Preparation And Functional Study Of Human Vascular Endothelial Cadherin CDH5 Monoclonal Antibody

Objective:The aim of this study was to prepare the human vascular endothelial cadherin CDH5 monoclonal antibody(m Ab) by hybridoma technique, to perform the functional study of m Ab and explore the relationship between CDH5 and tumor vessel angiogenesis, which will provide the theory and tool basis for further study. Moreover, we also established the“sandwich” enzyme-linked immunosorbent assay(ELISA) using above CDH5 m Ab,which could be employed in disease diagnosis and prognosis in clinical practice.Methods:The N-terminal segment of CDH5 was obtained by PCR amplification and constructed into the prokaryotic expression vector p ET-41 a. Recombinant protein was expressed and the soluble fragment was purified by Ni-NTA method. The purity of the protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) followed by Coomassie Blue staining.Using hybridoma technique, the monoclonal antibody recognizing CDH5 protein was produced, and the specificity of the antibody was verified by ELISA, Western blot,immunofluorescence and immunohistochemistry. The entire extracellular region segment of CDH5 was amplified by PCR and inserted into the prokaryotic expression vector p ET-41 a. After obtaining of recombinant proteins, Western blot and synthetic peptide dot-blot(dot blot) and competitive ELISA was employed for a preliminary and detailed epitope analysis, respectively.Then the m Ab was used in immunohistochemistry to examine the protein level of CDH5 in various types of tumor tissue. Furthermore, in vitro 3-D Matrigel angiogenesis experiment was set up to explore the effect of this antibody on tumor angiogenesis and vascular mimicry(Vascular mimicry, VM). Cell proliferation experiment by CCK-8 assay and cell adhesion were used to function verification of antibody on tumor cells. The impact of antibody on PI3K/AKT/β-catenin signaling pathway was verified by Western Blot.Moreover, we also selected the higher affinity antibodies and determine the best combination of antibodies using a checkerboard titration method for sandwich ELISA establishment. After that, the antibody was labelled by horseradish peroxidase(HRP) and used for human plasma CDH5 level detection.Results:1. The recombinant plasmid of CDH5 N-terminal-p ET-41 a vector was constructed and the fusion protein was expressed and purified by Ni-NTA affinity chromatography. SDS-PAGE and Coomassie blue staining results showed a strong band at a molecular weight of about70 k Da, indicating that the recombinant protein was successfully expressed and purified.We then use these protein for mice immunization and the monoclonal antibody was obtained in large scale.2. The ELISA kit was set up and the highest titer of 2C11 against CDH5 was 1:8000.Western Blot, immunofluorescence and immunohistochemistry suggested monoclonal antibodies 2C11 could specifically recognize native CDH5 protein, which was located in the junctions of endothelial cells and the presence of expression in melanoma. Furthermore,The recombinant plasmid of CDH5-N-terminal-p ET41 a vector was then constructed, the fusion protein was produced, and the 2C11 antibody was verified to recognize the amino acid sequence of CDH5-N-terminal-p ET41 a between 351-456. Dot Blot method further showed that 2C11 epitope was LDREVVPWYNLTVEA.3. Moreover, Immunohistochemistry revealed that the expression of CDH5 in human renal cell carcinoma, colorectal cancer and lung cancer tissues; in vitro 3-D Matrigel angiogenesis experiments confirmed the inhibition of the monoclonal antibody in tumor angiogenesis(P<0.05); cell adhesion experiments showed that the monoclonal antibody had the ability to reduce cell adhesion effect(P<0.05); CCK8 results showed that m Ab2C11 could inhibit cell proliferation(P<0.05); Western Blot experiments showed decreased P-AKT and β-catenin levels(P<0.05).4. By screening the pairing, select the 4E6 and 9A6 monoclonal antibody successfully established human plasma soluble CDH5 double antibody sandwich ELISA method, the detection limit was 24.7 pg/ml, intra-assay coefficient of variation(CV) of 4.1%-7.7%,inter-assay CV 8.7%-10.8%, the average recovery was 96.7%.Conclusion:(1) The monoclonal antibody 2C11 specifically recognizing endogenous CDH5 protein was produced and characterized by Western blot, immunofluorescence and immunohistochemistry, the epitope recognized by 2C11 was successfully identified.(2) Immunohistochemistry showed the presence of CDH5 on kidney cancer, colon cancer, melanoma, while the screened analysis confirmed the expression of CDH5 in lung adenocarcinoma cell line, and monoclonal antibody 2C11 exhibited the ability to inhibit tumor cell proliferation. Functional analysis of m Ab had suggested that 2C11 could inhibit tumor angiogenesis in vitro and in vivo, which was involved in the development of VM due to the important of CDH5 in the VM. Moreover, the m Ab 2C11 could affect cell proliferation which was further involved in the lung cancer development.(3) Established a double antibody sandwich ELISA kits which have good sensitivity and specificity, which can be used as a new detection index for tumor diagnosis to detect human plasma soluble CDH5 content.