Development of a solid-phase blocking ELISA for the detection of neutralizing antibodies against foot-and-mouth disease virus serotype O

Abstract:

[Objective] To evaluate the level of neutralizing antibodies (NA) against foot-and-mouth disease virus (FMDV), a solid-phase blocking enzyme-linked immunosorbent assay (ELISA) based on bovine monoclonal antibodies was developed. [Methods] In this study, the bovine monoclonal antibody (mAb) E32 was used as the capture antibody and a biotinylated bovine mAb C4 as the detection antibody. Both mAbs had been produced in our laboratory. The mAb E32 is a cross-reactive antibody against FMDV and the mAb C4 is an intraserotype broadly neutralizing antibody against FMDV serotype O. With the two mAbs, a solid-phase blocking ELISA for detecting neutralizing antibody (NA-SPBE) against FMDV serotype O was developed. The sensitivity, specificity, repeatability, cross-reactivity, and correlation with virus neutralization test (VNT) of this assay were assessed. [Results] The optimum working concentrations of antibody E32, FMDV-inactivated antigen, and biotinylated C4 were 0.5 μg/mL, 0.25 μg/mL and 0.06 μg/mL, respectively, and the optimum dilution of streptavidin-HRP was 1:40 000. When 1.35 log₁₀ was used as the cut-off value, the sensitivity and specificity of the assay were determined as 97.14% and 98.84%, respectively. There was no cross reactivity with the antibodies specific to bovine viral diarrhea virus (BVDV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), peste des petits ruminant virus (PPRV), or FMDV serotypes A and Asia1. The intra-batch and inter-batch repeatability of the assay showed the coefficient of variation less than 10%. The detection of 160 serum samples demonstrated a correlation (r=0.807 5, P<0.000 1) between the titers obtained by NA-SPBE and VNT. [Conclusion] NA-SPBE can detect the neutralizing antibodies against FMDV serotype O and provides powerful technical support for evaluating the efficacy of FMDV serotype O-inactivated vaccine.