Immunological analysis of mucin molecules expressed by normal and malignant mammary epithelial cells-猫眼课题宝

正常和恶性乳腺上皮细胞表达的粘蛋白分子的免疫学分析

DOI：

发表时间：

1987

期刊：

International Journal of Cancer

影响因子：

6.4

通讯作者：

中科院分区：

医学1区

文献类型：

作者：

A. B. Griffiths;J. Burchell;S. Gendler;Angela M. Lewis;K. Blight;R. Tilly;J. Taylor‐Papadimitriou

许多现有的针对人乳脂球或针对癌细胞的单克隆抗体识别高分子量糖蛋白的表位。在一项比较ELISA分析中，许多这些抗体已被证明与人乳脂球的提取物发生反应。在培养的正常乳细胞和乳腺细胞系的比较免疫印迹中，发现所有细胞都与大分子结合，根据细胞来源的不同，大分子的分子量有一定的变化。广泛用于癌症诊断的HMFG - 2抗体也可以识别低分子量成分的表位。在T47D细胞中，这些可能小至80,000 Mr，并且通过电子显微镜可以显示该细胞系在高尔基体中积累HMFG - 2活性成分。使用HMFG - 2亲和柱，我们从2种乳腺癌细胞系MCF - 7和T47D中获得免疫纯化的HMFG - 2反应物质，并表明所有上述抗体在固相ELISA中与纯化的物质发生反应。除了高分子量成分外，免疫纯化的材料还含有低分子量成分，包括68,000 mr的糖蛋白，但在Western blot上不能被HMFG - 2抗体识别。我们已经使用这种免疫纯化的材料来产生新的单克隆抗体。所有这些抗体都能识别其他抗体所见的高分子量条带，但其中两种抗体也能识别MCF - 7和T47D印迹中68,000 Mr的条带。第二代抗体显示出与HMFG - 2相似的组织反应谱，其中一种抗体对甲醇-丙酮固定的乳腺癌切片的反应至少与HMFG - 2一样强烈。

Many existing MAbs raised against the human milk fat globule or against carcinoma cells recognise epitopes on high‐molecular‐weight glycoproteins. In a comparative ELISA assay a number of these antibodies have been shown to react with an extract of the human milk fat globule. In comparative immunoblots of cultured normal milk cells and breast cell lines, all were found to bind to large molecules which show some variation in molecular weight depending on the cell source. The HMFG‐2 antibody, which is widely used in cancer diagnosis, also recognises epitopes on lower‐molecular‐weight components. In T47D cells these may be as small as 80,000 Mr, and with electron microscopy this cell line can be shown to accumulate HMFG‐2‐reactive components in the Golgi apparatus. Using an HMFG‐2 affinity column we have immunopurified HMFG‐2‐reactive material from the 2 breast cancer cell lines MCF‐7 and T47D and shown that all of the above antibodies react in a solid‐phase ELISA with the purified material. In addition to high‐molecular‐weight components, the immunopurified material was found to contain lower‐molecular‐weight components including a glycoprotein of 68,000 Mrthat was not, however, recognised by the HMFG‐2 antibody on a Western blot. We have used this immunopurified material to generate new MAbs. All of these recognise the high‐molecular‐weight bands seen with the other antibodies, but 2 of them also recognise a band at 68,000 Mr in blots of MCF‐7 and T47D. The Second‐generation antibodies show a spectrum of reactivity on tissues similar to HMFG‐2 and one reacts at least as strongly as HMFG‐2 with methanol‐acetone‐fixed sections of breast cancers.

Production of functional human T-T hybridomas in selection medium lacking aminopterin and thymidine.