Monoclonal antibody to rat galanin: production, characterization, and in vivo immunoneutralization activity.

Abstract

A monoclonal antibody (MAb) to galanin was prepared by cell fusion of myeloma Fox-NY and spleen cells from Robertsonian mice immunized with rat galanin. Hybridomas producing high-affinity antibodies were cloned in pristine-primed Balb/c mice. The antibody was purified by affinity chromatography and concentrated to 12 mg IgG/mL by dialysis. Immunoreactivity of the antibody was screened by radioimmunoassay. Ascites fluid contained approximately 10 mg/mL IgG that belong to the subclass of IgG2a as determined by enzyme-linked immunoadsorbent assay (ELISA). The titer of this IgG2a antibody entitled #G65G was 1:10,000 and the ID50 for rat galanin was 1000 fmol/mL as determined by liquid phase radioimmunoassay. Immunohistochemistry showed that this galanin MAb stains densed, beaded processes distributed to the enteric plexuses, where they appear to encircle neuronal cell bodies, to the muscle layer, where they are particularly abundant in the circular muscle layer and in the deep muscular layer, and to the mucosa. In vivo capacity of immunoneutralization by this antibody was tested in male Sprague-Dawley rats fasted for 24 h and anesthetized with urethane. Systemic injection of protein A purified galanin antibody (6 mg/rat) decreased by 70% of the inhibitory effect of intravenous galanin (2 nmol/kg/h i.v.) on gastric acid secretion induced by intracisternal TRH analog. These results show that galanin antibody #G65G is useful for in vivo immunoneutralization of galanin effects and is a valuable tool for immunohistochemical localization of galanin in gastrointestinal tissues.