Yang Wuying, Wang Hong, Dong Jiexian, Xu Zhenlin, Shen Yudong, Sun Yuanming, Liu Xixia, Li Zhenfeng, Lei Hongtao, Du Xiaoyan
Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, China.
Protein Expr Purif. 2012 Jul;84(1):140-6. doi: 10.1016/j.pep.2012.05.001. Epub 2012 May 15.
Furaltadone is an illicit veterinary drug that shows toxic, carcinogenic, and mutagenic effects, as does its metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ)(1). Recombinant antibodies with desirable affinity and specificity that can replace polyclonal or monoclonal antibodies are important factors for effective AMOZ immunoassays. In the present study, a novel single-chain variable fragment (scFv) antibody against the 2-nitrobenzaldehyde derivative of AMOZ (NPAMOZ) was prepared and characterized. The scFv gene was cloned into the pET-22b(+) expression vector, and 6His-tagged scFv antibodies expressed as inclusion bodies in Escherichia coli BL21 (DE3), which were then purified by nickel nitrilotriacetic acid column chromatography. Characterization of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and a novel indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) showed that the scFv antibody was ∼27kDa and exhibited HRP-anti-His-tag antibody-recognized activity. The final purity, yield and mg of this scFv antibody after ultrafiltration concentration were 97%, 20% and 29.1mg, respectively. The icCLEIA indicated that the antibody competitively combined with NPAMOZ, exhibiting an IC(50) value of 1.46±0.01 ng/ml (n=6). Cross-reactivity studies revealed that the antibody showed desirable specificity to NPAMOZ and little reactivity to analogs except the parent furaltadone. In summary, these findings suggested that the prepared recombinant scFv antibody can be used for future immunoassay screening for AMOZ.
呋喃它酮是一种非法兽药，具有毒性、致癌性和致突变性，其代谢产物3-氨基-5-吗啉甲基-2-恶唑烷酮（AMOZ）也是如此(1)。具有理想亲和力和特异性的重组抗体可替代多克隆或单克隆抗体，是有效进行AMOZ免疫分析的重要因素。在本研究中，制备并表征了一种针对AMOZ的2-硝基苯甲醛衍生物（NPAMOZ）的新型单链可变片段（scFv）抗体。将scFv基因克隆到pET-22b(+)表达载体中，6His标签的scFv抗体在大肠杆菌BL21(DE3)中以包涵体形式表达，然后通过镍氮三乙酸柱色谱法进行纯化。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、蛋白质印迹和一种新型间接竞争化学发光酶免疫分析（icCLEIA）对目标蛋白进行表征，结果表明该scFv抗体约为27kDa，并表现出HRP-抗His标签抗体识别的活性。超滤浓缩后，该scFv抗体的最终纯度、产量和毫克数分别为97%、20%和29.1mg。icCLEIA表明该抗体与NPAMOZ竞争性结合，IC(50)值为1.46±0.01 ng/ml（n=6）。交叉反应性研究表明，该抗体对NPAMOZ具有理想的特异性，除母体呋喃它酮外，对类似物几乎没有反应性。总之，这些发现表明所制备的重组scFv抗体可用于未来AMOZ的免疫分析筛选。
