A Study On The Preparation, Identification And Application Of Anti-podoplanin Antibodies

Background:In the present, many reasons result in the occurrence and death rate of cancers stay on a high level, such as bad living environment, irregular diet and living habits, great work stress and the genetic factors. Tumor becomes a common and frequently-occurring disease,which is harmful to the human health. Tumor metastasis is the most important cause of cancer patients’ death, but so far the molecular mechanism of tumor metastasis is still unclear. In recent years, more and more evidences showed that platelets play an important role in tumor metastasis, some possible mechanisms are as follows: 1. Activated platelets release a lot of cytokines, including platelet-derived growth factor, etc. These cytokines can promote the tumor cells transfer and growth and help the blood vessel regeneration; 2.The tumor cells lead to platelet activation, the activation platelets released the ADP alkanes and synthetic thrombosis A2(TXA2) and result in integrin glycoprotein Ⅱ b/Ⅲ(GP Ⅱb/Ⅲ a) activation. The activation platelets in cancer patients is increasing, and promoting the above process and development; 3. The activation platelets adhere in the tumor cells surface, help tumor cells from the body’s immune attacking, at the same time, help tumor cells’ implantation, adhesion, which is conducive to the tumor metastasis.PDPN plays a key role in the relationship between platelet and tumor cells.Podoplanin belongs to the family of type-I transmembrane sialomucin-like glycoprotein’s which was originally reported as a specific lymphatic endothelial marker protein. s PDPN comprise an extracellular domain with abundant Ser and Thru residues as potential O-glycosylation sites, a single transmembrane portion, and a short cytoplasmic tail with putative sites for protein kinase C and c AMP phosphorylation. The EDxx VTPG segment in the extracellular domain, designated as the platelet aggregation stimulating(PLAG)domain, is critical for the activity of podoplanin. C-type lectin-like receptor-2(CLEC-2)was recently identified as an endogenous receptor of podoplanin on platelets, theinteraction between PDPN and CLEC-2 leads to the platelet activation. Recently, many reports showed that PDPN expressed in various tumors, including lung cancer, gastric cancer, intestinal cancer, squamous cell carcinoma, malignant mesothelioma, Kaposi sarcoma, angiosarcoma, testicular seminoma and brain tumors, etc. The high level of PDPN in tumor cells cause platelet activation, this may be the basis on promoting tumor metastasis and growth.Anti-PDPN monoclonal antibody can inhibit PDPN combining with CLEC-2, prevent the activation and aggregation of platelet, which can inhibit tumor metastasis, and anti-PDPN monoclonal antibody will be not caused the risk of bleeding, they have a great value in clinical application. The different anti-PDPN monoclonal antibodies against different antigen epitope, they can establish a double antibody sandwich ELISA method,which can be used to detect the soluble PDPN level in cancer patients plasma. The level of soluble PDPN in plasma plays a role in the diagnosis and prognosis of the cancer patients.Objective:This study aims to prepare the anti-PDPN monoclonal antibody by the technology of monoclonal antibody, to explore the relationship between PDPN and tumor metastasis;Establish a double antibody sandwich method, to detect the level of soluble PDPN n cancer patients plasma, to study the role of soluble PDPN in development of tumor.Methods:(1) Immune the BALB/c mice with the peptide composed of 22 amino acid(DTETTGLEGGVAMPGAEDDVVC) coupling with Keyhole limpet hemocyanin(KLH)as the immunogenic.(2) We construct the anti-PDPN monoclonal antibodies; then identify the specific binding ability of anti-PDPN antibody with PDPN antigen using the commercialization anti-PDPN antibody 18H5 as a positive control, through the enzyme linked immunosorbent(ELISA), Western Blot and Flow Cytometry.(3) The function study of anti- PDPN antibody in vitro: The interaction between PDPN and the CLEC- 2 expressed in the surface of platelet lead to the platelet activation and platelet aggregation. In tumor cell surface, the level of PDPN is high, so the tumor cells can induced the platelet aggregation, we incubated the tumor cells with the anti-PDPN antibody in advance, and then mixed with platelets(PRP), Platelet aggregation analyzer is used to inspect the aggregation rate. We analyze if the anti PDPN antibody caninhibit the platelet aggregation induced by PDPN.(4) The applications in the detection of the soluble PDPN level in human plasma: Two monoclonal antibodies, SZ-168 and SZ-163, can combined with different antigen epitope of PDPN, so we established the double antibody sandwich ELISA method, the method can be used to detect the level of soluble PDPN in cancer patients plasma, used to analyze the role of soluble PDPN in diagnosis of tumor development.Results:We immune BALB/c mice with the polypeptides composed with 22 amino acids coupling the KLH, Two antibodies were screened by indirect ELISA successfully, SZ163 and SZ168, the titer is 500ng/ml and 15ng/ml respectively, and then we identified the specific binding ability of anti-PDPN antibody with reducing PDPN antigen and nature PDPN antigen through the methods as enzyme linked immunosorbent(ELISA), Western Blot and Flow Cytometry.In addition, we detected the PDPN level in with antibody, SZ168 through the FCM,we found the PDPN level in NCI- H226 cells is high, so we detected the platelet aggregation and if the SZ168 can inhibit the platelet aggregation with NCI- H226 cells.Experimental results indicate that SZ168 can inhibit platelet aggregation induced by PDPN(10 μg/ml: inhibition rate-74.6%), 6 μg/ml: inhibition rate-66.1%), 3 μg/ml: inhibition rate-8.47%).Finally, we set up the double antibody sandwich ELISA method(SZ163 coated the board, horseradish peroxidase labelled the SZ168 as enzyme mark), examined the level of soluble PDPN in stomach/lung /colorectal cancer patients and normal human plasma,through the statistical analysis, the result shows that the level of soluble PDPN in tumor patients is higher than in normal human plasma(P < 0.01), in tumor patients with metastatic were higher than that with non-metastatic tumour patients(P < 0.05).Conclusion:(1) We prepared two strains monoclonal antibodies(SZ163 and SZ168) with high titer and specificity through the hybridoma technology. The specificity can be detected by the ELISA, Western Blot and flow cytometry.(2) The study of PDPN in vitro showed that the high expression of PDPN in tumor cells can induce platelet aggregation, SZ168 mixed with tumor cells in advance and then reacted with the platelets, the result displayed SZ168 can inhibit platelet aggregation.(3) The double sandwich ELISA method was used to detect the level of soluble PDPN in normal human and tumor patients’ plasma, the results show that the soluble PDPN as tumor markers, can prompt the tumor occurrence and metastasis.