基于双抗夹心的Cry1B毒素蛋白质TRFIA检测方法的建立与评价

摘要

An indirect sandwich time-resolved fluoroimmunoassay (TRF1A) for quantitative determination of Cry1B toxic protein was established by using, a Eu-N1 delate as a tracer and soluble anti-Cry1B single chain-variable fragments (scFvs) as capture antibody and anti-Cry1B 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 Cry1B detection was 1.000-800.000 ng/ml. There was no cross reaction between Cry1B-and Cry1Ab, Cry1Ac and Cry1C. The intra-assay coefficients of variation( CV) were 3.8% -6. 6% and the average recoveries for Cry1B were 80.5% -84. 8% in rice samples, the inter-assay CV were 2.2% -8. 5% and the average recoveries were 84.1% -88. 4% for Cry1B. The results showed that the sensitive and specific indirect sandwich CrylB-TRFIA developed in this study had a wide detection range and a good repeatability and stability.%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、2.000 μg/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.