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

A monoclonal antibody (MAb) to galanin was prepared by cell fusion of myelo ma Fox-NY and spleen cells from Robertsonian mice immunized with rat galani n, Hybridomas producing high-affinity antibodies were cloned in pristine-pr imed 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 IgG(2a), as determined by enzyme-l inked immunoadsorbent assay (ELISA), The titer of this IgG(2a), antibody en titled #G65G was 1:10,000 and the ID50 for rat galanin was 1000 fmol/mL as determined by liquid phase radioimmunoassay. Immunohistochemistry showed th at this galanin MAb stains densed, beaded processes distributed to the ente ric plexuses, where they appear to encircle neuronal cell bodies, to the mu scle layer, where they are particularly abundant in the circular muscle lay er and in the deep muscular layer, and to the mucosa, In vivo capacity of i mmunoneutralization by this antibody was tested in male Sprague-Dawley rats fasted for 24 h and anesthetized with urethane, Systemic injection of prot ein A purified galanin antibody (6 mg/rat) decreased by 70% of the inhibito ry effect of intravenous galanin (2 nmol/kg/h i.v.) on gastric acid secreti on induced by intracisternal TRH analog. These results show that galanin an tibody #G65G is useful for in vivo immunoneutralization of galanin effects and is a valuable tool for immunohistochemical localization of galanin in g astrointestinal tissues.