Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterization and use for identification of cholinergic neurons.
Choline acetyltransferase (ChAT) has been purified from pig brain to greater than 95% homogeneity (purification factor: 646 000, specific activity of the purified enzyme: 128 mumol acetylcholine formed/min/mg). Gel electrophoresis of the purified enzyme in the presence of sodium dodecylsulphate and beta-mercaptoethanol revealed a single protein band at 68 000 daltons. Immunoprecipitation and double immunodiffusion tests showed that antisera raised against this protein specifically recognize ChAT. A monoclonal antibody prepared against the enzyme specifically binds a protein from crude pig brain supernatants which has a mol. wt. of 68 000 and a specific activity of 153 mumol/min/mg. This antibody shows no species cross-reactivity. The specificity of the immunohistochemical localization of ChAT has been established by comparing the labeling of pig retina using the antiserum with that obtained using the monoclonal antibody. Both probes specifically identify the same retinal structures: labeled cell bodies are found in the inner nuclear layer and the ganglion cell layer, while a double band is stained in the inner plexiform layer. In rat spinal cord, the antiserum labels the motoneurons and the preganglionic sympathetic neurons, located in the intermedio-lateral nucleus, the intercalated region, and the central autonomic area.
D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000201:enzymology; D002795:Choline O-Acetyltransferase / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D007106:Immune Sera; D005779:Immunodiffusion; D051379:Mice; D009474:Neurons / Q000201:enzymology*; D012160:Retina / Q000201:enzymology; D013552:Swine
D000911:Antibodies, Monoclonal; D007106:Immune Sera; D002795:Choline O-Acetyltransferase