HYBRIDOMA Volume 20, Number 4, 2001 Mary Ann Liebert, Inc. A Monoclonal Antibody, MIN/3/60, that Recognizes the Sulpho-Lewis x and Sulpho-Lewis a Sequences Detects a Sub-Population of Epithelial Glycans in the Crypts of Human Colonic Epithelium WENDY LOVELESS, 1 TEN FEIZI, 1 MAURIZIO VALERI, 2 RICHARD DAY, 3 and SYLVIE BAY 1,4 ABSTRACT Monoclonal antibodies (MAbs) directed to Lewis x (Le x ) and related carbohydrate sequences have been in- valuable in anticipating biological roles for these oligosaccharides by detecting the remarkable changes that occur in their expression from the earliest stages of embryogenesis, through development and sequential stages of cell differentiation and maturation. A notable impact has been in the molecular dissection of ligand–re- ceptor interactions in key cell adhesion events at the initial stages of leukocyte recruitment in inflammation, and almost certainly in the metastasis of epithelial tumours. Antibodies that recognise Le x and the 39-sialyl forms were observed to identify leukocyte subsets; these were subsequently found to match those recognized by the leukocyte–endothelium adhesion molecules, the E- and P-selectins. We now describe a MAb (rat hy- bridoma MIN/3/60) raised to 39-sulpho-Le x , a carbohydrate sequence which, in vitro, is bound not only by the E-, L-, and P-selectins, but also by the cysteine-rich domain of the macrophage endocytosis receptor. We ob- serve that MIN/3/60 is bispecific, however; it binds 39-sulpho-Le a as well as 39-sulpho-Le x . Nevertheless, our exploratory studies reveal that it may be a useful histochemical reagent when used in conjunction with a mono- specific antibody to 39-sulpho-Le a . The MIN/3/60 antibody reveals a sub-population of epithelial glycans in the crypts of Lieberkühn in normal human colon. 223 INTRODUCTION M ONOCLONAL ANTIBODIES (MAbs) have played a major role in revealing the changes that occur in the expression of specific oligosaccharide sequences from the earliest stages of embryonic development, through sequential stages of cellular differentiationand maturation, and in anticipatingtheir roles as ligands for carbohydrate-binding receptors. (1) Arguably the greatest impact of oligosaccharidesequence-specificMAbs has been in highlighting the status of the fuco-oligosaccharides, Lewis x (Le x ), 39-sialyl-Le x and related sequences, as antigenic markers of leukocyte subsets. These findings served to focus research efforts on this family of oligosaccharides, and led to the establishment of their roles as ligating elements for the leukocyte-endothelium adhesion molecules, the selectins, which play crucial roles in leukocyte recruitment in inflamma- tion, and lymphocyte recirculation. (2–4) In in vitro experiments, the sulphated sequences 39-sulpho-Le x and 39-sulpho-Le a , as found on an epithelial mucin glycoprotein, were also shown to be bound by the selectins (5,6) and more recently by the cysteine- rich domain of the macrophageendocytosisreceptors, (7) but the physiological relevance of the binding remains to be deter- mined. As part of our studies of the in vivo distribution and func- tion of the 39-sulpho-Le x and 39-sulpho-Le a antigens, we have previously characterized (8,9) an antibody to 39-sulpho-Le a , which as predicted labels various human epithelial tissues, (10) known to synthesize type 1 as well as type 2 chains (11,12) rather 1 The Glycosciences Laboratory, Imperial College School of Medicine, Northwick Park Campus, Watford Road, Harrow, Middlesex, HA1 3UJ, UK. 2 The Hybridoma Unit, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, UK. 3 St. Mark’s Hospital, Watford Road, Middlesex, HA1 3UJ, UK. 4 Present address: Unité de Chimie Organique, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.