Background. Pseudotuberculosis remains a serious healthcare problem, which determines the expediency of developing the express methods for its early diagnosis. To detect the pathogen, we designed test system for dot-immunoassay (DIA) based on antibodies labeled with silver nanoparticles (SNPs) isolated from hyperimmune rabbit serum obtained against killed cells of  Yersinia pseudotuberculosis of O:1b serovariant.The aim. To assess the possibility of using dot-immunoassay for express identification of Y. pseudotuberculosis cultures isolated from clinical material and environmental objects at the initial stage of bacteriological study during laboratory diagnosis of the disease.Methods. We used the materials from the outbreak of pseudotuberculosis in the Krylovskaya Boarding School of the Bakcharsky district of the Tomsk region in 2021. Specific antibodies from hyperimmune rabbit sera obtained against Y. pseudotuberculosis 3704 particulate antigen of O:1b serotype were labeled with SNPs and used in DIA on nitrocellulose membranes with visualization of reaction results with a solution of a physical developer. The presence of the causative agent of pseudotuberculosis in the test material was inferred by the formation of gray spots of different intensity (from 4+ to 1+).Results. All Y.  pseudotuberculosis strains isolated using bacteriological method on  the second day of the study from clinical material obtained from sick people and environmental objects were detected in DIA at concentrations ≥ 3.1 × 104 microbial cells per milliliter (m.c./ml).Conclusion. The designed test system for dot-immunoassay using SNPs as a marker of specific antibodies for the detection of Y.  pseudotuberculosis in cultures isolated from swabs from vegetables and clinical material from patients, including those  with  mixed infection, allows us to  detect a specific corpuscular antigen with a high sensitivity (≥ 3.1 × 104 m.c./ml), providing express identification of isolated cultures at the initial stage of bacteriological study.

Aim. Construction of an immunologic test-system for detection of causative agents of entero-pathogenic yersinia {Yersinia pseudotuberculosis and Y. enterocolitica) by dot-immunoassay. Materials and methods. Nanoparticles of colloid silver sized 5 - 9 nm were used as a marker of specific antibodies. IgG fraction was isolated from commercial antisera to Y. pseudotuberculosis (0:1) and Y. enterocolitica (0:3 and 0:9). Testing of the obtained test-system was carried out on 20 strains of Y. pseudotuberculosis and Y. enterocolitica (10 of each species). Results. Dot-analysis had a specific character and detected enteropathogenic yersinia at a level of 5Т05 - 8T06 CFU/ml (100 - 1000 CFU in sample). Wherein cross-reaction with heterologic studied microorganisms - Escherichia coli, Salmonella typhimurium, Shigella flexneri, Yersiniapestis EV - was not observed. A possibility of simultaneous detection and serotyping of Y. enterocolitica is shown, that is necessary for confirmation of their epidemic significance. Conclusion. The developed test-systems allow to study micro volumes of the samples under study (1 pi), are express (1.5 - 2 h), highly sensitive and specific, technically simple and do not require the use of high-cost equipment, special training of the staff, may be successfully used in practical healthcare in laboratories with varying equipment levels.

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Abstract. Immuno-serological diagnostic tools particularly identifying pathogen antigens are the most important methods of pseudotuberculosis studies . The main immunodominant and species-specific antigens located in the surface structures of the bacterial cell are of practical interest. Thereby the aim of the work was to isolate and characterize biologically active surface structures of the pseudotuberculosis microbe. Here, the living cells of Y. pseudotuberculosis 3704 (O:1b) were lysed by using 9 M urea solution to extract antigens localized in the microbial surface structures. The subcellular fractions obtained such as outer membranes (OM), urea extract (UE) and isolated protein-lipopolysaccharide complex (PLPSC) are characterized by physical and chemical parameters. The protein content in the preparations ranged from 42% to 53%. The polypeptide band of the OM preparation, UE polypeptide and PLPSC for pseudotuberculosis microbe was presented by 14, 16 and 9 major polypeptides with molecular weight ranging from 13,9 kDa to 131,5 kDa, 13,5 kDa to 101,6 kDa, and 20,7 kDa to 66,6 kDa, respectively. Proteolytically active proteins and polypeptides were detected in isolated subcellular fractions (OM and UE) by using the radial enzyme diffusion test and substrate-gel electrophoresis found to be presented by 4 and 7 polypeptides with molecular weight ranging from 28,0 kDa to 118,0 kDa and 29,2 kDa to 97,7 kDa in the OM and UE preparation, respectively. The subcellular fractions obtained are capable to exhibit immunogenic activity after inoculation to experimental animals and antigenic activity while interacting with specific antibodies in the radial immunodiffusion (RID) assay and antibodies labeled with colloidal silver nanoparticles in dot immunoassay (DIA). OM and PLPSC preparations in DIA with immunoglobulins isolated from experimental antisera and labeled with colloidal silver nanoparticles were detected at a concentration of ≥ 0.12 μg / ml (dry weight), cells of strain Y. pseudotuberculosis 3704 at a concentration of ≥ 3, 9 × 106 m.c. / ml, which is similar to the results of DIA with immunoglobulins isolated from commercial pseudotuberculosis antiserum (St. Petersburg) and labeled with nanoparticles of colloidal silver. Thus, the subcellular fractions of pseudotuberculosis microbe isolated by using urea as a lysing and decontaminating agent retain their antigenic and immunogenic properties and enzymatic activity suggesting about their potential benefits for use to improve early diagnostics of pseudotuberculosis.