Galkin A. Biotechnology and bioanalytical standardization enzyme immunoassay diagnostic tests

The thesis presents the results of a scientific study of biotechnology of creation of highly informative ELISA diagnostic tests and bioanalytical parameters of standardization and validation of diagnostic products. Original sets of monoclonal antibodies to IgA, IgG and human IgE, horseradish peroxidase (HRP), the main protein of the outer membrane Ch. trachomatis was obtained, and the possibility of their use for the development of various modifications high informative ELISA and/or immunoaffinity chromatography was proved. The study of the biological properties of antibodies and epitope characteristics of the corresponding antigens was performed. Consistent pattern of humoral immune response in Balb/c and NZB mice to HRP was studied. Improved methods of obtaining of preparations of human IgE, Fc-fragments of IgG and IgA were developed. Optimal parameters of biotechnology production of recombinant Ch. trachomatis heat shock protein (rHSP-60) was determined. It was proved the stimulating effect of Clivia miniata and Zephyranthes grandiflora extracts on rHSP-60 biosynthesis in E. coli bacteria. The possibility of using the obtained immunosorbents, immunoconjugates, Ch. trachomatis rHSP-60 to develop highly informative ELISA kits was proved. It was shown that in the serum from Ch. trachomatis infected persons contains idiotypic antibodies with anti-HSP-60 activity, which is one of the causes of false negative results. It was proved high activity of the hybrid positive control for IgM-"capture" ELISA kits, as well as for the detection of IgM and IgA antibodies to Ch. trachomatis (indirect ELISA). The parameters of standardization and validation characteristics of serodiagnostics tests were justified. Scientific and methodological recommendations on the validation of different types of ELISA were formed.