Preparation And Clinical Application Of Specific Monoclonal Antibodies Against Recombinant Human Epidermal Growth Factor

Objective To produce hybridoma cell lines which can stably secrete monoclonal antibodies against recombinant human epidermal growth factor (rhEGF) and establish ABC enzyme linked immunosorbent assay (ABC-ELISA) for quantitatively detectining human EGF for studying the mechanism and clinical applications of EGF and providing a valuable tool to purify EGF by affinity chromatography and study how to diagnose and treat tumors. To preliminarily explore relation between EGF and tumor.Methods Balb/c mice were immunized with rhEGF. The cells of spleen of Balb/c mice which were not immunized with antigens were used as feed cells. Hybridoma technique and ELISA test were used to prepare and screen the hybridoma cells lines of monoclonal antibodies against rhEG.F. We analyzed their nuclear types and injected the hybridoma cells to abdominal cavity of Balb/c mice for ascites and obtained a mount of monoclonal antibodies from ascites. The monoclonal antibodies were respectively purified with Protein A-Sepharose from ascites and identified by SDS-PAGE. The subclasses and isotypes were identified by Mouse Monoclonal Antibody Isotyping Kit. The antigenic epitopes recongnized by EGF-B2,EGF-C7 and EGF-A8 were analyzed by the ELISA additivity test. One of antibodies was labeled with biotinyl- N-hydroxysuccinimide(BNHS) and the labling effect was examined by Dot-ELISA. Finally, The method of ABC-ELISA for quantitatively detecting human EGF was stablished preliminarily, which was applied to detect serum EGF of tumor patients and control.Results three hybridoma cell lines were obtained, named EGF-B2, EGF-C7 and EGF-A8 respectively, which produce monoclonal antibodies specifically against rhEGF. They own 92, 104 and 106 chromosomes respectively. They could steadily secrete antibodies after the cell lines had been continuously cultivated three months and repeatedly been frozen under -196℃ in liquid nitrogen and the monoclonal antibodies were stable in the condition of high tempreture, acid and base .The purities of IgG in ascites is over 95℅ through affinity chromatography. The isotypes of EGF-B2, EGF-C7 and EGF-A8 monoclonal antibodies were IgG1κ, IgG1λ and IgG3κand the affinity constant were 2.76×1010,3.2×109 and 1.45×109. EGF-B2 and EGF-C7 antibodies are against same antigen-binding epitopes on the surface of rhEGF and EGF-A8 is against different antigen-binding epitopes on the surface of rhEGF with EGF-B2 and EGF-C7 . It was evaluated that the three antibodies can bind to rhEGF specifically by ELISA and are made of only single protein band by western-blotting. The McAb EGF-B2 has been labeled with biotinyl- N-hydroxysuccinimide(BNHS) successfully and the lowest value of labled antibody which is detected by avidin biotin peroxidase complex (ABC) is 0.87pg. Applying the ABC-ELISA for quantitatively determining EGF in human serum of tumor patients and control , the result show that the EGF in serum of tumor patients is remarkably higer than control( P<0.05).Conclusion We have successfully prepared three hybridoma cell lines which stably secrete high-titer and high-specific monoclonal antibodies against rhEGF.The ABC-ELISA for quantitatively determining human EGF has high stability, specificity and sensitivity.It has the trend to replace radio-immunity analysis. Applying the ABC-ELISA for quantitatively determining EGF in human serum of tumor patients and controls, the results show that EGF is related with tumor of epithelial origin closely.