Purification Of The Anti-human Lung Cancer Monoclonal Antibody And Its Recognized Antigen

[Objective] The aim of this study to purify the anti-human lung cancer monoclonal antibody N-35from the cell culture supernatant of hybridoma p35, which were used by Protein A Sepharose CL-4B chromatographic column, and employ mcAb N-35as the ligand for the immunoaffinity chromatography. to catch the human lung cancer associated antigen N35protein which be recognized.[Method] Limited dilution and cloning culture of hybridoma cell P35, at the same time inoculated Yunnan Gejiu lung adenocarcinoma cancer cell GLC-82with logarithmic phase in to96wells plate, after GLC-82bespreading, we mounted and blocked these tumor cells; Prepared the positive mouse immunized serum anti-human lung cancer cells, using the cancer cell GLC-82plate to screen of hybridoma supernatants with ELISA. got the hybridoma cell line that secreted mcAb stably, cultured abundantly and cryopreserved. the potency and subtype of the mcAb was detected, identified the tissue specificity of monoclonal antibody by immunohistochemistry; After the supernatant was sufficiently collected. Protein A Sepharose CL-4B chromatographic column can be used to purify mcAb N-35. identified the purity and activity of N-35; The purified mcAb was coupled with CNBr activated SepharoseTM4B as the ligand for the immunoaffinity chromatography (IAC) to catch the lung cancer associated antigen from the lysate of lung cancer cells GLC-82. The purified antigen of the mcAb was identified by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and Western-blot. [Result] Screened more than30hybridoma cell lines that secreted monoclonal antibody N-35steadily, cryopreserved them respectively, the potency of mcAb was1:102, subtype was IgG2a, combination to lung cancer was specific; Purified the mouse anti-human lung cancer mcAb N-35, and extracted lung cancer associated antigen N35protein recognized by the antibody successfully.[Conclusion]100%of P35subclone can secreted mcAb N-35steadily through thrice cloning culture and sieve; The purified mcAb N-35was high potency and high purity by Protein A Sepharose CL-4B chromatographic column; The binding rate of mcAb N-35to CNBr activated SepharoseTM4B was fine. the immune chromatography column we prepared worked effectively. The purified antigen N35could be a new tumor-associated antigen (TAA).