基于双抗夹心的Cry1B毒素蛋白质TRFIA检测方法的建立与评价  

Development and evaluation of a double-antibody sandwich time-resolved fluoroimmunoassay(TRFIA) for detection of Cry1B

作　　者：徐重新[1,2] 陈莎莎[1] 张霄[2] 刘媛[2] 张存政[2] 黄鹰[1] 刘贤进[2] 

机构地区：[1]南京师范大学生命科学学院、江苏省微生物与功能基因组学重点实验室,江苏南京210023 [2]江苏省农业科学院食品质量安全与检测研究所农业部食品质量安全监控重点开放实验室,江苏南京210014

出　　处：《江苏农业学报》2013年第1期184-188,共5页Jiangsu Journal of Agricultural Sciences

基　　金：国家973计划项目(2012CB722505)

摘　　要：为了建立检测Cry1B毒素蛋白质的双抗夹心时间分辨荧光免疫分析(TRFIA)方法,利用稀土元素Eu3+作为示踪剂,以可溶性表达的抗Cry1B毒素蛋白质单链抗体(scFv)作为捕获抗体,以免疫兔血清获得的抗Cry1B毒素蛋白质多克隆抗体为检测抗体,建立检测Cry1B抗原的TRFIA技术,并对其进行方法学的综合评价。通过正交试验,获得的最佳单链抗体(Capture antibody)、多克隆抗体(Detection antibody)、Eu3+标记的二抗(Tracer antibody)浓度分别是2.500μg/ml、2.000μg/ml、1.000μg/ml。标准检测曲线灵敏度为0.170 ng/ml,线性测量范围为1.000～800.000 ng/ml。抗原类似物间无交叉反应。Cry1B毒蛋白质在大米中添加回收的批内变异系数为3.8%～6.6%、平均回收率为80.5%～84.8%,批间变异系数为2.2%～8.5%、平均回收率为84.1%～88.4%。表明本试验建立的双抗夹心Cry1B-TRFIA检测范围宽,特异性强,重复性和稳定性好。An indirect sandwich time-resolved fluoroimmunoassay （TRFIA） for quantitative determination of Cryl B toxic protein was established by using, a Eu-N1 clelate as a tracer and soluble anti-CrylB single chain-variable fragments （scFvs） as capture antibody and anti-CrylB polyclonal antibodies as detection antibody. The optimized working concentrations of capture antibodies,detection antibodies and the tracer were 2. 500 μg/ml, 2. 000 μg/ml, 1. 000 μg/ml, respectively. The detection limit of the assay was 0. 170 ng/ml, and the linear range for CrylB detection was 1. 000-800.000 ng/ml. There was no cross reaction between CrylB.and CrylAb, CrylAc and CrylC. The intra-assay coefficients of variation（CV） were 3.8%-6. 6% and the average recoveries for CrylB were 80.5%-84.8% in rice samples, the inter-assay CV were 2.2%- 8. 5% and ihe average recoveries were 84. 1% -88.4% for CrvlB. The results showed that the sensitive and snecifie indirect sandwich CrylB-TRFIA developed in this study had a wide detection range and a good repeatability and stability.

关 键 词：苏云金芽孢杆菌 时间分辨免疫分析 单链抗体 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]