Isolation And Identification Of The Lung Function Of Antigen And Its Correlation With Lung Cancer

Objective A monoclonal antibody 13C9, which had been targeted by screening of functional monoclonal antibody library against lung cancer, was adopted to recognize its specific antigen on multiple cultured-cell-types and tissues of lung cancer. Its function to the growth of lung cancer was detected both in vivo and in vitro. Furthermore the 13C9-specific antigen was identified. And the relationship between the expression of the antigen protein in lung cancer and clinical parameters of lung cancer was studied. This study may provide a clue for exploring valuable molecular targets and treatment agents for targeted therapy of lung cancer.Methods With immunofluorescence and immunohistochemistry techniques, the expression of 13C9-specific antigen was detected and localized on multiple cultured-cell-types and tissues of lung cancer. ELISA was applied to determine the production of 13C9 monoclonal antibody by culturing hybridoma cells and its subtype. Experiments of tumor treatment in vivo and vitro were carried to evaluate the inhibiting functions of the 13C9 antibody to the growth of lung cancer. Thereafter, the 13C9-specific antigen was optimized by dot blotting techque, extracted by RIPA technique. And with Western blotting, immunoprecipitation, MALDI-TOF-PMF mass spectrometry, the 13C9-specific antigen was identified. On the other hand, clinical specimens of lung cancer were collected and detected with immunohistochemistry. The correlation between the expression of the antigen protein in lung cancer and clinical parameters of lung cancer was studied.Results Results from immunofluorescence showed that among 12 fixed lung cancer cell lines, positive rate of the recognition of the antibody 13C9 to its antigen was above 90%, intensity was +++; with viable cells of lung cancer cell lines, the antibody 13C9-recognized antigen was demonstrated on the cell membrane. Immunohistochemical results showed that within 174 cases of paired tissues of lung cancer, positive rate of the 13C9-recognized antigen in lung cancer tissues was up to 87.4%, compared with the paired adjacent tissues which was just 16.7%,had a significant difference (t=3.5555E-48, P<0.01). The production of the antibody 13C9 by culturing hybridoma cells was 4.38ug/ml, belong to IgM, Kapa subtype, measured by ELISA. Transplant tumor inhibition experiment showed that the antibody 13C9 inhibit the lung tumor growth in mice by 63.29%, compared with control group (P=0.01, P<0.05). By dot blotting, the 13C9-recognized antigen protein was optimized and localized on the cell membrane. Then with RIPA extraction, immunoprecipitation and electrophoresis, the antigen protein band on the gel-line was excised and identified by MALDI-TOF-PMF. The molecular weight of the 13C9-recognized antigen protein was 226KD, and we named it as M protein. Conformation experiment was taken with commercial antibody, which demonstrated that M protein was recognized by both the antibody 13C9 and the commercial antibody. Investigation to the results from immunohistochemistry showed, that in 331 cases of lung cancer the expression of M protein in squamous cell carcinoma was higher than that in adenocarcinoma (P=0.000, P<0.01), the expression of M protein in T3+T4 stage was higher than in T1+T2 stage, (P=0.001, P<0.01). The expression of M protein in squamous cell carcinoma of 161 lung cancer patients was more intense with T stage advance, while in adenocarcinoma of 137 lung cancer patients, the expression of M protein was more intense with decreasion of tissue differentiation.Conclusion Results from this study demonstrated:①13C9 possesses characteristics of a functional monoclonal antibody,13C9-recognized antigen expressed in multiple cultured-cell-types and tissues of lung cancer, inhibitive effect of antibody 13C9 to the growth of the tumor in nude mice was obvious.②The antibody 13C9-recognized antigen which named M protein, may belong as a functional protein in lung cancer; its molecular weight was 226KD, localized on cell membrane.③There are possible correlation between the expression of M protein and the type as well as the clinical parameters of lung cancer:the expression of M protein in squamous cell carcinoma was higher than that in adenocarcinoma, the expression of M protein shows a positive correlation with T stage advance especially in lung squamous cell carcinoma, while in lung adenocarcinoma it shows a negative correlation with the advance of tissue differentiation. This study may provide new experimental data for further exploring and developing of new targets and new targeting agents in lung cancer treatment.