Legay F, Henry S, Tappaz M L
INSERM U 171 and CNRS UA 1195, Groupe de Neurochimie Fonctionnelle, Hôpital Ste Eugénie, Pav. 4 H, 1 Av. G. Clémenceau, 69230 St Genis Laval, France.
Neurochem Int. 1987;10(3):287-94. doi: 10.1016/0197-0186(87)90102-1.
Monoclonal antibodies against rat brain GAD have been produced and immunochemically characterized in comparison with a traditional anti-GAD antiserum (Oertel et al., Neuroscience6, 2689-2700, 1981). An immunopurified fraction in which GAD represented an estimated 5% of the total protein was used as immunogen. Out of 10 mice injected with this fraction, 6 appeared to be immunized: their sera immunoprecipitated quantitatively GAD activity. Three cell fusions were performed between spleen cells of the best immunized mice and SP2/OAg14 myeloma cells. Around 500 hybridoma were generated in each hybridization experiment. The culture medium of 13 hybridoma significantly trapped GAD activity. All immunoprecipitation curves established with the ascitic fluid obtained from the positive hybridoma, showed a lower titer, at least 50-fold, than the titer of the conventional antiserum. None of these ascitic fluids was able to stain directly any protein from a rat high speed supernatant after western blotting. However, the electrophoretical analysis of the proteins immunotrapped by any of the monoclonal antibodies, followed by western blotting and immunolabelling with the anti-GAD antiserum ("cross-immunoblotting") showed the same two stained monomers. They have the same molecular weight (respectively 59 and 62 kDa +/- 2 kDa) as those stained directly by the anti-GAD antiserum from a rat brain supernatant. Although all monoclonal antibodies showed a lower affinity then the conventional antiserum, which prevents them from being used directly in immunoblotting they permit to definitively establish that the two monomers immunolabelled by the conventional antiserum are constitutive subunits of the rat brain GAD.
