Podoplanin (PDPN), a 38 kDa transmembrane sialoglycoprotein from human, is expressed in lymphatic endothelial cells but not in vascular endothelial cells, and has been considered as a specific marker of lymph. In this study, the gene...
morePodoplanin (PDPN), a 38 kDa transmembrane sialoglycoprotein from human, is expressed in lymphatic endothelial cells but not in vascular endothelial cells, and has been considered as a specific marker of lymph. In this study, the gene encoding the extracellular part of PDPN (ePDPN) was synthesized and used to expressed fusion protein ePDPN-His and GST-ePDPN, respectively, in E.coli. The purified GST-ePDPN fusion protein was mixed with QuickAntibody-Mouse5W adjuvant to immune mice, and the antiserum titer was determined by indirect ELISA. A stable cell line named 5B3 generating anti-PDPN monoclonal antibody (mAb) was obtained by hybridoma technology. The isotype of 5B3 cell line was IgG 2b , and the chromosome number was 102 ± 4. The 5B3 mAb was purified successfully from ascites fluid through Protein G column, and its affinity constant was 2.94 × 10 8 L/mol. Besides, excellent specificity of the 5B3 mAb was further demonstrated in ELISA, western blot and immunohistochemistry experiments, suggesting that 5B3 mAb displays similar application value to D2-40, a commercial available antibody. Hence, the current study provides conclusive guidelines for preparation of other mAbs and their applications in immunohistochemistry diagnosis. Podoplanin (PDPN) is an O-linked transmembrane sialoglycoprotein that consists of 162 amino acids with molecular weight 38 kDa 1–3. Previous studies have shown that PDPN is a specific marker of lymphatic endothe-lial, and it is highly expressed in lymphatic endothelial cells but not in vascular endothelial cells 4. PDPN can also be found in other tissues types, including follicular dendritic cells, reticular cells, mesothelial cells, testicular germ cells and ovarian cells 5. Furthermore, PDPN has also been reported in lymphangiomas, Kaposi sarcomas, seminomas and epithelioid mesotheliomas hemangioblastomas 6. To date, PDPN contributes to the diagnosis of lymphatic endothelial cell-derived tumors and to the judgement of lymphatic invasion and metastasis in other tumor tissues by labeling the lymphatic endothelium 7. Therefore, it is important to develop methodologies for early diagnosis of lymphatic-related tumors using PDPN as a biomarker. Currently, IHC detection has been recommended as gold standard for tumor diagnosis by the World Health Organization (WHO). Hence, the major challenge in the present study is generation of a monoclonal antibody with high affinity and specificity against PDPN for IHC diagnosis. To date, several commercial mAbs against PDPN have been used, including D2-40, NZ-1, 18H5, LpMab-9 and LpMab-10 8–10. Among these mAbs, only D2-40 has been used for pathological diagnosis. However, dysgerminoma tissue as an antigen for producing the D2-40 faced challenges such as weak immune response by low concentration of PDPN protein, and many non-specific antibodies generated by numerous substances with the method 11. In fact, it's difficult for researchers to obtain carcinoma in situ like dysgerminoma tissue or to obtain cancer cell lines to immune mice. Furthermore, it's challenging to select a stable eukaryotic cell line expressing PDPN since the cells can be contaminated during an extended cell culturing process. In view to the current situation, it is urgent to establish a rapid, easy, cost-effective, feasible and reliable method such as expressing the PDPN antigen in E. coli, and producing a novel
