Objective of the study was to develop enzyme-immunoassay test-kit for the detection of Bacillus anthracis spores. Materials and methods. Microbial cultures from the State Collection of Microorganisms at the premises of Affiliated Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation and BALB/c mice were used in the research. Hybridization of B-lymphocytes with SP2/0-Ag14 myeloma cells was performed according to G. Kohler and C. Milstein procedure in De St. Fazekas and P. Scheidegger modification. Hybridomas were cultured in the peritoneal cavity of BALB/c mice. Ascitic fluids were isolated from mice, precipitated with ammonium sulfate and purified by means of ion-exchange chromatography for preparation of monoclonal antibodies. Specific activity of hybridoma’s supernatants, ascitic fluids, purified monoclonal antibodies was studied by «sandwich» ELISA. Specific components of test-kit were lyophilized in suitable cryoprotective medium. Results and conclusions.We have obtained new hybrid cell lines producing specific monoclonal antibodies against Bacillus anthracisspore antigens and ascitic fluids from which immunoglobulins were isolated. Optimum combinations of monoclonal antibodies as a sensitizer and a component of immunoperoxidase conjugates have been selected. Monoclonal antibodies 272E10G1-272F7A10 provide the highest sensitivity of ELISA for the detection of anthrax microbe spore antigens. Our enzyme-immunoassay test allows for identification of Bacillus anthracis spores in concentrations up to 5,0·105 spores per milliliter. No cross reaction with closely related saprophytes and other heterologous microorganisms in concentrations of 1,0·108 CFU per milliliter is observed.

The aim of the work was to obtain and characterize hybridomas producing monoclonal antibodies to antigens of coronavirus SARS‑CoV‑2, promising for the construction of diagnostic immunochemical tests. Materials and methods. Recombinant nucleocapsid and receptor binding fragment of spike protein of SARS‑CoV‑2 were used for immunization of BALB/c mice. Antigens were absorbed on aluminium hydroxide gel and injected subcutaneously to BALB/c mice at a 7-day-interval. Immune splenocytes and myeloma cells SP2/0-Ag14 were fused by polyethylene glycol 1450. Cell cultures producing specific antibodies against nucleocapsid and receptor binding fragment were selected applying indirect ELISA in 96-well plates sensitized by desired antigens. Clones of hybridomas were obtained using the method of limiting dilutions. Production properties were studied through in vitro cultivation in 24-well culture plates. Immune-ascitic fluids were collected during the cultivation of hybrid cells in peritoneal cavities of BALB/c mice. Monoclonal antibodies were purified by affinity chromatography on protein A sepharose sorbent, conjugated with horseradish peroxidase, and tested for the possibility to be used in sandwich ELISA for detection of inactivated SARS‑CoV‑2 coronavirus strain “Isolate B”. Results and discussion. As a result of hybridization and selection of clones, hybridomas producing monoclonal antibodies to nucleocapsid and receptor binding fragment of SARS‑CoV‑2 have been obtained. During the in vitro and in vivo cultivation the clones maintained the consistent proliferative and antibody producing activity. The application of monoclonal antibody 415D12 as a capture one and 411D12 antibody conjugated with horseradish peroxidase as a detector antibody in ELISA allows for identifying SARS‑CoV‑2 coronavirus at a minimum concentration of 1·103 PFU per ml.

Aim. Obtaining hybridomas, stable producing specific monoclonal antibodies against Burkholderia mallei and Burkholderia pseudomallei antigens. Materials and methods. The microbial cultures from State Collection of Microorganisms from the Branch of 48 CSRI of the Defense Ministry of Russian Federation (Kirov) and BALB/c mouse were used in research. Hybridization of B lymphocytes with SP2/0-Ag14 myeloma cells was performed by G.Kohler and C.Milstein procedure in De St. Fazekas and D.Scheidegger modification. The specific activity of immune sera, hybridoma supernatants, ascites and evaluating the diagnostic capabilities of monoclonal antibodies was studied by ELISA. Results. Hybridomas, producing monoclonal antibodies against causative agents of glanders and melioidosis antigens, were obtained and characterized. Obtained hybridomas are active and stable antibody producers after repeated in vitro and in vivo passaging. Immunoglobulins from obtained ascites were isolated. Antibodies provided the greatest sensitivity and specificity were selected. Conclusion. Monoclonal antibodies, producing by obtained hybridomas may be used for creating of immune biological tests.

Objective of the study was the development of immune-enzymatic monoclonal test-kit for detecting glanders and melioidosis agents. Materials and methods. We used microbial cultures and hybrid cell lines obtained from the collection of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation. Hybridoma cells were incubated in the peritoneal cavity of BALB/c mice. Preparations of glanders and melioidosis monoclonal antibodies were isolated from the ascetic fluids through precipitation with ammonium sulfate and purification by means of ion-exchange chromatography. Specific components of the test-kits were subjected to freeze drying in corresponding protective media. Study of diagnostic properties of the developed test systems was performed using ELISA. Results and conclusions. We have obtained preparations of monoclonal antibodies in vivo, as well as isolated and purified immunoglobulins from ascetic fluids. We also selected the pairs of monoclonal antibodies for manufacturing specific components. Experimental series of immune-enzymatic monoclonal test-systems allowing for specific detection of glanders and melioidosis causative agents in concentrations ranging from 0.5·106 CFU/ml and higher were made. The absence of cross-reactivity with closely related saprophytes and heterologous microorganisms in concentrations of 1,0·108 CFU/ml was shown. Demonstrated was the possibility in principle to differentiate between Burkholderia malleiand Burkholderia pseudomallei using ELISA. Test systems are promising for follow up state registration as medical products for in vitro diagnostics.

Objective of the study was the development of experimental ELISA tests and lateral flow immunoassays for detection of staphylococcal enterotoxins, A and B types.Materials and methods. Hybridomas, producing monoclonal antibodies against staphylococcal enterotoxins A and B from State Collection of the Affiliated Branch of the “48th Central Research Institute” of the Ministry of Defense of the Russian Federation, BALB/c mice and staphylococcal enterotoxins A and B were used in the research. Hybridoma cells were incubated in culture flasks and in the peritoneal cavity of BALB/c mice. Monoclonal antibodies were isolated from ascitic fluids through precipitation with saturated ammonium sulfate subsequently purified using ion-exchange chromatography. Obtained preparations of monoclonal antibodies were used for the construction of ELISA tests and immune-chromatographic reagent panels for the detection of staphylococcal enterotoxins A and B. Specific components of ELISA tests were lyophilized in protective media.Results and discussion. ELISA tests and lateral flow immunoassays which allow for detecting staphylococcal enterotoxins A and B at concentrations of 0.5 ng/ml and higher, including in food samples, have been constructed.