免疫分析
融合蛋白
赭曲霉毒素A
分子生物学
检出限
化学
免疫印迹
大肠杆菌
重组DNA
色谱法
生物
生物化学
真菌毒素
抗体
食品科学
基因
免疫学
作者
Jiaxin Cheng,Liwen Liang,Y. P. Liu,Min Ye,Xixia Liu,Yingyu Hou,Junfeng Shui,Danyang Li,Qin Wu,Huan Liu,Ping Su,Jin Xuan,Yuanliang Hu,Jianjun Hou
标识
DOI:10.1016/j.jfca.2023.105530
摘要
The ochratoxin A (OTA) nanobody-Glutathione S-transferase (GST) fusion protein preparation and one-step immunoassay for detection OTA in cereal were described in this study. We optimized the OTA nanobody codons and the soluble OTA nanobody-GST fusion protein was expressed in Escherichia coli (E. coli). The expression conditions were optimized and the high-yield fusion protein was purified by GST affinity method. Then, the one-step immunoassay was selected to detect cereal samples by a comparison among three immunoassay modes. SDS-PAGE and western blot analyses revealed that the molecular weight of OTA nanobody-GST fusion protein was about 41 kDa. The expression yield reached to 32.9%, and the purified fusion protein was 155 mg/L bacteria. The one-step immunoassay indicated that the average concentration required for 50% inhibition of binding (IC50) and the limit of detection (LOD) for OTA were 2.25 ± 0.08 ng/mL and 0.32 ± 0.06 ng/mL, and the linear range was from 0.63 ± 0.08 ng/mL to 10.62 ± 1.27 ng/mL. The recovery rate of spiked cereal was from 85.34% to 116.19%. The present findings indicated that the entire time of one-step immunoassay was 15 min. It could be further used to develop a rapid detection kit for detection of OTA.
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