Monoclonal Antibody to Rat α-CGRP: Production,Characterization,and In Vivo Immunoneutralization Activity

Spleen cells from a Robertsonian mouse immunized with rat α-CGRP were fused with FOXNY cells to induce hybridoma cells. Antibody activities were screened by radioimmunoassay, and hybridomas producing high affinity antibodies were cloned by limiting dilutions. Ascites were produced from the highest affinity clone in pristine-primed Balb/c mice. Ascites fluid contained approximately 20 mg/ml IgG which was of subclass IgG2_a as determined by immunodiffusion analysis. The titer of this IgG2_a antibody titled #4901, was 1:2,000,000 and the ID₅₀ for rat α-CGRP, rat β-CGRP and human α-CGRP were 350, 4000, and 4500 pg/ml respectively. Protein A purified CGRP antibody #4901 (5-10 mg/kg) completely abolished the portal release of somatostatin and the inhibition of gastric acid secretion induced by intravenous infusion of rat αCGRP (15-20 μg/kg/h) in anesthetized rats. The unpurified antibody (25 mg/kg) also prevented the fall in mean arterial blood pressure and the increase in heart rate caused by intravenous injection of rat α-CGRP. Immunohistochemistry showed that CGRP monoclonal antibody stains nerve fibers and endocrine-like cells in the pancreas, and neuronal elements in the gastrointestinal tract. These results show that CGRP monoclonal antibody #4901, which is relatively specific for rat α-CGRP, is useful for in vivo immunoneutralization of CGRP and is also an excellent reagent for immunohistochemical localization of α- and β-CGRP in mammals.