A Monoclonal Antibody to the Rat Nuclear Triiodothyronine Receptor: Production and Characterization

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Abstract
The nuclear T3 receptor (NTR) was affinity-labeled with bromoacetyl-[125I]T3 purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and used to immunize BALB/c mice. Spleen cells from one strongly immunoreactive mouse were fused with Sp2 mouse myeloma cells, and 328 hybridomas wee screened by a dot-blot immunoassay using as antigen, a preparation of NTR partially purified by diethylaminoethyl-Sephadex chromatography. Four positive cultures were thus found; three of which were confirmed by comparing Western blotting patterns with the electrophoretic mobility of the affinity-labeled NTR. One of these 3 hybridomas was further subcloned by limiting dilution and gave rise to the 2B3 clone, which produces an immunoglobulin of the immunoglobulin G1 subclass. Several lines of evidence indicated that the 2B3 monoclonal antibody was indeed directed against the NTR. The antibody recognized a protein with the same electrophoretic mobility as the affinity-labeled receptor. Thus, Western blotting revealed a predominant protein with a mol wt of 57,000 and a less abundant 45,000 component on sodium dodecyl sulfate gels, and multiple isoelectric variants of the 57,000 protein, with a predominant form at pI 6.2, were detected on two-dimensional gels. Incubation of the 2B3 antibody with the NTR labeled with [125I]T3 resulted in the formation of an antibody-receptor complex, as indicated by a shift of the radioactivity peak upon gel filtration on Sephacryl S-300. In contrast, control ascitic fluid did not change the elution profile of the labeled NTR. The 2B3 antibody is able to remove the T3-binding activity from rat liver nuclear extracts. Finally, in accordance with previous T3-binding experiments, expected amounts of NTR were found in pituitary, liver, brain, kidney, spleen, and testis with the use of the Western blotting technique and immunohistochemistry on frozen tissue sections. This antibody should prove useful in the characterization and purification of the NTR and also in the study of its distribution in different tissues and cell types.