Preparation And Preliminary Identification Of A Group Monoclonal Antibodies Against Lung Adenocarcinoma Cell Line H2009

Objective:To prepare monoclonal antibodies(McAbs) against lung adenocarcinoma cell line H2009,so as to acquire such an antigen or gene for lung cancer development, in the hope of clarifying the mechanism of lung cancer emerging and developing, with the possibility to utilize the antigen as the tumor marker for the early diagnosis of lung cancer or the angionesis-targeted drugs for lung cancer. Contents:1.The preparation of large monoclonal antibody libraryT against lung adenocarcinoma cell line H2009;2. The characterization of the acquired monoclonal antibody against lung adenocarcinoma cell line H2009.Methods:Immunize BALB/C mice with lung adenocarcinoma cell line H2009. Then utilize the spleen cells from the immunized mice with the titer reaching over10^6to fuze sp2/0cells and selectively cultured with HAT medium. The hybridomas and monoclonal antibodies were screened by cells ELISA,immunoflurescence and flow cytometry to get the positive hybrydomas, which is more positive for lung adenocarcinoma cell line H2009and less positive or negative for HBeipcs. Then utilize the method limited dilution to get the single hybridomas which could stably secret monoclonal antibodies. These McAbs were analysed for their antibody titre、class、subclass and affinity. The antigen specificity for McAbs were evaluated and confirmed by ELISA to find out whether they are binding to other lung cancer cell lines such as the lung adenocarcinoma cell lines A549, A2, SK-LU-I, Ca-Lu-1, lung squamas cell line SK-MES-1and lung large cell line801D.To extract the membrane protein from the lung adenocarcinoma H2009, A549, A2and perform SDS-PAGE and Western blotting or immunoprecipitation to identify the extracted membrane protein of interest with the monoclonal antibody such as9H4,11C10,7C1,2D6,11B6. The binding sites and capabilities of the McAbs were observed by ELISA additive assay. We also investigate and compare the methods to purify the monoclonal antibodies IgM subtype.Results:20hybridoma cell lines secreting monoclonal antibodies which is more positive for lung adenocarcinoma cell line H2009and less positive or negative for HBeipcs were obtained. About the immunoglobulin subclasses of all McAbs were IgM, κ. ELISA and cyto flowmetry showed the monoclonal antibodies reacted to other lung cancer cell lines such as the lung adenocarcinoma cell lines A549, A2, SK-LU-I, Ca-Lu-1, lung squamas cell line SK-MES-1and lung large cell line801D We also get the preliminary condition about the specific membrane protein that the positive monoclonal antibodies bind to.Conclusion:We successfully obtained20specific McAbs which is more positive for lung adenocarcinoma cell line H2009and less positive or negative for HBeipcs and the preliminary condition about the specific membrane protein that the positive monoclonal antibodies bind to. We believe that the IgM monoclonal antibodies purified from the ascites is much purifier than from the hybridoma cell cuture supernatanat Key words:cell immunization; Hybridoma; Monoclonal antibody;the purifying of IgM.