RU2422832C1 - Method of producing erythrocytic diagnostic histoplasmosis and coccidiodomycosis antigen - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: substance of the invention involves H.capsulatum G18 5 P cultivation in a mycelial growth phase on a Smith synthetic protein-free salt medium at temperature 28°C for 3 months then a biomass is separated by bulky filtration and sterilised through membrane filters that is followed by a sterility test, and then the antigens found in the culture medium are precipitated with acetone cooled to -20°C and dried. The erythrocytic diagnostic antigen is produced on the recovered antigen complex common for histoplasmosis and coccidioidomycosis.

EFFECT: higher stability and indirect hemagglutination reproducibility.

2 cl, 3 ex, 2 tbl

Description

The invention relates to medical microbiology, in particular to the production of diagnosticums used to detect antibodies to pathogens of histoplasmosis and coccidioidomycosis in an indirect hemagglutination reaction.

To determine antibodies to pathogens of especially dangerous mycoses, diagnosticums are known that were prepared using mutton erythrocytes treated with tannin [1, 4], followed by conjugation of them with a polysaccharide antigenic complex isolated from micromycete cells using the beta-naphthol method [2].

1. The closest analogue is the drug described in the article A. Lipnitsky et al. [1] (prototype).

However, the known preparations are distinguished by relatively low stability, insufficient reproducibility of the diagnosticum preparation method, as well as the complexity and duration of the process of isolating antigens from the cell mass of fungi - causative agents of especially dangerous mycoses [2].

Today, the creation of highly sensitive erythrocyte diagnostic kits suitable for use in emergency situations remains relevant.

The purpose of the invention is the selection of an antigenic complex that does not require additional purification steps common to causative agents of coccidioidomycosis and histoplasmosis, and the creation of an antigenic erythrocytic diagnosticum based on it, characterized by stability and reproducibility of the results.

The goal is achieved by using not polysaccharide antigens extracted from the cell mass of fungi as a sensitin (see prototype), but an extracellular complex common for causative agents of coccidioidomycosis and histoplasmosis, the method of isolation of which and erythrocyte sensitization are characterized by the fact that for optimal formation and extraction of extracellular antigens a culture of H. capsulatum G185 P in the mycelial phase of growth is cultivated on Smith synthetic protein-free salt medium at a temperature of 28 ° C in t 3 months, after which the biomass is separated by filtration through cotton-gauze filters and sterilized through membrane filters, sterility is monitored, and then the antigens in the culture medium are precipitated with a two-three-fold volume of acetone cooled to -20 ° C; the precipitate obtained is separated by centrifugation at 8000 g for 15 min, washed twice with the same proportions in new portions of chilled acetone and dried at 37 ° C, the optimal sensitizing dose of the isolated antigen is determined and the latter is used to obtain an erythrocytic diagnosticum.

The resulting antigen does not require lyophilization, it is relatively easy to accurately dose, does not contain disinfectant residues, is stored for a long time without loss of serological activity and ensures high reproducibility of the method. Thus, an extracellular antigenic complex prepared in sufficient quantities at one time can provide a standard diagnosticum with stable properties for several years.

As a liquid culture nutrient medium, Smith's synthetic salt medium is used [3]. Such a broth does not contain protein-peptone components of the nutrient medium, which allows one to obtain extracellular antigen for its further use without additional purification steps. The dry antigen for sensitization is dissolved in 0.01 M phosphate-buffered saline, pH 5.9. Conjugation is carried out in a water bath at a temperature of 45 ° C for 2-2.5 hours with constant stirring. Fixation is carried out by adding 1 vol.% Formalin with additional incubation under the same conditions for 30 minutes These parameters optimize the sensitization of red blood cells, improve the quality of the drug and, ultimately, increase the sensitivity of RIGA.

The method is illustrated by the following examples.

Example 1. The selection of the antigenic complex from the culture filtrate of H.capsulatum G185P. The cultivation of the strain is carried out in a synthetic salt medium Smith, prepared according to the following recipe:

1. L-aspargin 7.0 g 2. Ammonium chloride 7.0 g 3. Potassium phosphate monosubstituted 1.31 g 4. Sodium citrate 0.9 g 5. Magnesium sulfate 1.5 g 6. Iron citrate 0.3 g 7. Glucose 10 g 8. Glycerin 25 g 9. Water up to 1 l

The medium is sterilized in an autoclave at 120 ° C for 15 minutes.

The prepared medium is poured into 600-800 ml into bacteriological mattresses, where a culture of H. capsulatum G185P is introduced in the mycelial growth phase of 5 ml at a concentration of 5-10 × 10 ⁶ mt / ml according to the optical turbidity standard of GISK named after L. A. Tarasevich . Mattresses in a horizontal position are incubated in a thermostat at 26-28 ° C for 3 months. Biomass is separated from the medium by filtration through a cotton-gauze and membrane sterilizing filters. After the sterility control, the complex of antigens is precipitated from the filtrate by adding to the last 2-3 volumes of acetone chilled to -20 ° C. The precipitate was separated by centrifugation at 8000 g for 15 minutes, washed twice with new portions of chilled acetone and dried in an oven at 37 ° C.

