Lectin ELISA for the c-erb-B2 Tumor Marker Protein p185 in Patients with Breast Cancer and Controls

The search for diagnostic and prognostic factors in breast cancer has included several oncogenes, particularly c- erb -B2, which encodes a 185-kDa transmembrane glycoprotein receptor, denoted p185, with tyrosine kinase activity (1)(2). p185 is frequently investigated by immunohistochemical techniques. The extracellular domain is shed from tumors in vitro (3) and is detectable in serum by immunoassay (4); however, the considerable overlap between patient and control values renders its estimation of little value in individuals. However, many glycoproteins exhibit carbohydrate changes in cancer (5). Thus a test combining the concentration with changes in glycosylation might be more specific than either alone. We therefore investigated potential glycosylation changes in p185 in the serum of breast cancer patients and controls collected sequentially from a patient series immediately before surgery, using the convenient Lectin ELISA format (6). The protein was specifically captured immunochemically and quantified with one of two biotin-labeled lectins binding particular sugars on the protein: wheat germ agglutinin (WGA), with affinity for sialic acid and N -acetylglucosamine, and Concanavalin A (Con A), which binds preferentially to mannose. In preliminary experiments, we investigated these assays in lysates of SKBR3 breast cancer cells, which express p185, and normal lymphocytes.Monoclonal antibody raised against a synthetic peptide sequence of p185 extracellular domain (OM-11-954) was obtained from Cambridge Research Biochemicals. In immunoblotting studies, the antibody detects an ∼170-kDa protein in SKBR3 cell membranes blocked by preincubation with synthetic peptide. In immunocytochemical studies using frozen and paraffin-embedded material, staining was blocked by preincubation with the synthetic peptide. The antibody does not recognize the epidermal growth factor receptor. Human breast carcinoma cells, SKBR3, were a gift from Professor B.R.Westley, Pathology Department, University of Newcastle, Newcastle upon Tyne, United Kingdom. The tetramethylbenzidine peroxidase substrate (K-blue) was from Bionostics; all other reagents were from Sigma. Absorbances were measured …