Characterization of the antibody diversity of glycoprotein E2 domain B/C in pestivirus by using high-throughput single B cell technology.

Microfluidic technology is an advanced platform for isolating single B cells and constructing paired antibody chain libraries. In the present study, a customized immunization approach coupled with a high-throughput, microfluidic-based single B cell platform was utilized to identify and characterize a pool of monoclonal antibodies (mAbs) targeting the B/C domain of the classical swine fever virus (CSFV) E2 protein. A total of 4241 antigen-specific plasma cells were isolated and subsequently subjected to next-generation sequencing, which yielded 82 distinct naturally paired antibody sequences. Binding assays performed on 81 mAbs revealed that 38 mAbs effectively bound to the B/C domain of the CSFV QZ07 strain E2 protein. Among these, 14 mAbs specifically recognized the B/C domain of genotype 2 QZ07-E2, and 24 mAbs reacted with both genotype 2 QZ07-E2 and genotype 1 C-E2; within the latter group, 8 mAbs exhibited cross-reactivity with the B/C domain of other pestiviruses. Phylogenetic profiling of CDR sequences also classified these antibodies into three distinct clusters, which showed a strong correlation with binding profiles. These findings suggest the presence of novel, broadly reactive pan-pestivirus epitopes in the B/C domain. The integration of microfluidics with high-throughput sequencing in single B cell technology in this study not only accelerates the antibody discovery process but also provides a robust platform to investigate the antigenicity of key pathogen proteins for developing diagnostic assays and next-generation marker vaccines.