van Oers J W, Tilders J H, Berkenbosch F
Department of Pharmacology, Medical Faculty, Free University, Amsterdam, The Netherlands.
Endocrinology. 1989 Mar;124(3):1239-46. doi: 10.1210/endo-124-3-1239.
To produce a rat monoclonal antibody directed to rat CRF (rCRF), female Wistar rats were actively immunized with a rCRF-bovine thyroglobulin conjugate. Immunization resulted in the formation of CRF antibodies, as indicated by binding of [125I]iodo-rCRF and attenuation of the plasma corticosterone responses to ether stress. Rat spleen cells were fused with mouse myeloma P3 cells, and a hybridoma clone (PFU 83) was selected according to its capacity to bind [125I]iodo-rCRF and inhibit rCRF-induced ACTH release from cultured rat pituitary cells. PFU 83 antibodies (IgG2a subclass) are directed to the extreme C-terminal part (amino acids 38-39) of rCRF and bind with an affinity constant of 21 nM. In vitro, PFU 83 causes a parallel shift to the right of the rCRF dose-ACTH response curve, with an apparent affinity of 10 nM. PFU 83 neither binds nor blocks the ACTH-releasing activity of oCRF. Intravenous administration of PFU 83 ascites (generated in nude mice) to Wistar rats caused a dose-dependent inhibition of ether-induced ACTH secretion. Full blockade of the ACTH response to ether was found at a dose of 10 nmol PFU 83/rat. Based on the dynamics of rCRF binding and its in vitro and in vivo effects, we conclude that the rCRF-blocking bioactivity of PFU 83 is due to binding of PFU 83 to native rCRF and formation of a biologically inactive complex. Finally, we found that PFU 83 did not affect resting or ether-induced alpha MSH secretion, indicating that CRF does not play a major role in the control of alpha MSH secretion.
为制备针对大鼠促肾上腺皮质激素释放因子（rCRF）的大鼠单克隆抗体，用rCRF - 牛甲状腺球蛋白偶联物对雌性Wistar大鼠进行主动免疫。免疫导致形成CRF抗体，这通过[125I]碘 - rCRF的结合以及血浆皮质酮对乙醚应激反应的减弱来表明。将大鼠脾细胞与小鼠骨髓瘤P3细胞融合，并根据其结合[125I]碘 - rCRF和抑制rCRF诱导的培养大鼠垂体细胞释放促肾上腺皮质激素（ACTH）的能力选择了一个杂交瘤克隆（PFU 83）。PFU 83抗体（IgG2a亚类）针对rCRF的极端C末端部分（氨基酸38 - 39），并以21 nM的亲和常数结合。在体外，PFU 83使rCRF剂量 - ACTH反应曲线向右平行移动，表观亲和力为10 nM。PFU 83既不结合也不阻断人促肾上腺皮质激素释放因子（oCRF）的ACTH释放活性。向Wistar大鼠静脉注射PFU 83腹水（在裸鼠中产生）导致对乙醚诱导的ACTH分泌的剂量依赖性抑制。在剂量为10 nmol PFU 83 /大鼠时发现对乙醚的ACTH反应完全被阻断。基于rCRF结合的动力学及其体外和体内效应，我们得出结论，PFU 83的rCRF阻断生物活性是由于PFU 83与天然rCRF结合并形成无生物活性的复合物。最后，我们发现PFU 83不影响静息或乙醚诱导的α - 促黑素（α - MSH）分泌，表明CRF在α - MSH分泌的控制中不发挥主要作用。
