Polymorphic epithelial mucin from the sera of advanced breast cancer patients – isolation and partial characterisation

Abstract

The anti-breast carcinoma monoclonal antibody (MAb), NCRC-11 defines a polymorphic epithelial mucin (PEM) which is elevated in the circulation of advanced breast carcinoma patients. Here we describe the purification and partial characterisation of this component from patients' sera and its use in the production of a second generation MAb, C568 (IgM). Pooled sera was fractionated by immunoaffinity and size-exclusion chromatography and the purity of preparations assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Serum-derived PEM shows a similar pattern of electrophoretic mobility to PEM isolated from primary breast tumour tissue and migrates as several bands in 4% SDS polyacrylamide gels (Mr greater than 400,000). The epitope expression of PEMs isolated from either source is also similar, with both bearing topographically distinct determinants for several anti-mucin MAbs. The immunoreactivities of antibodies C568 and NCRC-11 were unaffected by boiling, reduction and alkylation, or by enzyme desialylation of PEM. Periodate oxidation and proteolytic digestion have suggested that the antigenic determinant for C568 is carbohydrate in nature whilst that of NCRC-11 is peptidic. In accord with the mucinous nature of the molecule, serum-derived PEM is susceptible to reductive beta-elimination, elutes in the void volume of a Sepharose CL-4B column and has a buoyant density of 1.45 g ml-1.