% reproducibility of the test (table 3).

Specificity or clinical specificity was established (9, 10) of the A series diagnosticum with 32 sera of individuals who applied to the clinic for examination. Negative results were obtained with twenty two serums at a dilution of 1: 5, with doubtful positive results (2+) were obtained with 9 sera at a dilution of 1: 5. One serum at a 1:10 dilution agglutinized 3+ sensitized PA erythrocytes, which was evaluated as a false-positive result (3.1%). A diagnostic titer of 1:20 was established for human sera at 100% specificity of RNHA.

Determination of specific activity or clinical sensitivity (9, 10) showed that in paired sera of 5 patients with anthrax skin form, antibodies to PA were detected using diagnosticum in titers 1: 20-1: 10240. The titer of antibodies in the sera of patients depended on the duration of the disease (table 4).

When checking the specificity or clinical specificity of RNGA with normal guinea pig sera, the diagnosticum did not react with six out of 10 tested animal sera at a dilution of 1: 5, with four serums at a dilution of 1: 5, dubious positive results were obtained. For guinea pig sera, a diagnostic titer of 1:10 is set at 100% specificity of RNGA.

The specific activity or clinical sensitivity of a diagnosticum with serum from guinea pigs immunized with PA with incomplete Freund adjuvant, as well as control animals that received only Freund's adjuvant according to the immunization schedule and survived after infection, was determined. Sera were taken on days 24-35 from animals that survived infection with 50-2000 spores of a virulent strain of the causative agent of anthrax. Positive results of rnga with animal sera were obtained in titers of 1: 10-1: 81920. The highest titers of RNGA (1: 20480-1: 81920) were established with the serum of guinea pigs immunized with PA and infected 2000 spores of B.anthracis 81/1. Low titers of RNGA (1: 10-1: 1280) were noted with the sera of non-immunized guinea pigs that survived after infection with 50 and 200 spores of B.anthracis 81/1, which indicated the development of an infectious process in animals. The causative agent of anthrax was not isolated from animals slaughtered on days 24-35. (Table 5).

The specificity of RNGA was confirmed by RTNGA with PA at a concentration of 10 μg / ml, which indicated the presence of antibodies in the control positive serum in the sera of infected guinea pigs and patients with anthrax in humans to antigen (PA) adsorbed on red blood cells (table 6).

When comparing the sensitivity of the indirect hemagglutination reaction with the erythrocyte anthrax antigenic diagnosticum and the enzyme immunoassay for detecting antibodies to the protective antigen of the anthrax microbe, almost the same sensitivity of both test systems was established.

When determining the specificity, false-positive results with normal serum of guinea pigs and humans in RNGA were 1: 5 and 1:10, respectively, in IFM 1: 160 and 1: 400 (optical density 0.4 or less).

The specific activity of both tests in the study of the sera of people suffering from cutaneous anthrax and guinea pigs who survived the infection turned out to be the same. The sera of patients with cutaneous anthrax in humans contained antibodies to PA, as determined in RNGA in titers 1: 20-1: 10240, in IFM 1: 400-1: 12800. With highly active serum from guinea pigs that survived the infection, the titers of RNGA and enzyme immunoassay systems were 1: 640-1: 81920 and 1: 6400-1: 81920, respectively.

Thus, the diagnostic erythrocyte anthrax antigenic for the detection of antibodies to the protective antigen, obtained using sodium alkyl sulfate as a conjugating component, is characterized by high reproducibility, sensitivity and specific activity and can be used to diagnose anthrax.

Given the almost identical sensitivity and specificity of the erythrocyte anthrax antigenic and enzyme immunoassay test systems for detecting antibodies to PA, the manufacturability, the production cost of an erythrocyte diagnosticum, the simplicity, short time of setting up and recording the results of RNGA, it is certainly preferable to use an erythrocytic diagnosticum.

Bibliography

1. Burdenko T.A. Development and use of RNGA for serodiagnosis of anthrax: Abstract. dis. ... cand. honey. sciences. - Chisinau, 1973. - 18 p.

2. Levi M.I., Jezepchuk Yu.V., Nemenova M.A. The use of protective antigen for the reaction of passive hemagglutination in anthrax // Zh. microbiol., epidemiol., immunobiol. - 1968. - No. 10. - S.132-134.

