乙酰肝素酶生物素-链亲和素系统时间分辨荧光免疫分析法的建立及应用  

Establishment of biotin-streptavidin time-resolved fluoroimmunoassay method for the measurement of heparanase

作　　者：朱宝[1] 谢国强[2] 肖华龙[2] 黄飚[3] 邵科晶[1] 许亚丰[1] 张艺[3] 

机构地区：[1]南京医科大学附属无锡人民医院核医学科,214023 [2]南京医科大学附属无锡人民医院医学检验科,214023 [3]卫生部核医学重点实验室

出　　处：《中华核医学与分子影像杂志》2014年第4期308-311,共4页Chinese Journal of Nuclear Medicine and Molecular Imaging

摘　　要：目的 建立测定乙酰肝素酶（HPA）的生物素-链亲和素系统（BSA） TRFIA并进行初步应用.方法 采用部分基因重组的鼠抗人HPA单克隆抗体包被微孔板条,加入HPA标准品和生物素标记的HPA,两者竞争性结合HPA单克隆抗体,再利用铕标记链亲和素（Eu^3＋-SA）作为示踪物,建立竞争法BSA-TRFIA.利用该方法检测健康人血清样本（n=32）和肿瘤患者血清样本（n=54）HPA水平,并比较TRFIA结果与ELISA结果的相关性.数据分析采用两样本t检验或t′检验、直线相关分析.结果 该法测定HPA的灵敏度为0.33 μg/L,批内和批间CV分别为5.29%和7.54%,平均回收率为105.5%,标准曲线范围为O-1 000 μg/L.健康人血清样本测定结果为（2.03±1.47）μg/L,患者血清样本为（22.13 ±7.38） μg/L（t′=19.388.P〈O．01）。该法测定值与ELISA结果相关（r=0．979，P〈0．01）。结论应用BSA-TRFIA建立的HPA测定法是一种灵敏度高，可测范围广，稳定，高效的定量检测方法。Objective To establish a novel TRFIA for the measurement of heparanase （HPA） in serum samples,and investigate its clinical application.Methods The micro-pore plate wells were first coated with partially recombinant murine anti-human HPA monoclonal antibody.Biotin-labeled recombinant HPA protein was then used to compete with HPA in serum samples,and the prepared europium （III）-labeled streptavidin （Eu^3＋-SA） was used as signal readout for establishing the BSA-TRFIA assay.Using this assay,the serum HPA levels in healthy subjects （n=32） and tumor patients （n=54） were measured.The results of BSA-TRFIA were compared with those of ELISA.Two-sample t test （or t＇ test）,and linear correlation analysis were used to analyze the data.Results The sensitivity of BSA-TRFIA for measuring HPA was 0.33 ug/L.The CV values for intra-batch and inter-batch were 5.29% and 7.54%,respectively.The average recovery rate was 105.5%.The standard curve range was 0-1 000 ug/L.The serum HPA level measured by the BSA-TRFIA method in healthy subjects was （2.03± 1.47） μg/L.In tumor patients,the HPA level was significantly higher：（22.13±7.38） μg/L （t＇=19.388,P〈0.01 ）. The HPA values measured by the BSA-TRFIA method were in good agreement with those by ELISA method （r= 0.979, P〈0.01）. Conclusion BSA-TRFIA is a highly sensitive and stable method with efficient quantization and wide dynamic range for the measurement of HPA.

关 键 词：荧光免疫测定 生物素 抗生蛋白链菌素 乙酰肝素酶 

分 类 号：R730.4[医药卫生—肿瘤]