Single B-Cell-Based Generation of Porcine Anti-CSFV Erns Monoclonal Antibodies and Application in a Blocking ELISA Assay

:Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is an acute, febrile, and highly contagious disease that has led to significant economic losses in the global swine industry. Although the attenuated lapinized CSF vaccine (C-strain) has effectively controlled CSF outbreaks in China since the 1950s, it remains challenging to serologically differentiate infected from vaccinated animals (DIVA). Currently, the application of E2 subunit vaccines allows for DIVA by detecting antibodies against the Erns protein. Therefore, this study aimed to develop a blocking ELISA for CSFV Erns antibody detection using porcine monoclonal antibodies (mAbs) derived from single B cell technology. Peripheral blood mononuclear cells (PBMCs) were isolated from immunized pigs, and single CD21+IgM−Erns-His tag+ B cells were sorted via flow cytometry. Using one-step PCR, full-length genes of porcine IgG heavy and light chains were amplified separately, yielding 11 porcine mAbs against the CSFV Erns protein. Among these, three mAbs (E0S3, E0S5, and E0S10) exhibited broad reactivity, while two (E0S1, E0S4) showed no cross-reaction with bovine viral diarrhea virus (BVDV). Using mAb E0S4 as the blocking antibody, a blocking ELISA was established and optimized. The assay demonstrated a detection limit of 1:128, no cross-reactivity with other swine viruses or BVDV, and intra- and inter-assay coefficients of variation below 10%. ROC curve analysis determined an optimal cut-off value of 48.4%, with high sensitivity and specificity. In conclusion, the developed blocking ELISA provides a reliable tool for high-throughput serological surveillance, facilitating the DIVA strategy and contributing to CSF eradication programs.