Development and evaluation of immunoglobulin-binding-proteins based ELISA for serological diagnosis of foot-and-mouth disease virus in domestic animals.

Foot-and-mouth disease (FMD) is a highly contagious viral disease that affects multiple livestock species and causes significant economic losses. Current serological assays rely on species-specific kits, which increases cost and complexity. We developed a universal multi-species ELISA using recombinant [AG]4, consisting of tandem repeats of immunoglobulin-binding domains from Staphylococcal protein A (SpA) and Streptococcal protein G (SpG), conjugated to HRP as a universal secondary antibody. To enable differentiation between infected and vaccinated animals (DIVA), we incorporated the 8BF antigen, which contains three linear B-cell epitopes from the FMDV 3B non-structural protein. The 8BF-4AG ELISA demonstrated high reproducibility (intra-batch coefficients of variation and inter-batch coefficients reproducibility less than 10 %) and specificity (no cross-reactivity with eight related pathogens). The detection results of clinical samples showed a high coincidence rate of 93.75 %-100 % with commercial competition ELISA kits. The results of 726 bovine sera, 82 porcine sera and 48 ovine sera from different farms showed FMD positivity rates ranging from 0 % to 11.11 %. This 8BF-4AG ELISA is a high-throughput, cost-effective tool for FMD surveillance with broad applicability in epidemiological studies and routine veterinary diagnostics.