title: Generation of monoclonal antibodies to native active human glycosyltransferases.
abstract: Complex carbohydrates serve a wide range of biological functions in cells and tissues. Their biosynthesis involves more than 200 distinct glycosyltransferases in human cells, and the expression, properties, and topology of these enzymes regulate the glycosylation patterns of proteins and lipids. Glycosyltransferases are ER-Golgi resident enzymes with slow turnover, which makes monitoring of protein expression a method more directly linked to enzyme function, than monitoring gene expression. In situ monitoring of expression and subcellular topology of glycosyltransferase proteins by immunological techniques using monoclonal antibodies therefore provides an excellent strategy to analyze the glycosylation process in cells. A major drawback has been difficulties in generating antibodies to glycosyltransferases and validating their specificities. Here we describe a simple strategy for generating and characterizing monoclonal antibodies to human glycosyltransferases. This strategy includes a process for recombinant production and purification of enzymes for immunization, a simple selection strategy for isolation of antibodies with optimal properties for in situ detection of enzyme expression, and a comprehensive strategy for characterizing the fine specificity of such antibodies.
authors.affiliation: Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen N, Denmark.
authors.forename: Malene Bech
authors.initials: MB
authors.lastname: Vester-Christensen
authors.forename: Eric Paul
authors.initials: EP
authors.lastname: Bennett
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
authors.forename: Ulla
authors.initials: U
authors.lastname: Mandel
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000917:Antibody Formation; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D005260:Female; D016695:Glycosyltransferases / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007114:Immunization / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude
chemical_list: D000911:Antibodies, Monoclonal; D016695:Glycosyltransferases