Development of Novel Anti-Rhinoceros Podoplanin Monoclonal Antibodies for Flow Cytometry, Western Blot, and Immunohistochemistry

Podoplanin (PDPN) is a type I transmembrane protein with a highly glycosylated extracellular domain, a transmembrane domain, and a short intracellular domain. PDPN expression is observed in normal tissue and cells, including lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. Therefore, anti-PDPN monoclonal antibodies (mAbs) in various animal species helped to identify PDPN-positive cells.We have developed anti-PDPN mAbs in more than 20 species. These anti-PDPN mAbs are applicable to flow cytometry, western blot, and immunohistochemistry. In this study, anti-rhinoceros PDPN (rhiPDPN) mAbs, PMab-315 (mouse IgG2a, kappa) and PMab-324 (mouse IgG2b, kappa) were established using the Cell-Based Immunization and Screening (CBIS) method. PMab-315 and PMab-324 strongly recognized the rhiPDPN-overexpressed Chinese hamster ovary K1 (CHO/rhiPDPN) and exhibited the same KD value (4.5 × 10−8 M) for CHO/rhiPDPN by a flow cytometry-based assay. Both mAbs can detect rhiPDPN in western blot and immunohistochemistry. These results suggest the usefulness of PMab-315 and PMab-324 for the pathological analysis of rhinoceros-derived tissues and cells.