Milton N G, Hillhouse E W, Nicholson S A, Self C H, McGregor A M
Department of Medicine, King's College School of Medicine and Dentistry, London.
J Mol Endocrinol. 1990 Oct;5(2):159-66. doi: 10.1677/jme.0.0050159.
Murine monoclonal antibodies against human/rat corticotrophin-releasing factor-41 (CRF-41) were produced and characterized for use in the immunological and biological characterization of CRF-41. Spleen cells from BALB/c mice immunized with CRF-41 conjugated to bovine gamma-globulin were fused with a BALB/c-derived non-secretor X-63 myeloma line. Hybridomas were selected for CRF antibody production by enzyme-linked immunosorbent assay, and positive hybridomas cloned twice. Three monoclonal antibodies were obtained (KCHMB001, KCHMB002 and KCHMB003) and characterized as IgG1, IgG1 and IgG2a isotypes respectively, with affinity constants for rat CRF-41 of 30, 53 and 34 nmol/l respectively. All three monoclonal antibodies recognize an epitope contained between residues 34 and 41 of the human/rat sequence. The antibodies were able to neutralize the ACTH-releasing activity of rat CRF-41, applied to rat pituitary fragments in vitro, in a dose-dependent manner. Isoelectric focusing showed that KCHMB003 detected bands of synthetic rat CRF-41 and rat [Met(O)21,38]-CRF-41 at pH 7.1 and 6.8 respectively. Use of KCHMB003 in a two-site enzyme-amplified immunoassay showed that this antibody recognizes both synthetic rat CRF-41 and immunoreactive CRF-41 in rat hypothalamic tissue extracts.
制备了抗人/大鼠促肾上腺皮质激素释放因子41（CRF-41）的鼠单克隆抗体，并对其进行了表征，用于CRF-41的免疫学和生物学特性研究。用与牛γ球蛋白偶联的CRF-41免疫的BALB/c小鼠的脾细胞与源自BALB/c的非分泌型X-63骨髓瘤细胞系融合。通过酶联免疫吸附测定法筛选产生CRF抗体的杂交瘤，并将阳性杂交瘤克隆两次。获得了三种单克隆抗体（KCHMB001、KCHMB002和KCHMB003），分别鉴定为IgG1、IgG1和IgG2a亚型，对大鼠CRF-41的亲和常数分别为30、53和34 nmol/l。所有三种单克隆抗体都识别在人/大鼠序列的34至41位残基之间包含的一个表位。这些抗体能够以剂量依赖的方式中和体外应用于大鼠垂体片段的大鼠CRF-41的促肾上腺皮质激素释放活性。等电聚焦显示，KCHMB003分别在pH 7.1和6.8处检测到合成大鼠CRF-41和大鼠[Met（O）21,38]-CRF-41的条带。在两点酶放大免疫测定中使用KCHMB003表明，该抗体识别大鼠下丘脑组织提取物中的合成大鼠CRF-41和免疫反应性CRF-41。
