Wang Quan, Liu Yingchun, Wang Mingyan, Chen Yongjun, Jiang Wei
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Minhang, Shanghai, 200241, China.
Anal Bioanal Chem. 2018 Jan;410(1):223-233. doi: 10.1007/s00216-017-0714-y. Epub 2017 Oct 30.
There is an urgent need for the rapid and simultaneous detection of multiple analytes present in a sample matrix. Here, a multiplex immunochromatographic test (multi-ICT) was developed that successfully allowed for the rapid and simultaneous detection of four major nitrofuran metabolites, i.e., 3-amino-2-oxazolidinone (AOZ), semicarbazide (SEM), 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), and 1-aminohydantoin (AHD), in fish samples. Four different antigens were separately immobilized in four test lines on a nitrocellulose membrane. Goat anti-mouse immunoglobulin (IgG) was used as a control. Sensitive and specific monoclonal antibodies (mAbs) that recognize the corresponding antigens were selected for the assay, and no cross-reactivity between the antibodies in the detection assay was observed. The free analytes in samples or standards were pre-incubated with freeze-dried mAb-gold conjugates to improve the sensitivity of the detection assay. The multi-ICT detection was accomplished in less than 15 min by the naked eye. The cutoff values for the strip test were 0.5 ng/mL for AOZ and 0.75 ng/mL for AHD, SEM, and AMOZ, which were all below the maximum residue levels set by the European Union and China. A high degree of consistency was observed between the multi-ICT method and commercially available enzyme-linked immunosorbent assay (ELISA) kits using spiked, incurred, and "blind" fish samples, indicating the accuracy, reproducibility, and reliability of the novel test strip. This newly developed multi-ICT strip assay is suitable for the rapid and high-throughput screening of four nitrofuran metabolites in fish samples on-site, with no treatment or devices required. Graphical abstract A multiplex immunochromatographic test (multi-ICT) was developed that successfully allowed for the rapid and simultaneous detection of four major nitrofuran metabolites (AOZ, SEM, AMOZ, and AHD) in fish samples.
迫切需要对样品基质中存在的多种分析物进行快速同时检测。在此，开发了一种多重免疫层析检测法（multi-ICT），该方法成功实现了对鱼类样品中四种主要硝基呋喃代谢物，即3-氨基-2-恶唑烷酮（AOZ）、氨基脲（SEM）、3-氨基-5-甲基吗啉代-2-恶唑烷酮（AMOZ）和1-氨基乙内酰脲（AHD）的快速同时检测。四种不同的抗原分别固定在硝酸纤维素膜上的四条检测线上。山羊抗小鼠免疫球蛋白（IgG）用作对照。选择了识别相应抗原的灵敏且特异的单克隆抗体（mAb）用于该检测，并且在检测分析中未观察到抗体之间的交叉反应。样品或标准品中的游离分析物与冻干的mAb-金偶联物预先孵育，以提高检测分析的灵敏度。通过肉眼在不到15分钟内完成multi-ICT检测。试纸条检测的临界值为AOZ 0.5 ng/mL，AHD、SEM和AMOZ为0.75 ng/mL，均低于欧盟和中国设定的最大残留限量。使用加标、实际污染和“盲样”鱼类样品时，multi-ICT方法与市售酶联免疫吸附测定（ELISA）试剂盒之间观察到高度一致性，表明新型试纸条的准确性、可重复性和可靠性。这种新开发的multi-ICT试纸条检测法适用于现场对鱼类样品中的四种硝基呋喃代谢物进行快速高通量筛查，无需处理或设备。图形摘要 开发了一种多重免疫层析检测法（multi-ICT），该方法成功实现了对鱼类样品中四种主要硝基呋喃代谢物（AOZ、SEM、AMOZ和AHD）的快速同时检测。