3. Buchanan T.M., Feeley J.C., Haues P.S., Brachman P.S. Anthrax in direct hemagglutina tion test // J. Immunol. - 1971. - Vol.l07.-P.1631-1636.

4. Patent No. 1239922, Russia, IPC 4 A61K 39/385. The method of obtaining erythrocyte anthrax / Zhanuzakov N.Zh., Zhiglov V.I., Shamardin V.A., Kuzmin Yu.A., Gert M.V ../; publ. 07/05/84., Bull.№14.

5. Patent No. 1698782, Russia, G01N 33/53, 33/556, A61K 39/07. A method of manufacturing anthrax erythrocyte diagnosticum / Bakulov I.A., Kotlyarov V.M., Nikolaychuk L.F. /; declared 07/11/89; publ. 12/15/91, Bull. No. 46.

6. Lamonica J.M., Wagner M.A., Echenbrenner M. Comparative secretome analyses of the Bacillus anthracis strains with variant plasmid contents. // Infect. Jmmun. - 2005 .-- Vol.73. - N.6. - P.3646-3658).

7. Barkova I.A., Lipnitsky A.V., Barkov A.M., Evteeva E.V. The use of immunoglobulins to individual extracellular antigens of Bacillus anthracis STI for the identification of anthrax microbe // Biotechnology 2005. - No. 2. - S. 91-95.

8. Johnson-Winegar A. Comparison of enzyme - linked immunosorbend and indirect hemagglution assays for determining anthrax antibodies // J. Clinic. Microbiol. - 1984. - Vol. 20. - Number 3. - P. 357-361.

9. Ristroph J.D., Ivins B. Elaboration of Bacillus anthracis antigens in new, delayed culture medium // Infect. Immunn. - 1989. - V.39. - No. 1. - P. 483-486.

10. Tkachev V.K., Vyatkina T.G. Assessment of the quality of the enzyme immunoassay system // Inform-method manual. ELISA diagnosis of syphilis. Koltsovo, 2005 .-- P.32-34.

11. Morse S.A., Bek-Sagyu K.M., Mardh P.A. Recommendations for laboratory diagnosis of ZPPPU // Journal. Sexually transmitted diseases. - 1998. - No. 4. - S.3-15.

“4+” and “3+” are the positive results of RIGA, respectively, when the erythrocytes settle down to the bottom of the hole in an even layer and when scalloped sedimentation of the edges.

Claims (2)

1. A method of obtaining a diagnosticum of an erythrocyte anthrax antigenic for detecting antibodies to a protective antigen, comprising treating formalized sheep erythrocytes with a conjugating drug followed by sensitization of red blood cells with an anthrax antigen, characterized in that the antigen obtained by culturing toxin B is used to sensitize red blood cells to designed to isolate anthrax toxin by concentrating acellular cu of the natural filtrate on ultrafilters excluding proteins of less than 10 kDa, and separation on an ultrafine Sephacryl S-300 in a 2.5 × 75 cm column, followed by selection of a fraction in the yield of 160-180 ml of eluate, which is concentrated on a PM10 ultrafilter to a protein content of at least 600 μg / ml, and determine the affiliation of the protein fraction to a protective antigen. 2. The method according to claim 1, characterized in that to obtain an erythrocyte antigenic diagnosticum, a volume of 10% of red blood cells is washed by centrifugation with a phosphate-buffered solution, the erythrocyte sediment is suspended to 2.5% of a suspension of red blood cells, to which an equal volume of sodium alkyl sulfate solution is added with a concentration 1.56 μg / ml, the erythrocyte suspension is incubated in a water bath with periodic stirring at 45 ° C for 10 min, the red blood cells are washed with a pH 7.2 phosphate-buffered solution, the precipitate is suspended in a pH 5.9 phosphate-saline solution with a protective anti with ene 300 μg / ml in a volume equal to the volume of 10% red blood cells, incubated in a water bath with periodic stirring at 45 ° C for 30 minutes, then placed in a refrigerator at 4 ° C for 18 hours, after 18 hours the supernatant was removed, the red blood cells were suspended in 2 ml of 1% formalin pH 7.2, incubated for 10 min at room temperature, after which the red blood cells are washed 2 times with phosphate-buffered saline, suspended to 1% concentration in phosphate-buffered saline pH 7.2 with bovine serum albumin 5 mg / ml, with 0.05% Tween 20, with 1% formalin.