Approaches to a markedly increased sensitivity of the radioimmunoassay for thyrotropin-releasing hormone by derivatization.

Antisera to thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) have previously been produced in rabbits by immunization with a conjugate having TRH linked to a carrier protein by means of dinitrophenylene (Dnp) moiety. Studies on the specificity of the antisera obtained suggested that the sensitivity of the radioimmunoassay for TRH may be increased substantially by prior conversion of the hormone in to dinitrophenylene derivatives. To test this possibility, several TRH-Dno derivatives were prepared by reaction of TRH with equimolar amounts of 1,5-difluoro-2,4-dinitrobenzene yielding Nim-(5-fluoro-2,4-dinitrophenyl)TRH. This intermediate was reacted with ammonia, histamine, tyramine or N alpha-acetyl-lysine methyl ester (N alpha Ac-Lys-OMe) to yield the respective unsubstituted and N-substituted Nim-(5-amino-2,4-dinitrophenyl)TRH derivatives: TRH-Dnp-NH2, TRH-Dnp-histamine, TRH-Dnp-tyramine and TRH-Dnp-N alpha Ac-Lys-OMe. Nim-(2,4-Dinitrophenyl)TRH was prepared similarly by reaction of TRH with 1-fluoro-2,4-dinitrobenzene. The products were isolated by means of high-performance liquid chromatography (HPLC) and were found to be pure by HPLC and thin-layer chromatography using several solvent systems. TRH-Dnp-histamine and TRH-Dnp-tyramine were labelled with 125I using the chloramine-T method. The labelled products were purified to homogeneity by ion-exchange chromatography on SP-Sephadex and adsorption chromatography on Sephadex LH-20, respectively, and were found by HPLC to be pure.