基于纳米颗粒的赭曲霉毒素A高灵敏酶联免疫检测方法的建立及应用  

Nanoparticle-based enzyme-linked immunosorbent assay for the sensitive detection of ochratoxin A

作　　者：章先 何珂 黄志伟 单颖[4] 曹统 谢珲[5] 宋厚辉 ZHANG Xian;HE Ke;HUANG Zhi-Wei;SHAN Ying;CAO Tong;XIE Hui;SONG Hou-Hui(College of Animal Science and Technology,College of Veterinary Medicine,Zhejiang A&F University,Lin’an,Zhejiang 311300,China;Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,Lin’an,Zhejiang 311300,China;Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology,Lin’an,Zhejiang 311300,China;College of Animal Sciences,Zhejiang University,Hangzhou,Zhejiang 310058,China;Zhejiang Academy of Medical Sciences,Hangzhou,Zhejiang 310015,China)

机构地区：[1]浙江农林大学动物科技学院动物医学院,浙江临安311300 [2]浙江省畜禽绿色生态健康养殖应用技术研究重点实验室,浙江临安311300 [3]动物健康检测互联网技术浙江省工程实验室,浙江临安311300 [4]浙江大学动物科学学院,浙江杭州310058 [5]浙江省医学科学院,浙江杭州310015

出　　处：《菌物学报》2020年第3期599-609,共11页Mycosystema

基　　金：浙江省自然科学基金(LQ17C170002);浙江农林大学人才项目(2016FR025);浙江农林大学学生科研训练项目(118-2013200166);浙江省重点研发计划(2018C02041)。

摘　　要：赭曲霉毒素(ochratoxins)主要是由青霉菌Penicillium和曲霉菌Aspergillus产生的有毒次级代谢产物,常见于发霉或发酵的农产品中,其中赭曲霉毒素A(ochratoxin A,OTA)毒性最强且最为普遍。OTA是粮食作物和饲料的重要污染物,在加工、储存或运输过程中均可产生,具有肾毒性和免疫毒性,可通过蓄积作用发挥毒性效应,对人类和动物健康造成严重威胁。本研究通过将OTA单克隆抗体包被于纳米磁珠(magnetic nanoparticles,MNPs)表面,获得具有免疫活性的磁珠抗体复合物(MNPs-Anti OTA),并制备生物素标记的偶联抗原OTA-BSA-Bio,后续采用链酶亲和素标记的纳米金颗粒(Strep-HRP-AuNPs)催化底物进行信号检测,最终建立了OTA高灵敏检测方法(MNPs-bs-AuNPs-ELISA)。在最优条件下,经计算该方法检测下限(IC10)为0.01ng/mL,检测区间(IC20-IC80)为0.02-0.73ng/mL,半数抑制率(IC50)为0.13ng/mL。与OTA类似物OTB、OTC交叉反应性为4.3%和8.1%,对其他常见真菌毒素AFB1、ZEN、FB1、DON、CIT和PAT均无交叉反应。玉米、面粉和大豆样本中的加标回收率可达85.6%-115.7%,对天然样本中OTA含量的检测结果表明,该方法与LC-MS/MS相关性良好。本研究建立的MNPs-bs-AuNPs-ELISA可满足谷物及饲料样本中OTA的快速、高灵敏度定量检测,成本较低,具有很好的应用前景。Ochratoxin A(OTA), a secondary metabolite produced by species of Aspergillus and Penicillium with multiple toxicity such as nephrotoxicity, teratogenicity and immunotoxicity, is gaining more and more attention worldwide. A detection technique possessing high sensitivity that would be operable with ease is preferred for determination of OTA levels in foods and feeds. In this study, a rapid and sensitive magnetic nanoparticlebiotin-streptavidin-Au nanoparticles-enzyme linked immunosorbent assay(MNPs-bs-AuNPs-ELISA) was prototyped and used for detection of OTA. The result proved that biotin-streptavidin enhanced the detection signal and thereby improved the sensitivity of the assay. The lower limit of detection by using MNPs-bs-Au NPs-ELISA was 0.01 ng/mL while the IC50 was 0.13 ng/mL. The detectable range was 0.02-0.73 ng/mL. Cross-reactivities to OTA analogues, ochratoxin B and ochratoxin C, were 4.3% and 8.1%, respectively. No cross-reactivity(<0.01%) was observed to other co-occurring mycotoxins(AFB1, ZEN, FB1, DON, CIT and PAT). The recovery rates in spiked samples of corn, wheat and soybean ranged 85.6%-115.7%, and the intra-day and inter-day relative standard deviations were <10%. Simultaneous analysis of commercially obtained samples(corn, wheat, and feedstuff) showed a good correlation between MNPs-bs-AuNPs-ELISA and liquid chromatography tandem mass spectrometry(LC/MS-MS). This new method can be used as a sensitive, simple and cost-effective assay to quantify the levels of OTA in cereal and feed samples.

关 键 词：赭曲霉毒素A 纳米磁珠 纳米金 生物素-亲和素系统 定量检测 

分 类 号：TS207.5[轻工技术与工程—食品科学]