摘要
目的建立人血清透明质酸(HA)化学发光酶免疫定量检测分析方法。方法以偶联了牛血清白蛋白的透明质酸包被,以辣根过氧化物酶标记透明质酸结合蛋白(HABP),以过氧化氢(H2O2)-鲁米诺(luminol)化学发光体系作为检测体系,采用竞争法的反应模式,优化筛选温育条件、抗体包被条件、酶标记物稀释度和发光反应时间等条件。同时进行回收实验、热稳定性等实验,并随机选取60例肝纤维化患者血清与进口试剂进行比对实验。结果所建立方法的检出限为6.12μg/L,批内和批间变异均小于10%。检测HA的临床高、中、低值血清回收率为分别为93.5%、95.6%和101.2%;在4℃和37℃条件下分别进行了3、5、7 d的稳定性考察,线性相关系数均>0.98,标准偏差<9%。比对实验分析证明2种方法差异无统计学意义(P=0.62>0.05)。结论成功建立定量HA微孔板化学发光酶免疫分析方法,该方法具有较高的准确性、灵敏度及良好的重复性,并与进口试剂检测结果一致。
Objective To establish chemiluminescence enzyme immunoassay（CLEIA） for quantitative analysis of hu man serum hyaluronic acid（HA）.Methods The quantitative CLEIA was established based on competitive technology.HA coupled with Bovine serum albumin was coated on microtiter plates and hyaluronic acid binding protein（HABP） labeled with horseradish perxidase（HRP） as the catalytic enzyme and the H2O2-luminol as the luminescence reagent.Several physical and chemical parameters were studied and optimized such as immunoreaction conditions,the dilution ratio of HABP-HRP,luminescence reaction time and so on.In order to evaluate the method,recovery test,heat stabi lization test and comparison test between import ELISA in 60 cases with hepatic fibrosis were carried out.Results The detection limit was 6.12 μg/L.Inter-assay and intra-assay RSD were both less than 10%.The recoveries of three different spiked concentration samples were 93.5%,95.6% and 101.2%.After stored at 4 ℃ and 37℃ for 3,5,7 days,the analysis showed correlation coefficient higher than 0.98 and RSD lower than 9%.The detected results with CLEIA closely corresponded to those with imported ELISA in 60 patients sera with liver fibrosis（P = 0.62 0.05）.Conclusion Established CLEIA for quantity determination of serum HA has high accuracy,sensitivity and repeatability.The test re sults consistent with the imported reagent.
出处
《中国医药导报》
CAS
2013年第24期162-163,165,共3页
China Medical Herald
基金
首都医学发展科研基金重点项目(编号2005-2038)
关键词
透明质酸
竞争化学发光酶免疫分析
肝纤维化
Hyaluronic acid
Competitive chemiluminescence enzyme immunoassay
Liver fibrosis
