Monoclonal antibody to rat alpha-CGRP: production, characterization, and in vivo immunoneutralization activity

Spleen cells from a Robertsonian mouse immunized with rat alpha-CGRP were fused with FOX-NY 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 IgG2a as determined by immunodiffusion analysis. The titer of this IgG2a antibody titled #4901, was 1:2,000,000 and the ID50 for rat alpha-CGRP, rat beta-CGRP and human alpha-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 alpha CGRP (15-20 micrograms/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 alpha-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 alpha-CGRP, is useful for in vivo immunoneutralization of CGRP and is also an excellent reagent for immunohistochemical localization of alpha- and beta-CGRP in mammals.