Human podoplanin (hPDPN), which binds to C‐type lectin‐like receptor‐2 (CLEC‐2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer‐associated fibroblasts indicates poor prognosis. Human lymphatic endothelial cells, lung‐type I alveolar cells, and renal glomerular epithelial cells express hPDPN. Although numerous monoclonal antibodies (mA bs) against hPDPN are available, they recognize peptide epitopes of hPDPN. Here, we generated a novel anti‐hPDPN mA b, LpMab‐21. To characterize the hPDPN epitope recognized by the LpMab‐21, we established glycan‐deficient CHO‐S and HEK‐293T cell lines, using the CRISPR/Cas9 or TALEN. Flow cytometric analysis revealed that the minimum hPDPN epitope, in which sialic acid is linked to Thr76, recognized by LpMab‐21 is Thr76–Arg79. LpMab‐21 detected hPDPN expression in glioblastoma, oral squamous carcinoma, and seminoma cells as well as in normal lymphatic endothelial cells. However, LpMab‐21 did not react with renal glomerular epithelial cells or lung type I alveolar cells, indicating that sialylation of hPDPN Thr76 is cell‐type‐specific. LpMab‐21 combined with other anti‐hPDPN antibodies that recognize different epitopes may therefore be useful for determining the physiological function of sialylated hPDPN
