Immunological analysis of mucin molecules expressed by normal and malignant mammary epithelial cells

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Abstract
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.