Abstract
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.
Highlights
Original articlesКачественные показатели экспериментальных бруцеллезных антигенных препаратов, предназначенных для клеточных тестов in vitro
Objective of the study was to identify the qualitative indicators of experimental batches of brucellosis antigen preparations designed for cellular tests in vitro
Qualitative composition was assessed through ion exchange liquid chromatography with refractometric detection
Summary
Качественные показатели экспериментальных бруцеллезных антигенных препаратов, предназначенных для клеточных тестов in vitro. Цель работы – определение качественных показателей экспериментальных серий бруцеллезных антигенных препаратов, предназначенных для клеточных тестов in vitro. Количественное определение белков экспериментальных серий полученных препаратов WsAg и PPBC проводили методом специфической флуориметрии. В результате исследований определены физико-химические характеристики, изучены хроматографические профили и состав белковых фракций, а также проведена апробация изготовленных экспериментальных серий бруцеллезных антигенных препаратов. Анализируя охарактеризованный белковый и полисахаридный состав полученных образцов WsAg, можно сделать заключение о невозможности использования препарата WsAg для клеточных тестов, так как экспериментально доказана вероятность проявления неспецифических реакций лимфоцитов in vitro. Полученные характеристики позволят обеспечить надлежащее качество при производстве разработанного экспериментального препарата PPBC, предназначенного для клеточных тестов in vitro, с учетом современных правил валидации и стандартизации.
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