Preparation Of Monoclonal Antibody Against Podoplanin And Its Application In Immunohistochemistry

Podoplanin(PDPN),a 38 kDa transmembrane sialoglycoprotein from human,is expressed in lymphatic endothelial cells but not vascular endothelial cells,so it's regarded as a marker of lymphatic endothelial.To date,only D2-40 antibody has been used for pathological diagnosis,but the D2-40 antibody is expensive and was produced outside of China.Therefore,we aim to construct vectors to express PDPN for immunization and generate monoclonal antibody for IHC diagnosis by cell fusion.In this study,the gene sequence for extracellular region of PDPN was chosen to be synthesized after codon optimization,and inserted into vector pET-28a and pGEX-6P1 to express fusion protein PDPN-His 6 and GST-PDPN,respectively.GST-PDPN recombinant protein mixed with QuickAntibody-Mouse5W adjuvant was injected into BALB/c mice,and the titer of antiserum was determined using PDPN-His 6 as coating antigen by ELISA method.After cell fusion and screening,a stable monoclonal cell line named 5B3 generating antibody against PDPN was obtained.The iostype of 5B3 cell line is IgG2b and the chromosome number is 102±4.After anti-PDPN monoclonal antobody(mAb)was purified from ascites fluid by Protein G method,the titer of 5B3 mAb reached 1.28×105 with the affinity constant of 2.94×108 L/mol.After the 5B3 mAb was stored at 4? or 37? for different days,the result showed that the mAb still have a high stability.Furthermore,high specificity of the mAb was appeared in ELISA,Western Blot and immunohistochemistry(IHC),especially in sections of human tissue,including lung tissue,mesothelioma,seminoma,submucosal lymphatic vessels and thyroid,suggesting that the 5B3 mAb can be used in the classification of lymphatic invasion of primary tumors.Hence,anti-PDPN mAb for IHC diagnosis was successfully developed,and it was beneficial to reduce the cost of medical testing and break the monopoly of foreign countries,and provide guidance for the treatment and prognosis of related tumors.