Woodbury R G, Brown J P, Yeh M Y, Hellström I, Hellström K E
Proc Natl Acad Sci U S A. 1980 Apr;77(4):2183-7. doi: 10.1073/pnas.77.4.2183.
BALB/c mice were immunized with a human melanoma cell line, SK-MEL 28, and their spleen cells were fused with mouse NS-1 myeloma cells. Hybrid cells were tested in an indirect 125I-labeled protein A assay for production of antibodies that bound to surface antigens of SK-MEL 28 melanoma cells but not to autologous skin fibroblasts. One hybridoma, designated 4.1, had the required specificity. It was cloned and grown in mice as an ascites tumor. The monoclonal IgG1 antibody produced by the hybridoma was purified from the ascites fluid and labeled with 125I. The labeled antibody bound, at significant levels, to approximately 90% of the melanomas tested and to approximately 55% of other tumor cells, but not to three B-lymphoblastoid cell lines or to cultivated fibroblasts from 15 donors. Immunoprecipitation and sodium dodecyl sulfate gel electrophoresis were used to detect the target antigen in 125I-labeled cell membranes of both cultivated cells and tumor biopsy samples. A protein with a molecular weight of 97,000 was identified. This protein, designated p97, was present in both cultured cells and biopsy material from melanomas and certain other tumors, but it was not detected in eight different samples of normal adult epithelial or mesenchymal tissues obtained from five donors.
用人类黑色素瘤细胞系SK - MEL 28免疫BALB/c小鼠，然后将其脾细胞与小鼠NS - 1骨髓瘤细胞融合。在间接125I标记蛋白A试验中检测杂交细胞，以确定是否产生能与SK - MEL 28黑色素瘤细胞表面抗原结合但不与自体皮肤成纤维细胞结合的抗体。一种命名为4.1的杂交瘤具有所需的特异性。它被克隆并在小鼠体内作为腹水瘤生长。从腹水中纯化该杂交瘤产生的单克隆IgG1抗体，并用125I进行标记。标记抗体能与约90%检测的黑色素瘤以及约55%的其他肿瘤细胞显著结合，但不与三种B淋巴母细胞系或来自15名供体的培养成纤维细胞结合。采用免疫沉淀和十二烷基硫酸钠凝胶电泳法检测培养细胞和肿瘤活检样本的125I标记细胞膜中的靶抗原。鉴定出一种分子量为97,000的蛋白质。这种蛋白质命名为p97，存在于黑色素瘤和某些其他肿瘤的培养细胞及活检材料中，但在从5名供体获得的8份不同正常成人上皮或间充质组织样本中未检测到。
