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Development of a two-site immunoradiometric assay for rat/human corticotrophin-releasing factor. Application to the measurement of interleukin-1 beta-stimulated production of hypothalamic CRF in vitro.

Authors
  • Hagan, P1
  • Tilders, F
  • Poole, S
  • Bristow, A F
  • 1 Division of Endocrinology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Herts, UK.
Type
Published Article
Journal
Journal of Immunological Methods
Publisher
Elsevier
Publication Date
Mar 15, 1993
Volume
160
Issue
1
Pages
11–18
Identifiers
PMID: 7680697
Source
Medline
License
Unknown

Abstract

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-1 beta, a putative regulator of CRF secretion, and CRF was measured in hypothalamic homogenates and conditioned media. Interleukin-1 beta dose-dependently stimulated synthesis and secretion of CRF, demonstrating the applicability of the immunoradiometric assay, and confirming previous reports that interleukin-1 beta can directly stimulate CRF secretion from the rat hypothalamus in vitro.

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