Establishment and application of a blocking ELISA based on a monoclonal antibody against the F1L protein of Orf virus for antibody detection

Abstract:

Background Contagious ecthyma is a zoonotic disease caused by infection with the Orf virus (ORFV). It is prevalent in sheep and goat flocks worldwide and seriously impacts the development of the sheep and goat farming industry. Objective To establish and evaluate a blocking enzyme-linked immunosorbent assay (ELISA) for detecting ORFV antibodies. Methods A monoclonal antibody (mAb) against the ORFV F1L protein was prepared and conjugated with horseradish peroxidase (HRP). With the F1L protein expressed in a baculovirus system as the detection antigen and the HRP-conjugated F1L mAb as the detection antibody, a blocking ELISA for ORFV antibody detection was established after optimization of the reaction conditions. The applicability of this method was assessed by testing 100 serum samples in parallel with an indirect immunofluorescence assay (IFA). Results The HRP-labeled F1L mAb exhibited a high titer and stable properties. The F1L protein was successfully expressed in the soluble form, with the purified F1L protein showing high purity and good reactivity. The optimal conditions for the blocking ELISA were as follows: coating concentration of F1L protein at 1.0 μg/mL and dilution of the HRP-conjugated antibody at 1:2 000. The cut-off values A and B were determined as 37.47% and 28.40%, respectively. Samples with an inhibition ratio greater than 37.47%, less than 28.40%, and between the two values were considered positive, negative, and doubtful, respectively. The intra-assay coefficients of variation (CV) did not exceed 4.49%, and the intra-assay CV did not exceed 6.28%, indicating good repeatability. The blocking ELISA showed the limit of detection of 1:128 for ORFV-positive serum, demonstrating higher sensitivity than indirect IFA. The method showed good specificity, with no cross-reactivity with antibodies against other common pathogens. Clinical sample testing revealed a 97.87% concordance rate between this method and indirect IFA. Conclusion A blocking ELISA based on the F1L protein is successfully established and can be used for the detection of ORFV antibodies.