Hagan P, Tilders F, Poole S, Bristow A F
Division of Endocrinology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Herts, UK.
J Immunol Methods. 1993 Mar 15;160(1):11-8. doi: 10.1016/0022-1759(93)90003-p.
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.
下丘脑激素促肾上腺皮质激素释放因子（CRF）是一种高度保守的、由41个氨基酸残基组成的肽，其N端区域很少诱导抗体产生，这阻碍了双位点免疫测定法的发展。一种合成的N端肽CRF1-20-Cys-Tyr-NH2通过半胱氨酸硫醇基团与牛血清白蛋白偶联，并用于制备针对CRF N端的特异性抗体。同样的肽通过半胱氨酸硫醇基团与活化的巯基琼脂糖偶联，用于亲和纯化针对CRF N端的特异性抗体，这些抗体与放射性碘化的针对CRF C端的单克隆抗体一起用于开发一种针对CRF的特异性、灵敏的双位点免疫放射测定法。为了测试该测定法的实用性，用白细胞介素-1β（一种假定的CRF分泌调节因子）在体外刺激下丘脑，并在下丘脑匀浆和条件培养基中测量CRF。白细胞介素-1β剂量依赖性地刺激CRF的合成和分泌，证明了免疫放射测定法的适用性，并证实了先前的报道，即白细胞介素-1β可以在体外直接刺激大鼠下丘脑分泌CRF。
