Use of iodine-125 in radioimmunoassay of small molecules (application to testosterone)

The preparation of radioligands bearing radioactive iodine for the assay of small molecules has heretofore required a mixed anhydride synthesis. For many potential users, this step has been a barrier to the adoption of a very useful method. In this thesis a solution is offered in the form of activated (N-hydroxysuccinimide) esters. One step of thin layer chromatographic purification would afford pure radioligand. In order to demonstrate the general approach, one molecule, testosterone, bearing two widely separated different functional groups was arbitrarily chosen. A second aspect is concerned with the use of iodohistamines in radioimmunoassay. To date no systematic study of this alternative to iodophenols has been reported. Authentic standards were synthesized at the macroscale level; their structures were established by their method of synthesis and by their properties. Our coupling method (using N-hydroxysuccinimidyl esters) was easier and faster than the mixed anhydride procedure used by all previous workers. Maximum coupling was obtained at pH 8.5 at 40 for 30 minutes. At pH 7.0 the secondary amide bond formed is unstable in aqueous media and its formation accelerates hydrolysis of the activated esters. Two types of antisera were used for RIA; (1) anti-T-17β-succinyl-BSA antiserum and (2) anti-T-3-CMO-RSA antiserum. Two different systems were studied: (1) the homologous system in which the radioligand and the antigen have the same type of chemical bridge and (2) the heterologous system in which the bridges are different. Testosterone was used as the non-labeled hormone