The article discusses the selection of optimal conditions for the lyophilization of poly- and monoclonal peroxidase conjugates used in the direct version of the enzyme immunoassay intended for the cholera toxin detection in Vibrio cholerae O1, O139 strains. Effective stabilizers included in protective media comprise 1 % sucrose, 1 % sodium thiosulfate, 0.5 % egg albumin, or 0.5 % bovine serum albumin. Their use increases the shelf life, increases stability, and contributes to the preservation of high sensitivity and specificity of poly- and monoclonal peroxidase conjugates. Evaluation of the serological activity of antitoxic conjugates in ELISA after lyophilization in a protective medium with stabilizers showed that it remains at the initial level. The results of testing lyophilized conjugates during storage allow us to speak about the possibility of their use for two years without changing the main indicators.

As a result of the studies, the method of direct solid-phase enzyme immunoassay (ELISA test) was optimized for detecting toxigenic strains of Vibrio cholerae O1 and O139 serogroups based on monoclonal and polyclonal antitoxic immunoglobulin peroxidase conjugates. The study detected the following optimal parameters for the analysis: sensitization of a tablet by toxicodermia drugs diluted in phosphate-saline buffer (PBS) pH 7.4, up to titers of 1:2-1:4, their adsorption on the solid phase of the tablet for 2 hours at 37° C, blocking of free binding sites by the 1% solution of bovine serum albumin (BSA) for 30 minutes (at 37°C); contact time of the investigated sample with mono- or polyclonal conjugates peroxidase - 30 minutes (at 37°C). Tests on homologous and heterologous strains showed a high specificity of the enzyme immunoassay and the possibility of its use for detecting toxigenic Vibrio cholerae.

The aim of the work was to study surface antigenic determinants of V. cholerae R-variant strains using enzyme immunoassay and a panel of monoclonal antibodies (MAbs).Materials and methods. 60 strains of V. cholerae R-variant isolated from ambient environment objects in the territories of the former USSR and the constituent entities of the Russian Federation over a 30-year period (1988–2019) were investigated in the slide agglutination reaction with cholera diagnostic sera, enzyme immunoassay (ELISA) using the panel of MAbs specific to membrane proteins and a set of reagents “Monoclonal diagnostic immunoglobulins labeled with horseradish peroxidase, dry, for serological identification of V. cholerae O1 and O139 (in vitro) through ELISA and dot-ELISA”.Results and discussion. The analysis of the surface structures of V. cholerae R-variant strains with atypical agglutinability has been carried out applying enzyme immunoassay. It showed that individual strains with different amounts of O-antigen are registered among the studied strains identified at isolation as V. cholerae R-variant (the optical density range is from 0.261±0.002 to 1.312±0.003). Epitopes of specific O-antigen were found in some “conservative” strains (30 %) that are agglutinated only with RO serum, and in several strains (20 %) that do not have the wbeT gene that determines its synthesis, and lost agglutinability with all diagnostic cholera sera, including RO. The protein epitopes recognized by complementary MAbs are represented with varying frequency in the composition of surface antigens of R-vibrios; a decrease in their representation or absence on the cell surface correlates with the modification or loss of R-LPS and is accompanied by a negative agglutination reaction.

Aim. Elucidation of the role of extrachromosomal elements of heredity in manifestations of toxic properties of Yersinia pestis. Materials and methods. The study was carried out in vaccine strain Y. pestis EV76 (pMTl, pCDl, pPCPl) and non-plasmid variants of vaccine EV76 (pMTl\ pCDl', pPCPl') and virulent 231 (рМТГ, pCDl’, pPCPl') strains of Y. pestis. Presence of functionally active form of lipopolysaccharide (LPS) in the incubation medium of the bacteria was evaluated via toxicity of supernatant of Y. pestis for intact animals (infection-toxic shock) and mice sensitized by D-GalN. Results. 37°C cultures of Y. pestis EV76 containing a full amount of plasmids were established to release LPS into the environment. Non-plasmid variants of both vaccine and virulent strains of Y. pestis pMTl', pCD Г, рРСР 1 do not have this ability. Separation of LPS from cell wall was detected in live bacteria of plague infectious agent. This process is assumed to be coupled with translocation of proteins coded by pMTl, pCDl, pPCPl plasmids from the cell into the environment. Conclusion. Functional interconnection between extrachromosomal elements of heredity and toxic activity of Y. pestis LPS is established for the first time.

Literature and own data on mechanisms of realization of lipopolysaccharide (LPS) toxic potential of Yersinia pestis in the conditions of a macroorganism are analyzed. 2 modifications of LPS are examined - temperature dependent changes of chemical structure of polymers and a change in their conformation under the effect of micro- and macroorganism factors. A special attention is paid to comparative study of toxic and immune modulating properties of the specified LPS forms. Both LPS forms are concluded to activate TLR4/MD2 receptor, inducing synthesis of 2 types of cytokines - pro-inflammatory and interferons. However, dominance of their signal pathways and cross-regulation of the transduced signal are mirrored, and as a result the initial form of LPS initiates interferon synthesis, and conformationally changed - pro-inflammatory cytokines. Results of the experiments are summarized in 2 schemes of signal transfer by TLR4/MD2 receptor under the effect of 2 forms of Y. pestis LPS. Variations of cytokine-inducing properties of the initial and conformationally-altered forms of Y. pestis LPS corresponds to the immune response of the organism at each stage of the infectious process: late inflammatory response by interferon type is characteristic for intra-cellular cycle of plague development, and pro-inflammatory cytokine hyper-production is observed at the terminal stage of infection-toxic shock.