Development of Fc-specific multi-biotinylated antibodies via photoreactive tandem AviTag repeats for the ultrasensitive determination of ochratoxin A
Development of Fc-specific multi-biotinylated antibodies via photoreactive tandem AviTag repeats for the ultrasensitive determination of ochratoxin A
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DOI：
10.1016/j.foodcont.2021.108525
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发表时间：
2022
期刊：
影响因子：
6
通讯作者：
Xiao-Yi Gao;Chong-Mei Xu;Xiao-Kun Zhang;Meng-Ran Li;Xiao Gong;Hong-ming Yang;Jin-Bao Tang
中科院分区：
文献类型：
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作者：
Xiao-Yi Gao;Chong-Mei Xu;Xiao-Kun Zhang;Meng-Ran Li;Xiao Gong;Hong-ming Yang;Jin-Bao Tang
赭曲霉毒素A （OTA）是一种常见的食品污染物，严重威胁着消费者的安全与健康。本研究采用fc特异性多生物素化抗体，建立了一种间接竞争性生物素-链亲和素扩增酶联免疫吸附试验（icBA-ELISA），用于OTA的敏感检测。首先利用氨基酰基-tRNA合成酶/抑制因子tRNA技术构建了一个携带光交联剂（对苯甲酰苯丙氨酸，Bpa）与AviTag串联重复序列融合的fc结合蛋白（z结构域），即ZBpa-(AviTag)n。生物素分子与光反应性生物素化ZBpa-(AviTag)n介导的抗体共价结合，得到fc特异性多生物素化抗体（IgG - *Z-Bnconjugates），用于建立icBA-ELISA。IgG - * z - b5偶联物对OTA的半最大抑制浓度为0.0519 ng mL - 1，检出限为0.00944 ng mL - 1。与化学偶联的IgG -生物素偶联物相比，灵敏度提高了4.8倍和4.7倍。因此，所建立的icBA-ELISA法在食品中各种毒素的检测中具有显著的应用潜力。
Ochratoxin A (OTA) is a common food contaminant that seriously threatens the safety and health of consumers. Here, an indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (icBA-ELISA) was developed for the sensitive detection of OTA by employing Fc-specific multi-biotinylated antibodies. An Fc-binding protein (Z-domain) carrying a photo-cross-linker (p-benzoylphenylalanine, Bpa) fused with tandem AviTag repeats, viz. ZBpa-(AviTag)n, was first constructed using the aminoacyl-tRNA synthetase/suppressor tRNA technique. Biotin molecules were covalently conjugated to antibodies mediated by the photoreactive biotinylated ZBpa-(AviTag)n, resulting in the Fc-specific multi-biotinylated antibodies (IgG−*Z-Bnconjugates) that were used to establish icBA-ELISA. Among the IgG−*Z-Bn-based icBA-ELISA, the IgG−*Z-B5conjugates exhibited a half-maximal inhibitory concentration of 0.0519 ng mL−1and a limit of detection of 0.00944 ng mL−1for OTA. The sensitivity was improved 4.8 and 4.7 times compared with that of the chemical-coupled IgG−biotin conjugates. Thus, the proposed icBA-ELISA for the sensitive detection of OTA has remarkable potential applications for the detection of various toxins in food.