Development of novel anti-rhinoceros podoplanin monoclonal antibodies for flow cytometry, western blotting, and immunohistochemistry

Podoplanin (PDPN) is a type I transmembrane glycoprotein characterized by a highly glycosylated extracellular domain, a single transmembrane domain, and a short intracellular tail. PDPN is physiologically expressed in various normal tissues and cell types, including pulmonary type I alveolar epithelial cells, renal podocytes, and lymphatic endothelial cells. In addition to its physiological roles, PDPN has emerged as a valuable biomarker in cancer research. Consequently, the development of anti-PDPN monoclonal antibodies (mAbs) across a range of animal species has facilitated the identification and characterization of PDPN-expressing cells. To date, we have generated anti-PDPN mAbs for over 20 species, suitable for applications, such as flow cytometry, Western blotting, and immunohistochemistry. In the present study, we established two anti-rhinoceros PDPN (rhiPDPN) mAbs—PMab-315 (mouse IgG2a, kappa) and PMab-324 (mouse IgG2b, kappa)—using the Cell-Based Immunization and Screening method. Both mAbs bound to rhiPDPN-overexpressed Chinese hamster ovary K1 (CHO/rhiPDPN) and exhibited an identical KD value (4.5 × 10⁻⁸ M) in a flow cytometry-based assay. Furthermore, PMab-315 and PMab-324 effectively detected rhiPDPN in both Western blot and immunohistochemical analyses. These findings highlight the potential utility of PMab-315 and PMab-324 as robust tools for the pathological investigation of rhinoceros-derived tissues and cells.