DEVELOPMENT OF A 2-SITE IMMUNORADIOMETRIC ASSAY FOR RAT HUMAN CORTICOTROPIN-RELEASING FACTOR - APPLICATION TO THE MEASUREMENT OF INTERLEUKIN-1-BETA-STIMULATED PRODUCTION OF HYPOTHALAMIC CRF INVITRO

The hypothalamic hormone corticotrophin-releasing factor (CRF) is a highly conserved, 41-residue peptide, the N terminal region of which rarely induces antibody production, which has hindered the development of two-site immunometric assays. A synthetic N terminal peptide, CRF1-20-Cys-Tyr-NH2, was conjugated to bovine serum albumin through the cysteine thiol group, and used to prepare N terminal directed CRF-specific antibodies. The same peptide, conjugated through the cysteine thiol group to activated thiol-Sepharose, was used to affinity purify N terminal CRF-specific antibodies, and these were used in conjunction with a radioiodinated C terminal directed monoclonal anti-CRF antibody for the development of a specific, sensitive two-site immunoradiometric assay for CRF. To test the utility of the assay, hypothalami were stimulated in vitro with interleukin-1beta, a putative regulator of CRF secretion, and CRF was measured in hypothalamic homogenates and conditioned media. Interleukin-1beta dose-dependently stimulated synthesis and secretion of CRF, demonstrating the applicability of the immunoradiometric assay, and confirming previous reports that interleukin-1beta can directly stimulate CRF secretion from the rat hypothalamus in vitro.