Production of a specific antiserum to rat brain glutamic acid decar☐ylase by injection of an antigen-antibody complex

Abstract

Production of an antiserum specific to rat brain l-glutamic acid decar☐ylase is described, featuring the injection of an antigen-antibody complex. Partial purification of glutamate decar☐ylase was first achieved through differential centrifugation, ammonium sulfate fractionation, chromatography on Sephadex G-150, preparative isoelectric focusing in sucrose gradient and polyacrylamide gel electrophoresis for the production of a preliminary polyvalent, so called ‘trapping’ glutamate decar☐ylase antiserum in sheep. In rat brain homogenate supernatant this antiserum maximally inhibited glutamate decar☐ylase activity by 80% and totally precipitated enzyme activity on centrifugation. This antiserum, however, was polyvalent, as in crossed immunoelectrophoresis it detected four antigens in rat brain homogenate supernatant and three antigens in a partially purified preparation of glutamate decar☐ylase. One of these three precipitin lines could be radioactively labelled by 2-[ 3H]-γ-acetylenic γ-aminobutyrate, an irreversible inhibitor of glutamate decar☐ylase. Injection of this antigen-antibody-precipitin line into a new non-immunized sheep yielded a new antiserum, which slightly inhibited but maximally precipitated 85% of the glutamate decar☐ylase activity in rat brain homogenate supernatant. In crossed immunoelectrophoresis the latter antiserum detected one antigen in the partially purified preparation of glutamate decar☐ylase. In crossed immunoelectrophoresis with intermediate gel the antiserum altered the mobility of a single antigen in brain homogenate supernatant. Thus, according to precipitating characteristics, the second antiserum appears to be specific to glutamate decar☐ylase.