Abstract
Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.
Highlights
Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in normal tissues including renal podocytes, type I lung alveolar cells, and lymphatic endothelial cells [1,2]
P3U1, Chinese hamster ovary (CHO)-K1, and CHO/horse PDPN (horPDPN) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and FHK-Tcl3.1 was cultured in Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Inc.) [32]
Two BALB/c mice were immunized with CHO/horPDPN cells (1 × 108), which were intraperitoneally (i.p.) administered together with Imject Alum (Thermo Fisher Scientific Inc.)
Summary
Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in normal tissues including renal podocytes, type I lung alveolar cells, and lymphatic endothelial cells [1,2]. The expression of human PDPN (hPDPN) has been reported in several malignant tumors, including oral squamous cell carcinomas [11], esophageal cancers [12], lung cancers [13], malignant mesotheliomas [14,15], osteosarcomas [16,17,18], chondrosarcomas [17], malignant brain tumors [19,20,21,22], and testicular tumors [23]. An anti-horse PDPN (horPDPN) mAb, PMab-202 was recently established by immunizing mice with synthetic peptides of horPDPN, it was not useful for immunohistochemical analysis [32]. We immunized mice with CHO/ horPDPN cells and established hybridomas that could produce mAbs against horPDPN
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