Establishment of a monoclonal antibody PMab-225 against alpaca podoplanin for immunohistochemical analyses

Abstract Podoplanin( PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPNhave been established in our previous studies. However, mAbs against alpaca PDPN(aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for producing anti -aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamsterovary (CHO)-K1 cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using flow cytometry. One of the mAbs, PMab-225 (IgG 2b , kappa), specifically detectedCHO/aPDPN cells via flow cytometryand recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells, colon lymphatic endothelial cells, and kidney podocytesvia immunohistochemistry. These findings demonstrate that PMab-225 antibody is useful to investigate the function of aPDPN via different techniques.