赭曲霉毒素A模拟抗原表位及噬菌体展示酶联免疫吸附分析法的建立  

Mimic Epitope of Ochratoxin A and Its Application in Phage Enzyme-Linked Immunosorbent Assay

作　　者：王吕[1] 熊斯诚 邹旭强[2] 陈超超[1] 邵辉锋 陈雪岚[1] 

机构地区：[1]江西师范大学功能有机小分子教育部重点实验室,生命科学学院,南昌330022 [2]南昌大学食品学院,南昌330047

出　　处：《分析化学》2015年第6期856-861,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.31160323);功能有机小分子教育部重点实验室开放基金(No.KLFS-KF-201414)资助项目~~

摘　　要：以驴抗鼠二抗包被微孔板,以捕获方式包被抗赭曲霉毒素A（OTA）单克隆抗体,利用噬菌体随机七肽库筛选OTA模拟抗原表位,并以其替代检测抗原,建立了基于噬菌体展示技术的酶联免疫吸附分析（Phage ELISA）检测OTA的方法。结果表明,筛选获得的模拟OTA抗原表位七肽氨基酸序列为GMSWMMA。基于模拟表位噬菌体建立的Phage ELISA方法半数抑制浓度（IC50值）为（0.15±0.02）ng/m L,检测OTA的线性范围为0.03~0.50 ng/m L,OTA的检出限为0.03 ng/m L,且Phage ELISA与其它4种常见真菌毒素（黄曲霉毒素B1、伏马毒素B1、脱氧雪腐镰刀菌烯醇及玉米赤霉烯酮）无交叉反应。大米样品OTA加标实验表明,批內加标回收率为97.0%~115.2%,批间加标回收率为107.2%~123.1%,与ELISA试剂盒检测结果比较,无显著性差异。The donkey anti-mouse IgG was firstly coated onto the microplates, and then anti-ochratoxin A monoclonal antibodies （anti-OTA mAbs） bound with the IgG were used to select the mimic epitope of OTA antigen. The enzyme-linked immunosorbent assay （ELISA） for OTA was developed based on the selected filamentous phage. The results showed that the amino acid sequence of the mimic epitope of OTA was GMSWMMA. The proposed phage ELISA exhibited a dynamic linear range for the detection of OTA from 0.03 ng/mL to 0.50＂ ng/mL with a half inhibition concentration （ ICs0 ） of （0.15＋0.02） ng/mL, with the detection limit of 0.03 ng/mL. The Phage ELISA showed high specificity toward OTA, with negligible cross reactivity with other four common mycotoxins. The recoveries of OTA spiked rice samples for intra-assay were 97.0%-115.2% , and those for inter-assay were 107.2%-123.1%. Meanwhile, the results of OTA spiked real rice samples obtained by phage ELISA and commercial ELISA exhibited no significant difference.

关 键 词：赭曲霉毒素A 噬菌体展示肽库 模拟表位 酶联免疫吸附法 

分 类 号：O652[理学—分析化学]