免疫亲和富集-荧光光谱法检测牛血清白蛋白的研究

Facile Separation and Detection of Bovine Serum Albumin by Immunoaffinity Enrichment-Fluorescence Spectroscopy

本文通过戊二醛法合成免疫磁球(IMMs),采用荧光电镜、透射电镜和振动样品磁强计对其进行表征。合成的IMMs为单分散的球形结构,粒径约为510 nm,具有超顺磁性(饱和磁化强度为40.44 emu/g)。基于此,以牛血清白蛋白(BSA)作为蛋白质模型,建立了免疫亲和富集-荧光光谱法用于目标蛋白质的分离与检测。考察并优化了影响免疫亲和富集的因素。在最佳实验条件下,IMMs对目标蛋白质的最大吸附容量为107.7μg/g,可重复使用10次以上。将该方法应用于人血清基质中BSA的测定,加标回收率不低于89.3%,由基质辅助激光解吸电离飞行时间质谱仪测得分离后的BSA纯度较高。结果表明,该方法适用于人血清样品中蛋白质的选择性分离与检测。

In this study,immunomagnetic microspheres （IMMs） were prepared using glutaraldehyde as a crosslinker and characterized by confocal laser scanning microscopy, transmission electron microscope, and vibrating sample magnetometer. The as-prepared magnetic materials showed a unique core-shell structure, dimensional homogeneity, and strong magnetic responsiveness. The immunoaffinity enrichment-fluorescence spectroscopy method was established using bovine serum albumin（BSA） as a model protein for the selective separation and detection of target proteins. Under optimal immunoaffinity enrichment conditions, the maximum adsorption capacity of the target analyte was 107.7ptg/g. The proposed method was successfully applied for the capture of BSA from spiked human serum with satisfactory recoveries and the IMMs could be reused for at least ten cycles. The proteins separated by IMMs were of high purity, which was confirmed by matrix-assisted laser desorption ionization time offlight mass spectrometry. This work would hopefully serve as an effective method for the selective separation and detection of target proteins from complex biological samples.