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The polymorphic epithelial mucin (PEM) appears to be the target molecule for many monoclonal antibodies (MAbs) which react with tumour-associated and epithelium-specific antigens. PEM contains a large domain made up of 20 amino-acid tandem repeats which are highly immunodominant as many of the antibodies reactive with this molecule recognize epitopes within this area. Using overlapping peptide octamers, we have precisely mapped the epitopes of 4 MAbs reactive with the tandem repeats including one, SM-3, which shows enhanced tumour specificity. We report that the core of the SM-3 epitope corresponds to the continuous amino acid sequence Pro-Asp-Thr-Arg-Pro. We also show that the epitopes recognized by 3 other antibodies, which show reactivity with normal and malignant tissues, map to within this area of the tandem repeat. However, none of these epitopes contain the proline found at the amino end of the SM-3 determinant. These results are consistent with the idea that, in the cancer-associated mucin, premature termination of the carbohydrate side-chains results in the exposure of the SM-3 epitope.

Three hybridomas producing monoclonal antibodies (IgG), reacting with components of the human mammary milk fat globule have been isolated. When tested for binding to a wide range of human cell lines and strains, all three antibodies show negative reactions with fibroblasts, lymphoblastoid cells, and a large number of epithelial cell lines of non-breast origin. Two of the antibodies (1.10.F3 and 3.14.A3) reacted with seven out of eight breast cancer lines tested, and with epithelial cells cultured from human milk. The other antibody (3.15.C3) reacted with only two of the breast cancer cell lines.

A series of human urinary mucin-like glycoproteins, previously detected using lectins to stain gels after electrophoresis, and showing genetic polymorphism (Karlsson et al., 1983) can also be detected using the tumour-binding monoclonal antibodies, Ca1, Ca2, Ca3, HMFG1, and HMFG2. The evidence from immunoprecipitation and immunoadsorbant chromatography experiments is that the epitopes recognized by these antibodies are carried on the same molecules as the lectin-binding determinants. The discovery that the antibodies bind specifically to a family of molecules which show genetic polymorphism provides a powerful new tool for the analysis of the material expressed aberrantly in cancer.

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 Mr that 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.