Enzyme immunoassays for Thyroliberin (TRH) and TRH-elongated peptides in mouse and rat hypothalamus

Abstract

Enzyme immunoassays (ElAs) for Thyroliberin (TRH) and TRH-elongated peptides were developed. Three haptens <E-H-P-NH 2 (TRH). <E-H-P-OH (TRH-OH), and S-K-R-Q-H-P-G-K-R-F (P10) were conjugated by the use of different heterobifunctional cross-linking agents either to sun-flower globulin as carrier or to acetylcholinesterase as tracer. For a same hapten, the same chemical group in the peptide was used to prepare the immunogen and the enzyme conjugate. These ElAs were performed with a second antibody solid phase technique using acetylcholinesterase as label. They permited the measurement of TRH and TRH-elongated peptides with a sensitivity threshold of 10fmol/well for TRH and 2fmol/well for P10. TRH ElA only detected authentic TRH whereas TRH-OH ElA detected TRH and TRH peptides elongated on C terminal part. Anti-P10 serum was specific of TRH peptides elongated both on C and N terminal parts and no cross reactivity was observed with TRH. Using these assays, TRH and TRH-elongated peptides were determined in crude or chromatographed mouse and rat hypothalamus tissular extracts. Several TRH extended forms were identified by P10 ElA, whereas TRH-OH ElA permitted detection of both TRH and TRH-elongated peptides in chromatographed extracts. Authentic TRH was measured by TRH ElA both in crude and chromatographed hypothalamic extracts. These assays can permit the study of the processing and maturation of TRH.