Immunohistochemical Characterization of a Panel of 56 Antibodies with Normal Human Small Intestine, Colon, and Breast Tissues

Abstract

The epithelial mucin MUC1 is heavily but differently glycosylated depending on the origin and developmental status of the tissue, which greatly influences the reactivity of monoclonal antibodies (MAbs). A partial characterization of their epitopes is possible by mild, carbohydrate-specific periodate oxidation of tissue sections prior to immunostaining. Using this strategy, we have evaluated 56 MAbs submitted to the ISOBM TD-4 (MUC1) Workshop. Paraffin sections from normal human small intestine, colon and breast were immunostained at different defined antibody concentrations either directly or after oxidation with 20 mM periodate at pH 5 for 30 min (PO). In addition, monolayers of T-47D breast cancer cells without PO treatment were examined in immunofluorescence. The array of observed reactivities allowed us to classify the MAbs as follows. Fourteen antibodies were found to detect MUC1 largely independent of the degree of glycosylation, and are therefore classified as pan-MUC1 MAbs (Group A). Twenty-four MAbs were nonreactive with one or more types of the examined epithelia, but became reactive after PO of the tissue sections. We have called these differentiation-dependent MUC1 MAbs (Group B). They might be especially valuable in histological tumour diagnosis. According to their differential staining behaviour towards untreated small intestine, colon, and breast tissue sections, we divided these MAbs into 4 subtypes (Group B1 through Group B4). A further group of six MAbs detected PO-sensitive carbohydrate epitopes (Group C). A seventh antibody apparently also belongs to Group C by immunohistological criteria, although its corresponding epitope was not PO-sensitive. Three further MAbs are still unclear in their specificity, and another 2 are not MUC1-specific (Group D). Six preparations were found nonreactive with the examined tissues; 4 of these were also negative with T-47D cells. Generally, a broad spectrum of different immunohistological patterns has emerged which appears to be widely independent of the type of epitope (sequence versus conformational, length of sequence) and the relative affinities determined in vitro.