The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084, China.
Talanta. 2010 Sep 15;82(4):1472-7. doi: 10.1016/j.talanta.2010.07.023. Epub 2010 Jul 16.
A competitive immunoassay for estradiol (E2) based on secondary antibody format was established. The donkey anti-rabbit IgG was used as the secondary antibody to coat micro-plates, and the horseradish peroxidase (HRP)-luminol-H(2)O(2) chemiluminescent system with high sensitivity was chosen as the detection system. The addition of sodium trichloroacetate (CCl(3)COONa) in the enzyme buffer as a replaceable packing material can realize directly analysis of E2 in human serum without extraction, which improved reproducibility and resolution of the assay. Additionally, the method showed specific recognition of estrogen, without cross-reaction for the major steroids (estrone (E1), estriol (E3), dihydrotestosterone (DHT), androstenedione, testosterone (T)) commonly found in human serum. The chemiluminescence immunoassay with secondary antibody can be applied to detect E2 with good precision at concentrations as low as 1.48 pg mL(-1). The proposed method has been successfully applied to the determination of E2 in 97 human sera and showed a good correlation compared with the commercially radioimmunoassay (RIA) kit with a correlative coefficient of 0.9881. This method has exhibited great potential in the fabrication of diagnostic kit and can be used in the clinical analysis of E2 in human serum.
建立了一种基于二级抗体形式的雌二醇（E2）竞争免疫分析方法。驴抗兔 IgG 被用作包被微板的二级抗体，选择高灵敏度的辣根过氧化物酶（HRP）-鲁米诺-H2O2 化学发光系统作为检测系统。在酶缓冲液中添加三氯乙酸钠（CCl3COONa）作为可替换的填充材料，可以实现人血清中 E2 的直接分析，而无需提取，从而提高了分析的重现性和分辨率。此外，该方法对雌激素具有特异性识别，与人血清中常见的主要甾体（雌酮（E1）、雌三醇（E3）、二氢睾酮（DHT）、雄烯二酮、睾酮（T））无交叉反应。基于二级抗体的化学发光免疫分析可以应用于检测浓度低至 1.48 pg mL-1 的 E2，具有良好的精密度。该方法已成功应用于人血清中 E2 的测定，与商业放射免疫分析（RIA）试剂盒相比具有良好的相关性，相关系数为 0.9881。该方法在诊断试剂盒的制备中具有很大的潜力，可用于人血清中 E2 的临床分析。
