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
A. Gordon et al., 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, Thyroid, 4(1), 1994, pp. 87-92
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.