The obtained dry antigenic complex is used in determining the optimal sensitizing dose of antigen and obtaining a diagnosticum.

Example 2. Preparation of a diagnosticum of erythrocytic histoplasmosis antigenic. Determination of the optimal sensitizing dose of the antigenic complex.

Determination of the optimal sensitizing dose of the antigenic complex is carried out using formalized, tanized sheep erythrocytes. The precipitate obtained from 5 ml of a 2.5% suspension of red blood cells is suspended in the same volume of 0.01 M phosphate-buffered solution, pH 5.9, to which weighed portions of 50, 100, 200, 400 μg / ml of dry antigenic complex, respectively . Conjugation is carried out in a water bath at a temperature of 45 ° C for 2-2.5 hours with constant stirring. Fixation is carried out by adding 1 vol.% Formalin with additional incubation under the same conditions for 30 minutes After washing three times, the red blood cells are brought to a concentration of 2.5 vol.% In a 0.15 M sodium chloride solution, pH 7.2, and used in the indirect hemagglutination reaction. The optimal sensitizing dose was 100 μg / ml, which determined the sensitivity of RIGA with control histoplasmosis serum series 2-1 in titers 1: 20480 with clear negative controls.

The preparation of the working series of the diagnosticum is carried out according to the above method with large volumes of reagents. In this case, the optimal sensitizing dose is increased by 2 times (up to 200 μg / ml). The resulting diagnosticum is packaged in ampoules in the form of a suspension of 10 vol.% In a protective drying medium and lyophilized.

Example 3. Determination of the specific activity of the diagnosticum

Histoplasmosis and coccidioidomycosis goat immune sera were obtained by multi-cycle immunization of producers. Disinfected cells of the mycelial phase of the pathogens H. capsulatum and C. immitis served as immunization antigens. When setting RIGA, the serum was diluted 1:10 with a 0.15 M sodium chloride solution, titrated twice in a volume of 0.4 ml in 1 vol.% NKS and one drop of 2.5 vol.% Suspension of diagnosticum was introduced. The results are presented in table 1.

The data in the table indicate the possibility of using a diagnosticum to determine the level of antibodies in immune sera both to the causative agent of histoplasmosis (in titer up to 1: 40960) and to the causative agent of coccidioidomycosis (with some decrease in titer).

Example 4. Determination of the specificity of the diagnosticum

To assess specificity, we used immune sera from laboratory animals (rams, goats, horses) containing antibodies to glanders, melioidosis, plague, and the serum of people with mycotic infections (candidiasis or invasive aspergillosis) and normal human serum.

Heterologous sera were diluted 1:10 with a 0.15 M sodium chloride solution, titrated in two steps in a volume of 0.4 ml and used to form RIGA in the macro variant. The results are presented in table 2 and indicate that the diagnosticum manufactured by the proposed method is able to detect group-specific antibodies to pathogens of histoplasmosis and coccidioidomycosis in a titer of 1: 160 and above. Titers with a higher concentration up to 1:80 are due to cross-reacting antibodies to heterologous antigens that have no diagnostic value.

Information sources

1. Lipnitsky A.V., Valkova E.R., Valkov B.G. Development and application of the reaction of passive hemagglutination and the reaction of neutralization of antibodies in deep mycoses. Diagnosis of especially dangerous infections. - Rostov on the Don. - 1968. - P.54-57 (prototype).

2. Drozdov A.I. Methods for the isolation of antigens from pathogenic fungi (Toolkit). - Leningrad, 1971, 33 p.

3. Smith CD. Nutritional factors that are requaired for the grows and sporulation of Histoplasma capsulatum. / In ′ Histoplasmosis ′. Proceeding of the Suond national conference / Ed. By A. Balows. - Springfild. - 1971. - P.64-70.

4. Valkova E.P., Lipnitsky A.B. Serological parameters in experimental deep mycoses. // J. Microbiol. epidemiol. immunobiol. - 1970. - No. 12. - S. 55-59.

Table 1 The results of determining the specific activity of the diagnosticum No. p / p Producer number Series (immunization cycle) Type of serum Diagnostic Series Caption in Riga The control one 2 one Histoplasmosis 2 1: 40960 - 3 1: 20480 - four 1: 20480 - 2 2 3 Histoplasmosis 2 1: 5120 - 3 1: 1280 - four 1: 5120 - 3 2 four Histoplasmosis 2 1: 10240 - 3 1: 5120 - four 1: 10240 - four 16 one Histoplasmosis 2 1: 2560 - 3 1: 2560 - four 1: 2560 - 5 16 2 Histoplasmosis 2 1: 10240 - 3 1: 2560 - four 1: 10240 - 6 16 3 Histoplasmosis 2 1: 10240 - 3 1: 5120 - four 1: 10240 - 7 eighteen one Histoplasmosis 2 1: 5120 - 3 1: 2560 - four 1: 2560 - 8 eighteen 3 Histoplasmosis 2 1: 40960 - 3 1: 5120 - four 1: 10240 - 9 eighteen 5 Histoplasmosis 2 1: 40960 - 3 1: 20480 - four 1: 20480 - 10 twenty one Histoplasmosis 2 1: 1280 - 3 1: 1280 - four 1: 1280 - twenty 2 Histoplasmosis 2 1: 5120 - eleven 3 1: 1280 - four 1: 2560 - 12 twenty 3 Histoplasmosis 2 1: 1280 - 3 1: 320 - four 1: 2560 - 13 one one Coccidioidomycosis 2 1: 1280 - 3 1: 320 - four 1: 1280 - fourteen one 3 Coccidioidomycosis 2 1: 640 - 3 1: 320 - four 1: 640 -

