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.

A Human Immunoglobulin G1 Antibody Originating from an in Vitro-Selected Fab Phage Antibody Binds Avidly to Tumor-Associated MUC1 and Is Efficiently Internalized

“…23 In flow cytometric analysis we compared PH1-IgG1 with HMFG1 that is reported to recognize a different glycosylation-sensitive epitope. 20,24 The binding pattern on tumor cell lines did not differ significantly between both antibodies except for the OVCAR-3 cell line, which was stained less by HMFG1 probably because of the different epitope recognition. On colon cancer cell lines, both antibodies hardly showed any binding.…”

High Density O-Glycosylation on Tandem Repeat Peptide from Secretory MUC1 of T47D Breast Cancer Cells

“…The material under study can be regarded as representative, because 1) the cell line T47D is a well established model with respect to O-glycosylation of MUC1 (3)(4)(5) and to the binding of MUC1-specific antibodies (25), and 2) it comprised all MUC1 glycoforms secreted by T47D cells. The latter statement is founded on consideration of the binding characteristics of anti-MUC1 antibody BC3, which recognizes a broad spectrum of glycoforms of the tandem repeat peptide (9,23).…”

Overexpression and altered glycosylation of MUC1 in malignant mesothelioma

“…Some of these antibodies had been previously characterised by workshops organised by the International Society of Oncodevelopmental Biology and Medicine (ISOBM) in terms of the epitope recognised within the VNTR and whether that recognition was dependent upon the glycosylation status of the epitope. These antibodies were assigned to various classes or clusters by the ISOBM workshops based upon their staining profile on normal breast tissue samples (summarised in Table 2) (Cao et al, 1998;Hanisch, 1998).…”

“…Though patient numbers were limited, mesothelioma (Cao et al, 1998;Hanisch, 1998 MUC1/EMA in malignant mesothelioma J Creaney et al patients with a sarcomatoid or mixed histology had lower serum concentrations of CA15-3 than mesothelioma patients with a predominately epitheliod histology ( Figure 4B). Median CA15-3 concentration in patients with benign asbestosrelated disease (either asbestosis and/or pleural plaques) was 38 ± 6 kU l À1 (range, 14 -155 kU l À1 ); 28% (9 out of 32) of patients with asbestos-related benign disease had CA15-3 levels above the upper limit of normal.…”