Khomenko Y. Scientific and experimental confirmation of dotimmunoassay technology for brucellosis and leucosis of

Critical components for dot blot immunoassay for diagnosis of brucellosis and bovine leukemia were chosen, technology of production of chemical and biological components was developed: method for purification of B. аbortus LPS was improved; an expediency of using of the fusion recombinant protein, which includes both 24 kDa and 51 kDA proteins of bovine leucosis virus, was justified; optimal concentration of antigens in the dot blot immunoassay were determined; the protocol of G. T. Hermanson (1996) was optimized to bind colloidal gold nanoparticles to recombinant protein G Streptococcus spр.; the technology of conjugation of protein G Streptococcus spр. with carbon nanoparticles was developed; an expediency of using of High Impact Polystyrene (HIPS) was justified; original method of drip sorption for immobilization of B. аbortus and bovine leucosis virus antigens was developed. The protocol for dot blot immunoassay was optimized. In particular, it was determined that the use of phosphate-salt buffer (pH 7.2–7.4) containing 0.75 % gelatin, 2.5 M urea and 0.01 % benzoic acid as a serum diluent completely eliminates the nonspecific binding of dot blot immunoassay components. Dot blot immunoassay based express protocol for discrimination of Br. аbortus and Yersinia Enterocolitica was established. Sensitivity and specificity of developed test kits for diagnosis of brucellosis and bovine leucosis is 100 %, which is in conformity with World Organisation for Animal Health requirements. Coefficient of reproducibility for test kits is below 10 % (CV<10 %), which is right for such test kit. It has been proved that the specificity, sensitivity and reproducibility of developed dot blot immunoassay for diagnosis of brucellosis and bovine leukemia are in conformity with World Organisation for Animal Health requirements: specificity and sensitivity are 100 %, reproducibility satisfies modern requirements for such type of test kits (CV<10 %).