Development of an indirect ELISA against Orf virus using two recombinant antigens, partial B2L and F1L

In this study, an indirect ELISA method for recombinant proteins based on truncated dominant antigenic epitopes of B2L and F1L genes of ORFV was established. A series of conditions and its performance were comprehensively evaluated. The optimized ELISA reaction conditions were: the optimal coating amount of antigen was 0.25μg/mL, 5% skim milk powder was closed for 1hour, the optimal dilution of serum was 1:200, the optimal incubation time of the rabbit anti-goat IgG was 1:8000, the optimal color development time of TMB was 15mins, and the threshold value of negative-positive was 0.358. The method specifically detects anti-ORFV antibodies and does not cross-react with positive sera for other common goat pathogenic bacteria antiserum. ORFV-positive sera were still positive after 1:512 dilution, with intra-batch coefficient of variation (CV) between 7.1% and 9.5% and inter-batch CV between 5.0% and 7.6%; 51% (92/180) of immunized goat serum samples were tested positive and 14.44% (14/63) of non-immunized goat serum samples were positive. The results show that the indirect ELISA antibody assay established in this study has good specificity, sensitivity and reproducibility, and provides a technical tool for clinical ORFV serum antibody detection and epidemiological investigation.PMID:38336349 | DOI:10.1016/j.jviromet.2024.114891
Source: Journal of Virological Methods - Category: Virology Authors: Source Type: research