In order to develop the most diagnostically informative methods for carrying out antigen-stimulated cellular tests in vitro a careful selection of stimulating agent (antigen) is required, possessing an adequate activating potential and providing specificity of the reaction.Objective of the study was to identify the qualitative indicators of experimental batches of brucellosis antigen preparations designed for cellular tests in vitro.Materials and methods. Initially we produced antigen complexes of brucellosis microbe on the basis of the vaccine strains of three epidemically significant Brucella species (B. abortus, B. melitensis, B. suis). Quantitative determination of WsAg and PPBC proteins of experimental preparation series was performed applying capillary electrophoresis. Qualitative composition was assessed through ion exchange liquid chromatography with refractometric detection.Results and discussion. We have specified physical-chemical features, investigated chromatographic profiles and composition of protein fractions, as well as tried the produced experimental batches of brucellosis antigen preparations. After analyzing the defined protein and polysaccharide composition of the obtained WsAg samples, one can conclude that WsAg preparation cannot be used for cellular tests as the probability of non-specific lymphocyte reaction manifestation in vitro was experimentally proven. By contrast, complex brucellosis antigen preparation PPBC has an expressed specific activity and specificity under in vitro conditions and the prospects to be used when developing methodological approaches for laboratory diagnosis of brucellosis and assessment of de facto immunity rate in risk contingents after vaccination. The obtained parameters will allow for proper quality provision when manufacturing the developed experimental PPBC preparation designed for cellular tests in vitro, taking into account modern validation and standardization regulations.

Introduction: Plague is endemic in a number of states around the world. Despite all public health measures taken to eradicate plague, the disease persists and even reappears in some countries. Today, traditional serological methods searching for the capsular antigen of the plague microbe are widely used to monitor the presence of Y. pestis in the environment. Yet, for the causative agent of the plague, the possibility of eliminating plasmids and maintaining of the ability to cause an infectious process by atypical strains is not excluded, which reduces the diagnostic value of preparations based on the detection of species-specific antigens of the plague microbe. Our objective was to develop biotechnology for the production of immunodiagnostic drugs for detection of the plague bacteria (capsular and capsule-free forms). Materials and methods: When performing the work, 27 strains of microorganisms of pathogenicity groups I–IV of different genera and species were used. Results: The development of immunodiagnostic preparations for the detection of Y. pestis in environmental samples is described. The presented experimental preparations allow detection of typical and antigenically modified plague microbe strains with a positive and/or negative expression of fraction 1. To evaluate the effectiveness of the designed diagnosticums, both laboratory and field testing was performed demonstrating positive results in the study of both artificially and naturally contaminated samples. The possibility of using the plague immunomagnetic sorbent providing selective concentration of material with a low pathogen content and purification of samples from possible contamination with extraneous microflora during epizootological examination was also confirmed. Conclusions: We constructed promising diagnostic preparations that help identify Y. pestis strains in the studied objects regardless of the cultivation temperature and plasmid profile.

The aim of the work was to implement the risk management strategies in the manufacturing and use of medical products for in vitro diagnostics by the example of the experimental series of the reagent kit “Lyophilized erythrocyte antigenic tularemia diagnosticum”.Materials and methods. We tested experimental series of the reagent panel “Lyophilized erythrocyte antigenic tularemia diagnosticum”. To carry out the identification, assessment and analysis of risks regarding the considered medical product, failure mode and effects analysis (FMEA) was proposed and adapted under production conditions. Identification of risks associated with manufacturing and control of medical products for in vitro diagnostics was carried out using technological regulations, standard operational procedures and manufacturing notes.Results and discussion. The main outcome of the study is the development of a corrective actions system aimed at reducing the risks and ensuring consistent monitoring. The proposed schemes for carrying out the risk management process can be used as standard ones in the design and development of medical products for in vitro diagnostics, taking into account the specifics of each individual manufacturing. The reporting documents developed within the framework of the system are applicable during the inspection of good manufacturing practice and in terms of completing the registration profile of a diagnostic product with subsequent registration in the healthcare system of the Russian Federation.

Anthrax poses a pressing issue for veterinary medicine and public health in many countries, including the Russian Federation, which necessitates the improvement and development of new, sensitive and specific diagnostic tools.The aim of the work was to create an experimental peroxidase conjugate for the detection of specific antibodies to the anthrax pathogen and to optimize the conditions for performing enzyme immunoassay (ELISA).Materials and methods. The peroxidase conjugate was constructed using horseradish peroxidase and Staphylococcus aureus protein A (Sigma-Aldrich, USA). Bacterial antigens isolated from strains of Bacillus anthracis 55ΔTPA-1Spo, B. anthracis Sterne 34 F2 were used as sensitizing agents. The developed experimental batches of the conjugate were tested in ELISA for the ability to bind antibodies in the blood sera of anthrax patients and vaccinated individuals. The sensitivity, specificity, and accuracy of the method were calculated using the built-in functions of the ROCR software package.Results and discussion. The peroxidase conjugate to detect specific antibodies to the anthrax pathogen in the study of clinical material has been developed; conditions for the ELISA performance have been optimized. To interpret the results of the study, a threshold value of the positivity coefficient was used, below which the result was considered negative, and at an equal or higher value, positive. The test demonstrated significant differences in the “positivity coefficient” indicator for the “Healthy”/“Sick” and “Healthy”/“Vaccinated” groups, while the differences between the “Sick”/“Vaccinated” groups were statistically insignificant. The maximum accuracy of the method was observed at blood serum dilutions of 1:250 and 1:500. 100 % intra-run, run-to-run and series-to-series reproducibility has been established for all positive samples. The sensitivity and specificity of the experimental peroxidase conjugates were 100 and 95.8 %, respectively, and the accuracy was 97.6 %.

The article presents the materials on the study of stability of the major quality parameters of five experimental series of standard sample of magnosorbent from Stavropol Antiplague Institute of Rospotrebnadzor (registration No. 007-9388-2015). Persistence of stability of the magnosorbent standard sample parameters has been confirmed, without changes in appearance and without loss of absorption intensity through the whole period of observations. Applying a standard sample of magnosorbent as a magnetic matrix will allow to optimize the production process obtaining precise and comparable results, considerably improving the biological parameters of newly-developed or already produced magnoimmunosorbent products for diagnostics. The results of the performed study of stability of the assessed parameters in the process of storage allow to recommend a 3-year warranty shelf life of the magnosorbent standard sample.