"Custom" synthesis of radioligands for RIA through activated esters. I. Testosterone

The current method labeling small molecules for radioimmunoassay by coupling iodohistamine to haptens through a mixed anhydride reaction is unacceptable to clinical laboratories. Therefore, we propose the use of a simple two-step procedure: treatment of 125I-2-iodohistamine with the activated ester of a small molecule followed by thin layer chromatography to remove unlabeled ligand. Only one radioactive substance, 125I-2-iodohistamine, need be stocked, and the availability of labeled ligands is limited only by the number of nonradioactive activated esters. This principle is illustrated by the use of testosterone. N-Hydroxysuccinimidyl esters of testosterone hemisuccinate and of testosterone-3-carboxymethyloxime were coupled to 2-iodohistamine, 125I-2-iodohistamine or to 125I-2,5-diiodohistamine. Optimum conditions required reaction of 20-50 fold molar excess of ester in 75 microL of tetrahydrofuran with iodohistamine in 75 microL of buffer at pH 8.5 for 30 min at 4 degrees. The reaction mixture was applied directly to a pre-absorbent TLC plate coated with silica gel and run in the system, benzene:ethanol:acetic acid, 75:24:1 (v:v:v). The desired radioligand was eluted in 85% yield.