Continuation of table 1 No. p / p Producer number Series (immunization cycle) Type of serum Diagnostic Series Caption in RNGA The control Coccidioidomycosis 2 1: 320 - fifteen one four 3 1: 160 - four 1: 320 - Coccidioidomycosis 2 1: 10240 - 16 9 one 3 1: 5120 - four 1: 5120 - Coccidioidomycosis 2 1: 5120 - 17 9 5 3 1: 2560 - four 1: 5120 - Coccidioidomycosis 2 1: 2560 - eighteen 9 10 3 1: 640 - four 1: 1280 - Coccidioidomycosis 2 1: 640 19 9 fifteen 3 1: 320 - four 1: 2560 - Coccidioidomycosis 2 1: 2560 - twenty 9 twenty 3 1: 640 - four 1: 1280 - Coccidioidomycosis 2 1: 2560 - 21 17 one 3 1: 640 - four 1: 1280 - Coccidioidomycosis 2 1: 2560 - 22 17 3 3 1: 1280 - four 1: 2560 - Coccidioidomycosis 2 1: 2560 - 23 17 6 3 1: 640 - four 1: 1280 - Coccidioidomycosis 2 1: 2560 - 24 19 one 3 1: 640 - four 1: 1280 - Legend: - negative result RIGA. Note. The data are taken from the tables of the "Act of commission tests of the diagnosticum of erythrocyte histoplasmosis antigenic dry", Federal State Health Institution VolgogradNIPCHI of Rospotrebnadzor dated June 05, 2009

table 2 Diagnostic specificity results No. p / p Producer number Series (immunization cycle) Type of serum Diagnostic Series Caption in RNGA The control 5574 (goat) Melioid 2 1:80 - one 2 3 1:40 - four 1:80 - 5 (goat) Melioid 2 1:40 - 2 fourteen 3 1:20 - four 1:40 - 7 (goat) Melioid 2 1:20 - 3 21 3 - - four 1:20 - 6518 (ram) Sapnaya 2 - - four four 3 - - four - - 6518 (ram) Sapnaya 2 - - 5 12 3 - - four - - 2801 (goat) Sapnaya 2 - - 6 9 3 - - four - - 10 (ram) Plague 2 1:40 - 7 four 3 1:20 - four 1:40 - 10 (ram) Plague 2 - - 8 9 3 - - four - - Coquette (horse) a commercial Plague 2 1:20 - 9 3 - - four - - Patient serum Clinic. diagnosis: candidiasis 2 40 - 10 3 twenty - four 40 - Patient serum Clinic. diagnosis: candidiasis 2 40 - eleven 3 1:20 - four 1:40 - Patient serum Clinic. diagnosis: candidiasis 2 1:20 - 12 3 1:20 - four 1:20 - Patient serum Clinic. diagnosis: aspergillosis 2 1:20 - 13 3 1:20 - four 1:20 - Normal human serum - 2 1:20 - fourteen 3 - - four 1:20 - Legend: - negative result RNGA. Note. The data are taken from the tables of the "Act of commission tests of the diagnosticum of erythrocyte histoplasmosis antigenic dry" FGZU Volgograd NIPCH Rospotrebnadzor dated June 05, 2009

Claims (2)

1. A method of producing an erythrocyte antigenic histoplasmosis and coccidioidomycosis diagnosticum, comprising isolating an antigenic complex and sensitizing red blood cells by it, characterized in that for optimal formation and isolation of extracellular antigens, H. capsulatum G185 P culture in the mycelial growth phase is cultured at synthetic temperature without Celica at synthetic temperature without bacteria 28 ° C for 3 months, after which the biomass is separated by filtration through cotton-gauze filters, sterilized through membrane filters, after four o carry out sterility control and then precipitate the extracellular antigens in the culture medium cooled to -20 ° C with a two-three-fold volume of acetone, the resulting precipitate is separated by centrifugation at 8000 g for 15 min, washed twice with new portions of chilled acetone and dried at 37 ° C, determine the optimal sensitizing dose of the isolated antigen and use it to obtain a red blood cell diagnosticum. 2. The method according to claim 1, characterized in that the sensitization of red blood cells is carried out in 0.01 M phosphate-buffered saline pH 5.9 at a temperature of 45 ° C for 2-2.5 hours, followed by fixation by adding 1 to the reaction mixture vol.% formalin with incubation for 30 minutes