Direct Quantification of Protein Antigens in Subunit Plague and Rickettsial Vaccine Preparations

Abstract

The aim of the work was to put forward the methods for direct quantitative determination of the content of Yersinia pestis and Rickettsia raoultii protein antigens in preparations and various prototypes of subunit vaccines. Materials and methods. Y. pestis LcrV and Caf1 antigens enclosed in the substance of the molecular microencapsulated plague vaccine (MMPV) and separately, in microcrystals of amino acids co-precipitated with plague proteins were used as model antigens. R. raoultii Adr2, OmpB24, and YbgF antigens were adsorbed on the prototype substance of the rickettsia vaccine. The release of plague antigens from MMPV microcapsules was carried out through successive treatment of the latter with organic solvents, methylene chloride and methanol, respectively; the carrier microcrystals were dissolved in 0.1 M sodium citrate buffer at pH 6.0. The antigen content in the prototype substance of the rickettsial vaccine was determined by measuring the amount of proteins not bound to the alumogel. Quantitative parameters characterizing the content of antigens in the substances and prototypes of vaccine preparations were calculated by processing digital images of polyacrylamide gels obtained by electrophoresis of protein antigen fractions extracted from carriers. Results and discussion. Methods for direct extraction and subsequent quantitative analysis of Y. pestis LcrV and Caf1 antigens from subunit vaccine preparations based on amino acid microcrystals and polylactide microcapsules that do not cause protein degradation have been studied. A different nature of the binding of LcrV and Caf1 in the substances of microcrystals has been established, while the proportion of antigens released from microcrystals has been quantified only in case of their complete dissolution. It was found that at low concentrations of LcrV and Caf1 proteins extracted from microcrystals, it is necessary to concentrate the extracts with subsequent removal of salts for their reliable visualization. It has been confirmed that 10 μg of plague antigens and proteins of R. raoultii in a dose volume of 200 μl of suspension is sufficient for quantitative analysis using electrophoretic method. The prospects of other physicochemical methods alternative to direct extraction of antigens for evaluating the composition and quality of vaccine preparations are discussed.