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

Abstract

Summary 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 > 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-II is peptidic. In accord with the mucinous nature of the molecule, serum-derived PEM is susceptible to reductive P-elimination, elutes in the void volume of a Sepharose CL-4B column and has a buoyant density of 1.45 g ml-'.

Factors Effecting the Measurement of Tumor-Associated MUC1 Mucins in Serum

“…The presence of this protein has not previously been described in PAGE studies of serum proteins using MUC1 VNTR binding antibodies [10][11][12][13][14][15][16][17]. However, lower Mr bands around 70 kD can be seen in the published immunoblots of breast cancer sera using anti body 139H2 [13] and have been found in the ascites of ovarian carcinoma patients [9], This protein was not reactive in a double-determi nant assay using VNTR binding antibodies and only weakly reactive in an inhibition assay.…”

Detection of polymorphic epithelial mucins in the serum of systemic breast cancer patients using the monoclonal antibody, NCRC-11

“…In addition, heterophile antibodies in some human sera that are cross-reactive with the murine monoclonal antibodies may cross-link the "capture" and "tracer" antibodies employed in immunoradiometric assays, and this would produce an elevated signal. The frequency of such antibodies in human sera is unknown but heterophile antibodies reactive with the NCRC-11 antibody have been detected during the purification of the PEM antigen from patients' sera by affinity chromatography using immobilised NCRC-11 antibodies [15].…”

“…Pooled serum samples from systemic breast cancer patients were fractionated by affinity chromatography using immobilised NCRC-11 antibodies to produce a partially purified preparation of NCRC-11-defined antigen. Analysis of the partially purified serum antigen was pefformed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and immunostaining with NCRC-11 antibodies as previously described [15].…”

“…The antigen was prepared in a reducing sample buffer and separated on a 4% acrylamide gel, After electrophoretic transfer to a nitrocellulose membrane, the antigen was immunostained with the NCRC-11 antibody as previously described [ 15]. The identification of antigen in two tracks of gel is shown.…”

Design of ligands for the purification of anti‐MUC1 antibodies by peptide epitope affinity chromatography

“…In malignancy, however, its expression is disorganized and upregulated so that its presence can be detected in the circulation of cancer patients (3,4). In malignancy, however, its expression is disorganized and upregulated so that its presence can be detected in the circulation of cancer patients (3,4).…”