Hill M, Hampl R, Lukác D, Lapcík O, Pouzar V, Sulcová J
Institute of Endocrinology, Prague, Czech Republic.
Steroids. 1999 May;64(5):341-55. doi: 10.1016/s0039-128x(99)00017-3.
Polyclonal antiserum against 3beta,17alpha-dihydroxypregn-5-en-20-one-19-O-(carboxymethyl )-oxime bovine serum albumin (17alpha-hydroxypregnenolone-19-CMO:BSA), was raised in rabbits. Its main structural determinants were the substituents on D-ring as demonstrated by its 107% cross-reaction with 17alpha-hydroxyprogesterone. This unspecificity was almost completely eliminated by addition of the excess of the cross-reactant directly to the analytical system. The contribution of the cross-reactant from the sample in such a system became negligible due to saturation of the populations of polyclonal antibodies recognizing the analyte as well as the cross-reactant. The possible interference of 17alpha-hydroxypregnenolone-3-sulfate was avoided by inserting ether extraction. The analytical system appeared to be stable to differences in cross-reactant concentrations even in samples from patients with pathologically elevated serum levels of 17alpha-hydroxyprogesterone. The radioimmunoassay was compared with the system using the unspecific antiserum alone, but after separation of the cross-reactants by HPLC. As demonstrated by parallel measurement of 125 samples of human plasma from both sexes and various ages either before and/or after adrenocorticotropin stimulation and 17 samples with elevated basal of human plasma 17alpha-hydroxyprogesterone levels, an excellent correlation was achieved between both methods. The method, based on a simple addition of the cross-reactant, avoids the time-consuming chromatographic separation and, in comparison with the other approaches for improving the specificity of polyclonal antisera, is efficient and rapid. Mathematical analysis of the relations in equilibrium demonstrates that such a simple approach is an efficient way for improvement of immunoassay specificity using some polyclonal antisera.
用兔制备了抗3β,17α - 二羟基孕 - 5 - 烯 - 20 - 酮 - 19 - O -（羧甲基）肟牛血清白蛋白（17α - 羟孕烯醇酮 - 19 - CMO:BSA）的多克隆抗血清。其主要结构决定因素是D环上的取代基，这通过其与17α - 羟孕酮107%的交叉反应得到证明。通过直接向分析系统中加入过量的交叉反应物，这种非特异性几乎被完全消除。由于识别分析物以及交叉反应物的多克隆抗体群体饱和，在这样的系统中来自样品的交叉反应物的贡献变得可以忽略不计。通过插入乙醚萃取避免了17α - 羟孕烯醇酮 - 3 - 硫酸盐的可能干扰。即使在血清17α - 羟孕酮水平病理性升高的患者样本中，分析系统对于交叉反应物浓度的差异似乎也很稳定。将该放射免疫分析与仅使用非特异性抗血清但通过高效液相色谱分离交叉反应物的系统进行了比较。通过对来自不同性别和年龄的125份人血浆样本在促肾上腺皮质激素刺激之前和/或之后以及17份人血浆17α - 羟孕酮基础水平升高的样本进行平行测量表明，两种方法之间具有极好的相关性。基于简单添加交叉反应物的方法避免了耗时的色谱分离，并且与其他提高多克隆抗血清特异性的方法相比，高效且快速。平衡关系的数学分析表明，这种简单方法是使用某些多克隆抗血清提高免疫分析特异性的有效途径。
