Production and characterization of monoclonal antibodies to insulin secretory granule membranes

Monoclonal antibodies to insulin secretory granule membranes were obtained following immunization of mice with granule membranes purified from a rat transplantable insulinoma. The specificities of the antibodies were investigated by using binding assays with different insulinoma subcellular fractions, by indirect immunofluorescence studies with intact and permeabilized cells, and by immunoblotting of granule membrane proteins fractionated by SDS/polyacrylamide-gel electrophoresis. Fifty-six antibodies were characterized initially, and 21 representative cell lines were cloned. The antibodies fell into four categories: (1) binding preferentially to secretory granules, and reacting with a component of approx. 80,000 Da on immunoblots (antigen designated SGM 80); (2) binding preferentially to secretory granules, and reacting with components of approx. 110,000 and 50,000 Da on immunoblots (antigen designated SGM 110); (3) binding preferentially to secretory granules but unreactive on immunoblots; (4) binding to membrane antigen(s) with a widespread intracellular distribution which included granules and plasma membranes. The antigens SGM 80 and SGM 110 were studied in more detail and both were shown to be integral membrane glycoproteins with antigenic determinants located on the internal face of the secretory granule membrane. These antigens were also present in normal rat islets of Langerhans and similar components were detected by immunoblotting in secretory granules from anterior pituitary and adrenal medulla. Proteins which were immunologically related to SGM 80 and SGM 110, but distinct in molecular size, were also identified in liver. It is concluded that secretory granules contain specific components which are restricted in subcellular location but widespread in tissue distribution. The antibodies obtained will be valuable reagents in the further investigation of the biogenesis and turnover of insulin secretory granules.