高灵敏度化学发光磁免疫法检测人绒毛膜促性腺激素.docVIP

高灵敏度化学发光磁酶免疫法检测人绒毛膜促性腺激素 作者：刘军涛 刘儒平 王蜜霞 刘春秀 罗金平 蔡新霞 【摘要】 将磁性微粒与抗体的偶联，通过优化偶联条件提高了偶联效率，制备了高灵敏度的磁微粒生物探针；采用3(2螺旋金刚烷)4甲氧基4(3邻氧酰苯基)1，2二氧杂环丁烷(AMPPD)碱性磷酸酶(ALP)化学发光体系，建立了化学发光磁酶免疫检测方法；对检测方法进行了优化和改进，提高了系统的灵敏度和检测速度，并对HCG样品进行相关检测。结果表明，HCG浓度在0.15～150 IU/L范围内，光强随HCG浓度增大而增加，两者之间线性关系良好，相关系数r为0.960；检出限可达0.15 IU/L; 相对标准偏差(RSD)小于5％; 检测总时间少于1 h。本方法可以应用于其它免疫分子的检测，在临床免疫检测方面具有广阔的应用前景。 【关键词】 化学发光，磁性微球免疫分析，人绒毛膜促性腺激素 　　Abstract A highly sensitive magnetic enzymelinked chemiluminescent immunoassay method was developed for the detection of human chorionic gonadotropin(HCG). The monoclonal antibody was covalently coupled on the surface of carboxylated magnetic beads to generate magneticbiotargeting; Alkaline phosphatase(ALP) was utilized as a labeled reagent of another monoclonal antibody, whereas 3(2spimadamantane) 4methoxy4(3phosphoryloxy)phenyl1,2dioxetane(AMPPD) was utilized as the chemiluminescent substrate. Based on this concept, a highly sensitive chemiluminescent immunoassay method was established to test HCG. Then, several modifications were made to optimize the method, and the detection sensitivity and procedure were improved accordingly. The detection of the assay could be fulfilled within 60 min and the test result of HCG concentration was linear over the range of 0.15 150 IU/L with good relativity(r=0.960). The relative standard deviation(RSD) were below 5% and the sensitivity of this method was 0.15 IU/L. The proposed method with wide linear range, simple operation and fast detection showed good prospect in practical application onsite. 　　Keywords Chemiluminescence, magnetic immunoassay, human chorionic gonadotropin 　　1 引 言 人绒毛膜促性腺激素(HCG)是由滋养叶层的合体滋养细胞分泌的一种糖蛋白激素，存在于孕妇的血液、尿、初乳、羊水和胎儿体内，对维持正常妊娠有重要意义[1,2]。妇女怀孕后，HCG浓度会按一定规律变化。医生可根据异常情况判断是否先兆流产、异位妊娠、葡萄胎等。某些妇产科疾病、术后治疗过程等都需要跟踪HCG指标变化[3]。目前，临床诊断中HCG检测方法有放射免疫分析[4]、电化学免疫分析[5] 、荧光免疫分析和化学发光免疫分析等[6,7]。其中化学发光磁酶免疫分析法将磁性分离技术、化学发光检测技术与免疫学方法三者相结合，综合了化学发光法灵敏度高、线性范围广、测定速度快以及免疫法的特异性高、准确性好等优点，还利用了磁性微粒在磁场可控运动的特点，可实现待测物的快速富集，具有更快的检