微板式化学发光酶免疫分析法临床测定人血清中孕酮  

Micro-plate Chemiluminescence Enzyme Immunoassay for Clinical Determination of Progesterone in Human Serum

作　　者：任世奇[1] 王栩 唐宝军[3] 胡国茂[3] 李振甲[3] 陈国南[1] 林金明[2] 

机构地区：[1]福州大学化学化工学院,福州350002 [2]清华大学化学系,北京100084 [3]北京科美东雅生物技术有限公司,北京100094

出　　处：《分析化学》2008年第6期729-734,共6页Chinese Journal of Analytical Chemistry

基　　金：国家863计划(No.2006AA02Z4A8);教育部创新团队(No.IRT0404)资助项目

摘　　要：将抗兔IgG(即二抗)物理吸附于聚苯乙烯微孔板上作为通用固相,通过免疫反应制备固相抗体。采用辣根过氧化物酶催化鲁米诺-过氧化氢化学发光体系,建立了一种高通量、简便、快速的化学发光酶免疫分析方法用于临床测定人血清中的孕酮。对各种影响因素如免疫试剂的稀释度、发光底物选择、发光反应时间及温育条件等进行了考察和优化,最终选定的实验条件:孕酮抗体和HRP标记物的最佳稀释度分别为1∶100000和1∶15000;选用Ⅱ号发光底物,发光反应10min后测定;37℃水浴条件下温育1h。对建立的方法进行了评价。该方法的灵敏度为0.08μg/L;批内和批间相对标准偏差均在15%之内;低、中、高3个不同浓度值样品的平均回收率分别为101%、101%和94.4%(在87.8%～108%之间)。使用本方法和经典的放射免疫法同时对36份人血清样品进行测定,结果显示,本方法与放射免疫分析方法相关性良好,其相关系数为0.9502。A high-throughput, simple and rapid chemiluminescence enzyme immunoassay （CLEIA）was developed for the clinical determination of progesterone in human serum, and the luminol-hydrogen peroxide was used as chemiluminescence system catalyzed by horseradish peroxidase （HRP）. The solid phase of anti-progesterone antibody was prepared through the immunoreaction between anti-progesterone polyclonal antibody with donkey anti-rabbit IgG, i.e. second antibody, which had been physically absorbed on the wells of poly-styrene microplate and was used as a universal solid phase. The effect of several factors, such as the dilution ratios of the immunoreagents, the chemiluminescent substrate, the chemiluminescence reaction time and incu- bation condition were examined and optimized. The optimal dilutions of anti-progesterone antibody and HRP-progesterone conjugate were 1：10000 and 1：15000, respectively. The substrate Ⅱ was chosen and the luminescence was determined after 10 min incubation. The immunoreaction was incubated in water bath at 37 ℃ for 1 h. The assay was evaluated with sensitivity as low as 0.08 μg/L. The RSD was less than 15% for both intra-and inter-assay precision. The recoveries of three different spiked concentration samples were 101%, 101% and 94.4%, respectively. This proposed method has been successfully applied to the clinical evaluation of progesterone in 36 human sera. The results showed a good correlation with the commercially available radioimmunoassay kit with a correlative coefficient of 0. 9502.

关 键 词：孕酮 人血清 化学发光酶免疫分析 辣根过氧化物酶 

分 类 号：R446.6[医药卫生—诊断学]