Production and characterization of polyclonal and monoclonal antibodies to rat brain l-glutamate decarboxylase

Specific monoclonal and polyclonal antibodies to rat brain glutamate decarboxylase (GAD) were produced and characterized. Polyclonal antibodies against GAD were raised in rabbits by injecting a total of 70-210 μg of purified GAD i.m. The specificity of anti-GAD serum was established from a variety of tests including Ouchterlony immunodiffusion, immunoelectrophoresis, immunoprecipitation, dot immunoassay, ELISA tests and Western immunoblottings. In immunodiffusion and immunoelectrophoresis tests using partially purified GAD preparations and anti-GAD serum a single, sharp precipitin line corresponding to GAD activity was obtained. Quantitative immunoprecipitation of GAD activity was achieved using anti-GAD IgG and Staphylococcus aureus. Specificity of the antiserum was further indicated from a dot immunoassay and ELISA tests in which the intensity of the reaction product was proportional to the amount of GAD protein present. In the Western immunoblotting experiments using partially purified GAD preparations only two protein bands corresponding to the position of the two subunits of GAD were stained by anti-GAD IgG, further supporting the specificity of polyclonal antibodies against GAD. In addition to polyclonal antibodies, several specific GAD-antibodies-producing clones were also obtained by the hybridoma technique. The specificity of monoclonal antibodies against GAD were established from the following criteria: positive on ELISA test using homogenous GAD as antigen; formation of GAD-anti-GAD IgG complex as indicated from gel filtration chromatography and sodium dodecyl sulfate polyacrylamid gel electrophoresis; and specific recognition of GAD subunit in a partially purified GAD preparation in Western immunoblotting test. Monoclonal antibodies were further characterized by immunohistochemical localization of known GABAergic neurons and their processes in the cerebellum and retina.