THE PRODUCTION OF A MONOCLONAL T-3-ANTIIDIOTYPIC ANTIBODY (T-3-MAAB) THAT MIMICS THE EFFECTS OF T-3 ON 2-DEOXY-D-GLUCOSE UPTAKE IN CHICK-EMBRYO HEART-CELLS

The Ig fraction of rabbit anti-T-3 antibody was injected into the sple ens of BALB/c mice. Four days later, the lymphocytes were recovered fr om their spleens and were fused with cells of the 653 myeloma cell lin e. Screening of the hybrid colonies was carried out in a T-3 RIA syste m. Positive colonies were those whose supernatant displaced I-125-labe led T-3 from its antibody. The positive cultures were recloned and one was injected ip into mice. The crude IgG fraction of the ascites flui d was affinity purified on an affigel-10 column containing a covalentl y bound rabbit anti-T-3-IgG. In order to eliminate possible endogenous T-3 contamination during the affinity purification, the column was st ripped with a 40 % solution of acetonitrile in 0.2 M acetic acid, neut ralized, and then the purification proceeded as described. The affinit y purified antibody was an IgG(2a) isotype. This monoclonal antibody ( T-3-MAAB) displaced labeled T-3 from its antibody in an RIA system. It also mimicked T-3 in the stimulation of [H-3]2-deoxy-D-glucose (2-DOG ) uptake in cultured chick embryo heart cells. After 6 h exposure, the dose-response curve of 2-DOG uptake to T-3-MAAB was shifted to the le ft by at least one order of magnitude when compared to the dose-respon se curve obtained with T-3. After 24 h exposure, T-3 had the expected additional stimulatory effect that was dependent on neosynthesis of pr oteins, while T-3-MAAB did not. Also at 24 h exposure, T-3-MAAB did no t stimulate the incorporation of labeled leucine and uridine into the heart cells while T-3 at an equivalent concentration did. The MAAB act ivity could be abolished by boiling, while boiling did not affect the activity of an equivalent concentration of T-3, thus excluding a T-3 c ontamination-mediated effect. We conclude, therefore, that (a) a monoc lonal hybridoma producing an antibody that mimics T-3 was established; (b) this antibody competed with labeled T-3 for an anti-T-3 antibody and, like T-3, stimulated sugar uptake into cultured chick embryo hear t cells; and (c) this antibody, unlike T-3, did not stimulate the neos ynthesis of proteins.