The Production of a Monoclonal T₃-Antiidiotypic Antibody (T₃-MAAB) That Mimics the Effects of T₃on 2-Deoxy-D-glucose Uptake in Chick Embryo Heart Cells

Abstract

The Ig fraction of rabbit anti-T3 antibody was injected into the spleens of BALB/c mice. Four days later, the lymphocytes were recovered from their spleens and were fused with cells of the 653 myeloma cell line. Screening of the hybrid colonies was carried out in a T3 RIA system. Positive colonies were those whose supernatant displaced 125I-labeled T3 from its antibody. The positive cultures were recloned and one was injected ip into mice. The crude IgG fraction of the ascites fluid was affinity purified on an affigel-10 column containing a covalently bound rabbit anti-T3-IgG. In order to eliminate possible endogenous T3 contamination during the affinity purification, the column was stripped with a 40% solution of acetonitrile in 0.2 M acetic acid, neutralized, and then the purification proceeded as described. The affinity purified antibody was an IgG2a isotype. This monoclonal antibody (T3-MAAB) displaced labeled T3 from its antibody in an RIA system. It also mimicked T3 in the stimulation of [3H]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 T3-MAAB was shifted to the left by at least one order of magnitude when compared to the dose-response curve obtained with T3. After 24 h exposure, T3 had the expected additional stimulatory effect that was dependent on neosynthesis of proteins, while T3-MAAB did not. Also at 24 h exposure, T3-MAAB did not stimulate the incorporation of labeled leucine and uridine into the heart cells while T3 at an equivalent concentration did. The MAAB activity could be abolished by boiling, while boiling did not affect the activity of an equivalent concentration of T3, thus excluding a T3 contamination-mediated effect. We conclude, therefore, that (a) a monoclonal hybridoma producing an antibody that mimics T3 was established; (b) this antibody competed with labeled T3 for anti-T3 antibody and, like T3, stimulated sugar uptake into cultured chick embryo heart cells; and (c) this antibody, unlike T3, did not stimulate the neosynthesis of proteins.

3,5,3′ Triiodo-L-thyronine Stimulates 2-Deoxy-D-Glucose Transport into L6 Muscle Cells Through the Phosphorylation of Insulin Receptorβand the Activation of PI-3k

“…Furthermore, we produced a T 3 anti-idiotypic monoclonal antibody that stimulated the uptake of 2-DOG uptake in cardiomyocytes in a manner similar to T 3 -RBC. This antibody lost its activity when boiled, while the activity of free T 3 was not affected by boiling (9). These experiments suggest that T 3 could interact with specific plasma membrane sites.…”

“…This antibody stimulated 2-DOG uptake in chick cardiomyocytes, but its action was not blocked by protein synthesis inhibitors. Because it was an IgG we suggested that it was acting through the cell plasma membrane (9). D'Arezzo et al (23) has shown that T 3 bound to agarose was as effective as free T 3 in activating the Naϩ/Hϩ exchanger in L6 myoblasts.…”