Ziai M R, Imberti L, Nicotra M R, Badaracco G, Segatto O, Natali P G, Ferrone S
Cancer Res. 1987 May 1;47(9):2474-80.
Monoclonal antibodies (MoAbs) 225.28, 657.9, and 902.5 recognizing distinct epitopes of the human high molecular weight melanoma associated antigen (HMW-MAA) were used to investigate the molecular and cellular heterogeneity of the HMW-MAA synthesized by human melanoma cells. Sequential immunodepletion and immunoprecipitation experiments showed that not all HMW-MAA molecules synthesized by a melanoma cell line express the antigenic determinants recognized by the three monoclonal antibodies. The majority of the HMW-MAA molecules expressed the epitope defined by MoAb 657.9 since this monoclonal antibody depleted the melanoma cell lysate of all antigen molecules recognized by the other two monoclonal antibodies. Depletion with MoAb 902.5 resulted in the removal of a large proportion of the HMW-MAA molecules precipitated by MoAb 657.9. The MoAb 225.28 depleted the cell lysate of only a fraction of the HMW-MAA molecules recognized by MoAb 657.9 and 902.5. Two-dimensional gel electrophoresis and peptide mapping analysis did not detect any significant difference among the HMW-MAA immunoprecipitated by the three monoclonal antibodies. The heterogeneity of the epitopes recognized by the three monoclonal antibodies is, at least partly, due to glycosylation of the antigen molecule, since treatment of melanoma cells with glycosidases differentially affects their ability to bind the three anti-HMW-MAA monoclonal antibodies. Fluorescent activated cell sorting analysis of the melanoma cells showed that the heterogeneity exhibited by the HMW-MAA is not due to the presence of different cell clones in the culture but reflects a differential distribution of epitopes on the HMW-MAA expressed on the surface of individual cells. Immunohistochemical staining of surgically removed benign and malignant lesions of melanocytic origin, of normal tissues, and of malignant lesions has shown a differential tissue distribution of the determinants recognized by the three monoclonal antibodies. Staining of melanoma cell lines and of surgically removed melanoma lesions with combinations of the three monoclonal antibodies did not cause any significant change of the percentage of stained cells but markedly increased the intensity of staining. These results indicate that combinations of monoclonal antibodies to distinct determinants of HMW-MAA can markedly increase the sensitivity of immunohistochemical techniques to detect melanoma cells.
识别人类高分子量黑色素瘤相关抗原（HMW - MAA）不同表位的单克隆抗体225.28、657.9和902.5，被用于研究人类黑色素瘤细胞合成的HMW - MAA的分子和细胞异质性。连续免疫去除和免疫沉淀实验表明，并非黑色素瘤细胞系合成的所有HMW - MAA分子都表达这三种单克隆抗体所识别的抗原决定簇。大多数HMW - MAA分子表达由单克隆抗体657.9所定义的表位，因为这种单克隆抗体能从黑色素瘤细胞裂解物中去除其他两种单克隆抗体所识别的所有抗原分子。用单克隆抗体902.5进行去除导致大量被单克隆抗体657.9沉淀的HMW - MAA分子被去除。单克隆抗体225.28仅从细胞裂解物中去除了一小部分被单克隆抗体657.9和902.5所识别的HMW - MAA分子。二维凝胶电泳和肽图谱分析未检测到这三种单克隆抗体免疫沉淀的HMW - MAA之间有任何显著差异。这三种单克隆抗体所识别的表位的异质性至少部分归因于抗原分子的糖基化，因为用糖苷酶处理黑色素瘤细胞会不同程度地影响它们与这三种抗HMW - MAA单克隆抗体结合的能力。对黑色素瘤细胞进行荧光激活细胞分选分析表明，HMW - MAA所表现出的异质性并非由于培养物中存在不同的细胞克隆，而是反映了单个细胞表面所表达的HMW - MAA上表位的差异分布。对手术切除的黑素细胞源性良性和恶性病变、正常组织以及恶性病变进行免疫组织化学染色，结果显示这三种单克隆抗体所识别的决定簇在组织中的分布存在差异。用这三种单克隆抗体组合对黑色素瘤细胞系和手术切除的黑色素瘤病变进行染色，并未导致染色细胞百分比有任何显著变化，但显著增加了染色强度。这些结果表明，针对HMW - MAA不同决定簇的单克隆抗体组合可显著提高免疫组织化学技术检测黑色素瘤细胞的敏感性。
