Young D A, Prout G R, Lin C W
Cancer Res. 1985 Sep;45(9):4439-46.
Monoclonal antibodies (McAbs) to human bladder carcinoma were generated by fusion of NS-1 mouse myeloma cells with spleen cells from BALB/c mice immunized with either cultured human bladder cancer cells or cells obtained from a fresh surgically removed bladder tumor. Four hybridomas which reacted strongly with bladder tumor cells and not to normal skin fibroblasts or urothelial cells were identified and cloned by limiting dilution to obtain monoclonality. One McAb, 3G2-C6, raised with cultured tumor bladder cells MGH-U1 (EJ) as the immunogen reacted more strongly to the bladder tumor lines tested than any of the other McAbs resulting from various fusion experiments. Hybridoma 3G2-C6 was found to secrete murine immunoglobulin G1 and to produce high titer ascites fluid when grown in BALB/c mice. Results from quantitative enzyme-linked immunosorbent assays on a panel of more than 35 cell lines demonstrated that McAb 3G2-C6 reacted with several bladder tumor cell lines 50 to 90 times more than with normal transitional urothelium. Two kidney and two testicular tumor lines also bound 10 times more 3G2-C6 than with normal cells. The 3G2-C6 antigen was only marginally detected on a number of other cancer and noncancerous cells tested such as breast and lung tumor cells, melanoma, fetal cells, and peripheral blood lymphocytes. To identify the antigen 125I-labeled membrane components from MGH-U1 cells were extracted with detergent, immunoprecipitated with Protein-A bound 3G2-C6, and analyzed by sodium dodecyl sulfate-gel electrophoresis. This revealed that McAb 3G2-C6 binds to a Mr 90,000 cell surface component. Indirect immunofluorescence microscopy with fluorescein isothiocyanate-anti-mouse immunoglobulin G also identified the antigen on the surface of cultured and fresh tumor cells and detected the antigen on 16 of 17 Grade 3 bladder tumor specimens as well as on some kidney and testicular tumor cells. This study confirms the potential of the hybridoma technique for producing McAbs capable of identifying tumor associated antigens which may be useful in the diagnosis and treatment of bladder cancer.
用培养的人膀胱癌细胞或手术切除的新鲜膀胱肿瘤细胞免疫BALB/c小鼠，取其脾细胞与NS-1小鼠骨髓瘤细胞融合，制备出抗人膀胱癌的单克隆抗体（McAbs）。鉴定出4株与膀胱肿瘤细胞反应强烈、与正常皮肤成纤维细胞或尿路上皮细胞无反应的杂交瘤，并通过有限稀释法进行克隆以获得单克隆性。以培养的膀胱肿瘤细胞MGH-U1（EJ）作为免疫原产生的一种单克隆抗体3G2-C6，与所检测的膀胱肿瘤细胞系的反应比各种融合实验产生的其他任何单克隆抗体都更强。发现杂交瘤3G2-C6分泌鼠免疫球蛋白G1，在BALB/c小鼠体内生长时可产生高滴度腹水。对一组35种以上细胞系进行的定量酶联免疫吸附测定结果表明，单克隆抗体3G2-C6与几种膀胱肿瘤细胞系的反应比与正常移行尿路上皮的反应强50至90倍。两种肾肿瘤细胞系和两种睾丸肿瘤细胞系与3G2-C6的结合也比正常细胞多10倍。在许多其他检测的癌细胞和非癌细胞如乳腺和肺肿瘤细胞、黑色素瘤、胎儿细胞及外周血淋巴细胞上，仅能微量检测到3G2-C6抗原。为鉴定该抗原，用去污剂提取MGH-U1细胞的125I标记膜成分，用与蛋白A结合的3G2-C6进行免疫沉淀，然后通过十二烷基硫酸钠-凝胶电泳进行分析。结果显示单克隆抗体3G2-C6与一种分子量为90,000的细胞表面成分结合。用异硫氰酸荧光素-抗小鼠免疫球蛋白G进行间接免疫荧光显微镜检查，也在培养的和新鲜的肿瘤细胞表面鉴定出该抗原，并在17份3级膀胱肿瘤标本中的16份以及一些肾和睾丸肿瘤细胞上检测到该抗原。本研究证实了杂交瘤技术在生产能够识别肿瘤相关抗原的单克隆抗体方面的潜力，这些抗原可能在膀胱癌的诊断和治疗中有用。
