Determination of critical factors causing cytotoxicity in the virus neutralization test

The fluorescent antibody virus neutralization (FAVN) test, an international standard method for serological testing for rabies, has been adopted by many countries. However, some dog serum samples inhibit the formation of cell monolayers by BHK-21 cells used in the test, resulting in failures to determine antibody titers. This inhibition of cell monolayer formation was defined as cytotoxicity. In this study, critical factors that induce cytotoxicity of the dog serum in BHK-21 cells were determined, and the effective ways to prevent cytotoxicity were also established. Specifically, some anticoagulants, anti-BHK-21 cell IgG antibodies, and serum storage at temperatures of >25°C were found to induce cytotoxicity. On the other hand, several treatments of the dog serum, including the absorption by BHK-21 cells or kaolin, incubation with trypsin-EDTA, and the use of collagen- or gelatin-coated plates, were shown to reduce cytotoxicity. Based on these results, the FAVN test may be modified to enhance its performance.