Epitope mapping of anti-mouse podoplanin monoclonal antibody PMab-1

Mouse podoplanin (mPDPN) is a type I transmembrane sialoglycoprotein, which is expressed on lymphatic endothelial cells, podocytes of the kidney, and type I alveolar cells of the lung. mPDPN is known as a platelet aggregation-inducing factor and possesses four platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 in the N-terminus and PLAG4 in the middle of the mPDPN protein. mPDPN overexpression in cancers has been reportedly associated with hematogenous metastasis through interaction with the C-type lectin-like receptor 2 of platelets. We previously reported a rat anti-mPDPN monoclonal antibody clone PMab-1, which was developed by immunizing the PLAG2 and PLAG3 domains of mPDPN. PMab-1 is very useful in flow cytometry, western blot, and immunohistochemical analyses to detect both normal cells and cancers. However, the binding epitope of PMab-1 remains to be clarified. In the present study, flow cytometry, enzyme-linked immunosorbent assay, and immunohistochemical analyses were utilized to investigate the epitope of PMab-1. The results revealed that the critical epitope of PMab-1 is Asp39 and Met41 of mPDPN. These findings can be applied to the production of more functional anti-mPDPN monoclonal antibodies.