Development of an immunoenzyme test system for diagnosis if rabies

Rabies remains one of the most serious and pressing problems for veterinary science and medicine. According to the data of Rosselkhoznadzor Information and analytical center, the Russian Federation has been an area of natural focal endemicity for many years [13]. An important stage in the control and eradication of rabies in its laboratory diagnosis by detection of virus antigen in the pathological material. The aim of the study was to design an enzyme-linked immunosorbent assay (ELISA) test kit intended for the laboratory diagnosis of rabies in agricultural, domestic, and wild carnivores. Obtaining specific components for the kit, the GNKI “Sheep” master seed strain of rabies virus, the standard CVS strain of rabies virus, and white mice of the BALB line were used. In order to obtain high-activity of specific immunoglobulins (Igs), the processes of antirabies serum production, immunoglobulin isolation, and purification have been improved as well as the conditions for immunoglobulin conjugation with peroxidase. It was found that the binding ratio should not to be lower than 0.45–0.6. A method of inactivation of rabies virus antigen has been developed, which eliminates the risk of being infected for laboratory workers and shows high specific activity in serological tests. The optimal concentrations of the reactants (immunoglobulin, antigen, conjugated) were determined; basic conditions for conducting the reaction were identified (pH of the carbonate/bicarbonate coating buffer, the optimum temperature, and duration of incubation) that ensure the required level of accuracy and sensitivity of the method. As a result of the conducted studies, a “Kit of preparations for the laboratory diagnosis of animal rabies in animals by ELISA method” was developed. The correlation between results obtained by the immunofluorescence (IF) method and the enzyme-linked immunosorbent assay (ELISA) methods was determined; the compliance coefficient constituted 98.8%, the sensitivity threshold of ELISA method being 3.3 Ig LD_50 / ml. The sensitivity, specificity, and reproducibility of the developed test system were determined through laboratory and production tests.