Pancino G, Charpin C, Osinaga E, Betaille B, Le Roy M, Calvo F, Roseto A
Division d'Immunocytologie Appliquée, Université de Technologie de Compiègne, France.
Cancer Res. 1990 Nov 15;50(22):7333-42.
Spleen cells from inbred Biozzi mice, immunized against the human breast cancer cell line T47D, were fused with murine myeloma SP2O cells to generate monoclonal antibodies. One of these, 1BE12, of IgM isotype, reacted with five of six human breast tumor cell lines, while no binding was detectable with normal lymphocytes, RBC, or fibroblasts. The antigen recognized by monoclonal antibody 1BE12 was localized on the surface of T47D and MCF7 cells and was detected in cell-free supernatants of cultures. The antigen was found also on the surface of milk secretory cells. Immunohistochemical staining of frozen and paraffin-embedded sections of human tissues showed apical polarized reactivity in normal breast glands, while in all breast cancers staining was either cytoplasmic or membranous and heterogeneously distributed. Immunostaining was also observed in some other normal epithelia, including salivary gland, gastroduodenal mucosa, exocrine pancreas, and cervix. The antigen was not detectable in secretory endometrium, whereas proliferative endometrium was strongly stained. Colon carcinoma, and cancers of the bladder and endometrium were strongly reactive. No staining was detected in melanoma, lymphoma, mesothelioma, non-small cell lung carcinoma, and thyroid, renal, and ovarian carcinomas. Lectin absorption of MCF7 membrane extracts reduced 1BE12 binding. A large reduction in 1BE12 reactivity was observed after digestion of T47D and MCF7 membrane extracts with proteases. Treatment with sodium periodate resulted in complete loss of antigenicity, while neuraminidase treatment did not affect 1BE12 binding. These findings suggest that the 1BE12 epitope is expressed on the carbohydrate moiety of a glycoprotein and does not contain sialic acid. Immunoblotting of the perchloric acid-soluble fraction of MCF7 membrane extracts after electrophoresis in 1% agarose detected the antigen as a high molecular weight species (Mr greater than 900,000). The antigen was purified by perchloric acid extraction of MCF7 membrane preparations followed by affinity chromatography on 1BE12 antibody coupled to Sepharose-4B and gel exclusion fast protein liquid chromatography. No reactivity of the purified material was found with monoclonal antibodies directed against human milk fat globule membrane-associated mucins HMFG1 and DF3.
用人类乳腺癌细胞系T47D免疫近交系Biozzi小鼠，取其脾细胞与鼠骨髓瘤SP2O细胞融合以产生单克隆抗体。其中一种IgM同型的单克隆抗体1BE12，能与六种人类乳腺肿瘤细胞系中的五种发生反应，而与正常淋巴细胞、红细胞或成纤维细胞未检测到结合。单克隆抗体1BE12识别的抗原定位于T47D和MCF7细胞表面，并在细胞培养的无细胞上清液中检测到。该抗原也存在于乳腺分泌细胞表面。对人体组织的冰冻切片和石蜡包埋切片进行免疫组织化学染色显示，正常乳腺腺管呈顶端极化反应，而在所有乳腺癌中，染色为细胞质或细胞膜性且分布不均。在其他一些正常上皮组织中也观察到免疫染色，包括唾液腺、胃十二指肠黏膜、外分泌胰腺和子宫颈。在分泌期子宫内膜中未检测到该抗原，而增殖期子宫内膜则被强烈染色。结肠癌、膀胱癌和子宫内膜癌有强烈反应。在黑色素瘤、淋巴瘤、间皮瘤、非小细胞肺癌以及甲状腺癌、肾癌和卵巢癌中未检测到染色。用凝集素吸收MCF7膜提取物可降低1BE12的结合。用蛋白酶消化T47D和MCF7膜提取物后，观察到1BE12反应性大幅降低。用高碘酸钠处理导致抗原性完全丧失，而用神经氨酸酶处理不影响1BE12的结合。这些发现表明，1BE12表位在糖蛋白的碳水化合物部分表达，且不含唾液酸。在1%琼脂糖中电泳后，对MCF7膜提取物的高氯酸可溶性部分进行免疫印迹，将该抗原检测为高分子量物质（Mr大于900,000）。通过对MCF7膜制剂进行高氯酸提取，然后在偶联到Sepharose - 4B的1BE12抗体上进行亲和层析以及凝胶排阻快速蛋白质液相色谱法来纯化该抗原。未发现纯化物质与针对人乳脂肪球膜相关粘蛋白HMFG1和DF3的单克隆抗体发生反应。
