Monoclonal Antibody to VIP: Production, Characterization, Immunoneutralizing Activity, and Usefulness in Cytochemical Staining

A very stable cell line has been generated that produces monoclonal antibody to VIP designated as CURE.V55. This hybridoma was produced by fusion of spleen cells from an immunized Robertsonian mouse containing the translocated 8.12 chromosome with FOX-NY myeloma cells that are APRT deficient. VIP monoclonal antibody producing cell line #55 was selected by limiting dilutions using thymocytes as feeder layers. Ascites fluid containing high concentration of VIP monoclonal antibody was produced from pristane-primed BALB/c mice. Ascites fluid contained approximately 20 mg/ml IgG and bound 50% of 2 fmol 125I-VIP at a final dilution of 1:50,000. Binding of this IgG1 antibody was inhibited by 50% at 5 nM concentration of either VIP 1-28 or VIP 7-28. Protein-A purified IgG of this antibody, used in a concentration of 30 mg/kg per rat (IV), completely reversed the inhibitory effect of gastric corpus contractions induced by intravenous injection of VIP (10 nmol/kg) in sodium pentothal-anesthetized rats. A control anti-keyhole limpet hemocyanin monoclonal antibody did not alter the stimulatory effect of VIP on gastric corpus contractions. Immunohistochemistry showed that this VIP monoclonal antibody stains neurons, and nerve fibers in human and rat gallbladder and the sphincter of Oddi as previously described with our polyclonal antiserum.