Use of monoclonal antibodies in the study of intestinal structure and function

The hybridoma technique, originally developed by G. Kohler & C. Milstein, is a powerful new experimental approach for analysis of complex biological systems, and is particularly suited for identification and study of surface-membrane antigens. This technique has been used for the production of monoclonal antibodies to intestinal brush border membrane proteins. Spleen cells, obtained from BALB/c mice immunized with purified brush border membranes, were fused with NSI mouse myeloma cells, and hybrids were selected with a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Hybridoma cultures were screened for production of specific antibodies by radio-immunobinding assays and by immunofluorescent staining of intestinal frozen sections. Selected hybridoma cultures were cloned twice and used for the production of large amounts of antibodies, which were characterized. Nineteen monoclonal antibodies have been prepared to date, about half of them specifically staining the brush border membrane of mature enterocytes. Ten of the antibodies specifically immunoprecipitate surface-membrane proteins, which were analysed by sodium dodecyl sulphate slab-gel electrophoresis, by two-dimensional slab-gel electrophoresis, and by specific enzyme assays. Two antibodies were found to be specific for sucrase-isomaltase, one for an aminopeptidase, two for an isoenzyme of alkaline phosphatase that is present exclusively in the proximal small intestine, and one for maltase-glucoamylase. These monoclonal antibodies, and others prepared by similar techniques from mice immunized with a wide variety of intestinal subcellular fractions, should prove invaluable tools for the study of the biosynthesis of cell-surface proteins, the fetal and postnatal development of specific intestinal functions, and the process of cell differentiation in the intestinal epithelium.