Linton E A, Lowry P J
Regul Pept. 1986 Mar;14(1):69-84. doi: 10.1016/0167-0115(86)90206-5.
We report the development of an immunoradiometric assay (IRMA) for the specific measurement of corticotrophin releasing factor (CRF-41) which uses two antibodies directed to opposite ends of the CRF-41 molecule. In this assay, 125I-labelled affinity purified rabbit anti-(CRF 36-41) immunoglobulin (IgG) and a guinea-pig anti-(CRF 1-20) serum are simultaneously added to 200 microliter volumes of standard or unknown. After 16 h incubation at room temperature, free and CRF-bound guinea-pig antibodies are precipitated using affinity purified sheep anti-(guinea-pig Fc region) IgG coupled to solid phase Dynospheres. Radioactive rabbit anti-(CRF 36-41) is only precipitated in tubes containing CRF-41, since the peptide acts as a link between the 125I-labelled rabbit IgG and the unlabelled guinea-pig CRF-specific antibodies. Precipitated counts are directly proportional to the concentration of CRF-41 in the sample. This CRF IRMA is compared with two radioimmunoassays (RIA) using the N- and C-terminal CRF antisera employed in the IRMA and found to be more sensitive, specific and rapid to perform. The CRF-41 content of rat and human hypothalamic extracts is the same whether measured by IRMA or conventional RIA. Sephadex G50 chromatography of rat hypothalamic extracts reveals two peaks, detected equally by IRMA and RIA, with a main peak in the elution position of synthetic CRF-41, and a smaller void peak. This is the case whether the hypothalamic extracts are prepared from adrenalectomised or sham-operated rats, non-stressed or subjected to ether stress. Re-chromatography of pooled void peaks under dissociating conditions gives the elution profile of synthetic CRF-41, indicating that the large molecular weight 'CRF-41' peak is not a CRF-41 precursor, but is due to CRF-41 associating non-covalently with large molecular weight proteins.
我们报道了一种免疫放射测定法（IRMA）的开发，该方法用于特异性测量促肾上腺皮质激素释放因子（CRF - 41），它使用了两种针对CRF - 41分子两端的抗体。在该测定中，将125I标记的亲和纯化兔抗 - （CRF 36 - 41）免疫球蛋白（IgG）和豚鼠抗 - （CRF 1 - 20）血清同时加入200微升的标准品或未知样品中。在室温下孵育16小时后，使用与固相Dynospheres偶联的亲和纯化羊抗 - （豚鼠Fc区）IgG沉淀游离的和与CRF结合的豚鼠抗体。放射性兔抗 - （CRF 36 - 41）仅在含有CRF - 41的管中沉淀，因为该肽充当了125I标记的兔IgG与未标记的豚鼠CRF特异性抗体之间的连接物。沉淀计数与样品中CRF - 41的浓度成正比。将这种CRF IRMA与两种放射免疫测定法（RIA）进行比较，这两种RIA使用了IRMA中使用的N端和C端CRF抗血清，发现该CRF IRMA更灵敏、特异且操作更快速。无论是通过IRMA还是传统RIA测量，大鼠和人类下丘脑提取物中的CRF - 41含量相同。对大鼠下丘脑提取物进行葡聚糖G50色谱分析显示有两个峰，IRMA和RIA检测到的情况相同，一个主峰在合成CRF - 41的洗脱位置，还有一个较小的空体积峰。无论下丘脑提取物是从肾上腺切除的大鼠还是假手术大鼠制备的，无论是未应激的还是受到乙醚应激的，都是这种情况。在解离条件下对合并的空体积峰进行再色谱分析，得到合成CRF - 41的洗脱图谱，表明大分子量的“CRF - 41”峰不是CRF - 41前体，而是由于CRF - 41与大分子量蛋白质非共价结合所致。
