Evaluation of a DAS-ELISA for quantification of foot-and-mouth disease virus 146S antigen during vaccine preparation

Abstract

The inactivated foot-and-mouth disease (FMD) vaccine's protection is dependent on the intact component of the FMD virus (FMDV) antigen, the 146S antigen particle. Sucrose density gradient (SDG) centrifugation is the standardised method for quantifying 146S antigen during FMD vaccine formulation. However, because it is operator-dependent, this approach is labour-intensive and produces varied outcomes. As a result, the polyclonal double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed as an alternative approach to quantify the intact 146S antigen of both FMDV South African Territories (Mahapatra and Parida) -1 & 2 serotypes. The polyclonal DAS-ELISA performance was compared to the SDG centrifugation test to evaluate the assay as an alternative technique for quantifying the intact 146S antigen of both SAT serotypes. In BHK-21 cells, the FMDV 146S antigen of both SAT serotypes was generated. For each serotype, sixteen samples were examined in duplicates using the SDG (10-30%) centrifugation and polyclonal DAS-ELISA at varied time intervals (0, 5, 10, 15 minutes) and dissociation conditions (Intact 146S, Temperature, pH, complete dissociation (CD)). The SDG method identified the intact 146S antigen particles at 254 nm using a Type 11 Optical Unit for a UA-6 absorbance detector, whereas polyclonal DAS-ELISA detected FMDV antigen particles at OD 450 nm using a microplate ELISA reader. The SDG was more specific to the Immunogenically intact 146S antigen, whereas polyclonal DAS-ELISA measured an equivalent reactivity to the intact 146S antigen and the 12S protein components in both SAT serotypes. The polyclonal DAS-ELISA technique was not suitable for quantifying the intact 146S antigen and so could not be used for quantification of the immunogenic 146S antigen component during vaccine production.