Preparation and specificity study of BTN2A1 monoclonal antibodies based on hybridoma technology

AimTo explore the preparation method of monoclonal antibodies targeting BTN2A1 and develop detection tools, providing experimental evidence for optimizing immunotherapy strategies.MethodsHigh-affinity BTN2A1 monoclonal antibodies were screened using hybridoma technology; antibody specificity for native conformational antigens was validated by flow cytometry; epitope mapping was performed via competitive ELISA; feasibility of a dual-antibody sandwich detection system was assessed based on antibody pairing experiments.ResultsFifteen high-affinity BTN2A1 monoclonal antibodies were successfully obtained (minimum half-maximal effective concentration: 0.002 862 g·L-1). Flow cytometry confirmed that all antibodies specifically recognized cell-surface native BTN2A1 in BTN2A1-overexpressing CHO cells and Daudi tumor cell lines with >95% binding positivity. Epitope analysis indicated all antibodies targeted the same epitope.ConclusionsThis study successfully generates a panel of high-affinity BTN2A1 monoclonal antibodies, providing essential tools for functional research. However, since all antibodies recognize an identical epitope, establishing a dual-antibody sandwich detection system is unfeasible. Further development of antibody combinations targeting distinct epitopes is thus required, laying the foundation for optimizing BTN2A1 immunoassays and therapeutic antibody development.