脱氧雪腐镰刀烯醇时间分辨免疫法的建立  

Ultrasensitive Time-resolved Fluoroimmunoassay of Deoxynivalenol

作　　者：高雷[1] 黄飚[2] 李秋萍[1] 张莲芬[1] 金坚[1] 

机构地区：[1]江南大学医药学院,工业生物技术教育部重点实验室,江苏无锡214063 [2]江苏省原子医学研究所,江苏无锡214063

出　　处：《食品科学》2009年第20期398-402,共5页Food Science

基　　金：国家“863”计划项目(2008AA10Z415)

摘　　要：目的:利用抗脱氧雪腐镰刀烯醇多克隆抗体及其人工抗原,采用时间分辨免疫荧光技术建立间接竞争脱氧雪腐镰刀烯醇时间分辨免疫检测方法(DON-TRFIA)。方法:以DON人工抗原(DON-BSA)包被微孔板,与DON标准或样品中的DON共同竞争结合抗DON多克隆抗体,然后用稀土离子Eu3+标记羊抗兔抗体进行示踪检测,并对建立DON-TRFIA进行方法学的考核。结果:该方法的灵敏度为0.01ng/ml,检测范围为0.01～100ng/ml,IC50为4.84ng/ml,批内、批间变异率均小于10%。不同样品添加回收实验表明小麦、玉米、啤酒、面包回收率分别为89.41%～123.2%、115.6%～123.6%、80.35%～118%、87%～92.45%。玉米样品检测结果表明DON-TRFIA与商品化DON-ELISA试剂盒结果高度相关,具有较好的一致性。结论:DON-TRFIA是目前已见报道文献中最灵敏的DON免疫检测法,具有很好的应用前景。Objective： An indirect competitive time-resolved fluoroimmunoassay （TRFIA） was established using anti-DON polyclonal antibody and coating antigen DON-BSA to detect deoxynivalenol in cereal. Methods： In an indirect TRFIA format, DON-BSA was coated onto a 96-well microplate and incubated with standard DON and DON polyclonal antibody, and a goat antirabbit IgG-Eu^3＋ conjugate was then used to evaluate the methodology of DON-TRFIA. Results： The ICs0 value was 4.84 ng/ml at a practical working concentration ranging from 0.01 to 100 ng/ml and the sensitivity for detection was 0.01 ng/ml. Both intra- and inter-assay standard deviations were less than 10%. The half-life time of reagents in assay was over 6 months at 4℃. The recovery rotes of deoxynivalenol in spiked wheat, corn, beer and bread were 89.41% - 123.2%, 115.6% - 123.6%, 80.35% - 118.0% and 87.00% - 92.45%, respectively. Additional analysis for corn samples confh＇rned that determination results using TRFIA method were in good agreement with those using ELISA method. Conclusion： The TRFIA method has the highest sensitivity for quantitatively analyzing DON in cereal and reveals excellent promising.

关 键 词：脱氧雪腐镰刀烯醇 时间分辨免疫分析 生物毒素 

分 类 号：S184[农业科学—农业基础科学]