基于骆驼科重链抗体可变区的谷物中甲萘威免疫检测方法的建立。
Development of an immunoassay for the detection of carbaryl in cereals based on a camelid variable heavy-chain antibody domain.
机构信息
Department of ecological science and engineering, Beijing Key Laboratory of Biodiversity and Organic Farming, College of Resources and Environmental Sciences, China Agricultural University, Beijing, China.
Department of basic veterinary medicine, Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing Laboratory for Food Quality and Safety, Beijing, China.
出版信息
J Sci Food Agric. 2019 Jul;99(9):4383-4390. doi: 10.1002/jsfa.9672. Epub 2019 Apr 3.
BACKGROUND
The variable domain of camelid heavy-chain antibodies (VHH) is increasingly being adapted to detect small molecules in various matrices. The insecticide carbaryl is widely used in agriculture while its residues have posed a threat to food safety and human health.
RESULTS
VHHs specific for carbaryl were generated from an alpaca immunized with the hapten CBR1 coupled to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) based on the VHH C1 and the coating antigen CBR2-BSA was developed for the detection of carbaryl in cereals. This assay, using an optimized assay buffer (pH 6.5) containing 10% methanol and 0.8% NaCl, has a half-maximum signal inhibition concentration of 5.4 ng mL and a limit of detection (LOD) of 0.3 ng mL for carbaryl, and shows low cross reactivity (≤0.8%) with other tested carbamates. The LOD of carbaryl using the VHH-based ELISA was 36 ng g in rice and maize and 72 ng g in wheat. Recoveries of carbaryl in spiked rice, maize and wheat samples were in the range of 81-106%, 96-106% and 83-113%, respectively. Relative standard deviations of repeatability and intra-laboratory reproducibility were in the range of 0.8-9.2% and 2.9-9.7%, respectively.
CONCLUSION
The VHH-based ELISA was highly effective in detecting carbaryl in cereal samples after simple sample extraction and dilution. © 2019 Society of Chemical Industry.
背景
骆驼科重链抗体（VHH）的可变区越来越多地被用于检测各种基质中的小分子。杀虫剂克百威在农业中广泛使用，但其残留对食品安全和人类健康构成了威胁。
结果
从用半抗原 CBR1 与血蓝蛋白偶联物免疫的羊驼中产生了针对克百威的 VHH。基于 VHH C1 和包被抗原 CBR2-BSA 开发了用于检测谷物中克百威的酶联免疫吸附测定法（ELISA）。该测定法使用优化的测定缓冲液（pH 6.5），其中含有 10%甲醇和 0.8%NaCl，半最大信号抑制浓度为 5.4ng/ml，检测限（LOD）为 0.3ng/ml，对其他测试的氨基甲酸酯的交叉反应性（≤0.8%）较低。基于 VHH 的 ELISA 检测克百威的 LOD 在大米和玉米中为 36ng/g，在小麦中为 72ng/g。在添加大米、玉米和小麦样品中的克百威回收率在 81-106%、96-106%和 83-113%范围内。重复性和实验室内部重现性的相对标准偏差分别在 0.8-9.2%和 2.9-9.7%范围内。
结论
基于 VHH 的 ELISA 非常有效地检测了谷物样品中的克百威，经过简单的样品提取和稀释。© 2019 化学工业协会。
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