Isolation, Purification and Evaluation of Serological Activity of Rabies Virus Antigens

Abstract

Objective of the study is to evaluate the serological activity of rabies virus antigens isolated from the brain tissue of mice by homogenization on FastPrep followed by ultracentrifugation. Materials and methods. Producer strain of the rabies virus “Ovechiy” GNKI. The rabies virus was isolated from the brain tissue of experimentally infected mice, followed by the study of the electrophoretic profile. The serological activity of the virus components was assessed by immunoblot and ELISA using specific anti-rabies sera.
Results and conclusions. In the course of comparing the methods of isolation and purification of the rabies virus antigen, it was found that most optimal one is to use a homogenization on FastPrep-24, followed by fractionation in a sucrose gradient. As a result of fractionation in a graded sucrose density gradient with a concentration of 15–50 % at 25000 g for 120 min, five fractions of the rabies virus components were obtained. The maximum purified protein fraction was from 15–20 % sucrose zone, which corresponded to a molecular weight of 67 kDa. The specific antigen activity of the fraction in ELISA reached up the titers of 1:1280 (Specificity coefficient 2.2).
Using immunoblot of antigens, obtained from the sucrose gradient in the range of 40–45 % and 20–35 % after ultracentrifugation, one major fraction of polypeptides (54 kDa) was detected, which showed the highest antigenic activity. The results obtained will be useful in the design of test systems for rabies screening and monitoring the effectiveness of anti-epizootic measures.