Purification of Common Lung Cancer Antigen recognized with Monoclonal Antibody TFS and Production of Monoclonal Antibodies of Second Generation by its antigen.

TFS-2是针对小细胞肺癌产生的单克隆抗体（MAb），可与所有组织类型的肺癌反应。最近，我们尝试用~（13）Tc标记的TFS-2对人肺癌进行放射免疫检测<99m>，然后对各种组织如骨、肺和肝中的原发性和转移性肺癌进行清晰的成像。（1）普通肺癌抗原的纯化：将转移到肝脏的小细胞肺癌块用作纯化来源。用含有0.5%NP-40的PBS溶解样品。将样品两次通过亲和色谱法（三甲苯基活化的Sepharose 4 B与MAb TFS-2偶联）。NP-40通过乙醇沉淀去除。SDS-PAGE显示两条蛋白带，分别为39 kDa和33 kDa。Westernblotting显示33 kDa条带与单克隆抗体TFS-2反应。直接转移到膜上分析了33 kDa的蛋白质，并测定了20个氨基酸残基。（2）肺癌共同抗原基因的分析：从小细胞肺癌细胞株NCI-1169中提取mRNA，构建表达质粒cDNA文库。将质粒转染到Cos-1细胞中，并通过标记的MAb TFS-2筛选在细胞表面上表达抗原的克隆<125>。分离出编码与单克隆抗体TFS-2反应的蛋白质的cDNA克隆。（3）第二代单克隆抗体的制备获得了几株与纯化抗原反应的杂交瘤细胞克隆。

Monoclonal antibody(MAb)TFS-2 was produced against Small Cell Lung Cancer and reacted with all tissue types of lung cancer. Recently we tried the radioimmunodetection of human lung cancer with ^<99m>Tc labeled TFS-2 followed by getting clear imaging of primary and metastatic lung cancers in the various tissues such as bone, lung, and liver.(1)Purification of Common Lung Cancer Antigen.Masses of metastatic small cell lung cancer to liver was used as source for purification. Samples were solubilized by PBS with 0.5% of NP-40. Samples were twice passed through Affinity Chromatography(tresyl-activated Sepharose 4B couppled with MAb TFS-2). NP-40 was removed by ethanol pi-ecipitatiori. SDS-PAGE showed 2 bands of protein, 39kDa and 33kDa respectedly. Band of 33 kDa reacted with MAb TFS-2 on the Western blotting. Protein of 33kDa was analyzed directly transferring to membrane and 20 amino acid residue was determined.(2)Analysis of Gene coding Common Lung Cancer Antigen.The MRNA was obtained from small cell lutig cancer cell line NCI-1169, and the expression CDNA library in plasmid was constructed from the MRNA. The plasmids were transfected to Cos-1 cells and clones expressing antigen on the cell surface were screened by ^<125>I labeled MAb TFS-2. A EDNA clone coding the protein reacted with MAb TFS-2 was isolated.(3)Production of Monoclonal antibodies of the second generation Several clones of hybridoma cell that reacted with purified antigen presently were obtained.