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.
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.
Hagan
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Division of Endocrinology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Herts, UK.
Tilders
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Poole
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Bristow
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AF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D003346:Corticotropin-Releasing Hormone / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism; D000939:Epitopes; D006168:Guinea Pigs; D006801:Humans; D007031:Hypothalamus / Q000187:drug effects*; D015592:Immunoradiometric Assay / Q000379:methods*; D007375:Interleukin-1 / Q000494:pharmacology*; D008297:Male; D008969:Molecular Sequence Data; D009924:Organ Culture Techniques; D010455:Peptides / Q000032:analysis / Q000138:chemical synthesis; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007375:Interleukin-1; D010455:Peptides; D003346:Corticotropin-Releasing Hormone