Abstract
Identifying immunoglobulin (Ig) genes from antigen-specific B cells is crucial for understanding immune responses and generating monoclonal antibodies for diagnostic and therapeutic purposes. Despite single B cell PCR-based mouse antibody development is well established, several practical challenges remain. Here, we present an optimized protocol for the sequencing and cloning of variable regions of antibodies from single antigen-specific mouse B cells, along with high-throughput antibody expression and characterization. This method builds upon existing techniques, incorporating laboratory refinements and detailed troubleshooting insights. By integrating fluorescence-activated cell sorting (FACS) with reverse transcription polymerase chain reaction (RT-PCR) to amplify immunoglobulin heavy and light chain genes, along with a 12-well format for antibody expression, our refined approach enables efficient monoclonal antibody production and functional screening, thereby accelerating the antibody discovery workflow across a range of experimental applications.