亲和色谱-氢化物发生原子荧光分光光度法纯化检测人血浆中的硒蛋白-P

开发了两步亲和色谱法:肝素-琼脂糖凝胶、Ni-琼脂糖凝胶色谱纯化人血浆中硒蛋白-P的方法,并采用氢化物发生-原子荧光分光光度法(HG-AFS)检测,成功搭建了硒蛋白-P的纯化检测平台。确定了亲和色谱纯化的最佳梯度洗脱条件,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)定性检测,得到了一定纯度的硒蛋白-P,其回收率达43.2%。HG-AFS方法的线性相关系数为0.999 1,检出限为0.09μg/L,日内精密度(RSD)为0.12%,日间精密度(RSD)为0.27%,加标回收率为95%～104%。该亲和色谱纯化方法简单易控、回收率高,HG-AFS检测灵敏度高,结果准确可靠。

Purification and Detection of Selenoprotein-P in Human Plasma by Affinity Chromatography-HG-AFS

A method was developed for the purification and detection of selenoprotein-P in human plasma.The sample was gradient eluted by two successive steps of affinity chromatography,i.e.,Heparin-Sepharose and Ni-Sepharose chromatography.The quality of the purified selenoprotein-P was detected by HG-AFS.The optimum gradient elution conditions were determined and the selenoprotein-P with a certain purity was obtained by SDS-PAGE.The recovery of the two steps of affinity chromatography reached up to 43.2%.The correlation coefficient of HG-AFS was 0.999 1,and the detection limit was 0.09 μg/L.The RSDs were 0.12% for within-day and 0.27% for inter-day.The recoveries were in the range of 95%-104%.The proposed method was simple and sensitive,and the results obtained were accurate.