Xin Tian-Bing, Liang Shu-Xuan, Wang Xu, Li Haifang, Lin Jin-Ming
College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China.
Anal Chim Acta. 2008 Oct 10;627(2):277-84. doi: 10.1016/j.aca.2008.08.020. Epub 2008 Aug 28.
A magnetic particles (MPs)-based chemiluminescence immunoassay (CLIA) with high sensitivity, specificity, and reproducibility was proposed for the evaluation of estradiol (E(2)) in human sera. The MPs coated with secondary antibody were used as dispersed solid phase for the immunoassay, and the horseradish peroxidase (HRP)-luminol-H(2)O(2) chemiluminescent system with high sensitivity was chosen as the detection system. The method showed specific recognition to E(2), without cross-reaction for the major steroids, including estrone (E(1)), estriol (E(3)), dihydrotestosterone (DHT), androstenedione, and testosterone (T), which was commonly found in human serum. The addition of sodium trichloracetate (Na-TCA) in the enzyme buffer as a blocking agent contributed to the realization of direct analysis of E(2) in human serum without extraction. Besides, the effects of several physicochemical parameters, including the dilution ratios of E(2)-6-HRP conjugate and anti-E(2) polyclonal antibody, immunoreaction time, chemiluminescent (CL) substrate volume, volume of MPs, and CL reaction time, were studied and optimized. The proposed method had a detection limit of 2.51pgmL(-1) with a larger working range of 15-1000pgmL(-1). The inter-assay and intra-assay coefficient of variation (CV) were both less than 15%. The average recoveries of three different spiked concentration samples were 93.3, 106 and 101%, respectively. The method has been successfully applied to the determination of E(2) in 105 human sera and showed a good correlation compared with the commercial radioimmunoassay (RIA) kit with a correlative coefficient of 0.9892. This method has exhibited great potential in the fabrication of diagnostic kit and could be used in the clinical analysis of E(2) in human serum.
提出了一种基于磁性颗粒（MPs）的化学发光免疫分析（CLIA）方法，用于评估人血清中的雌二醇（E(2)），该方法具有高灵敏度、特异性和重现性。用二抗包被的MPs作为免疫分析的分散固相，并选择高灵敏度的辣根过氧化物酶（HRP）-鲁米诺-H(2)O(2)化学发光体系作为检测系统。该方法对E(2)具有特异性识别，对人血清中常见的主要类固醇，包括雌酮（E(1)）、雌三醇（E(3)）、双氢睾酮（DHT）、雄烯二酮和睾酮（T）无交叉反应。在酶缓冲液中加入三氯乙酸钠（Na-TCA）作为封闭剂，有助于实现人血清中E(2)的直接分析而无需萃取。此外，研究并优化了几个物理化学参数的影响，包括E(2)-6-HRP偶联物和抗E(2)多克隆抗体的稀释比例、免疫反应时间、化学发光（CL）底物体积、MPs体积和CL反应时间。所提出的方法检测限为2.51pgmL(-1)，工作范围为15 - 1000pgmL(-1)。批间和批内变异系数（CV）均小于15%。三个不同加标浓度样品的平均回收率分别为93.3%、106%和101%。该方法已成功应用于105份人血清中E(2)的测定，与商业放射免疫分析（RIA）试剂盒相比显示出良好的相关性，相关系数为0.9892。该方法在诊断试剂盒的制备中显示出巨大潜力，可用于人血清中E(2)的临床分析。
