Preparation of a Chicken scFv to Analyze Gentamicin Residue in Animal Derived Food Products
Preparation of a Chicken scFv to Analyze Gentamicin Residue in Animal Derived Food Products
复制标题
制备鸡单链抗体以分析动物源性食品中的庆大霉素残留
DOI：
10.1021/acs.analchem.6b00426
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发表时间：
2016-04-05
影响因子：
7.4
通讯作者：
Li, Xinping
中科院分区：
文献类型：
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作者：
Li, Cui;He, Jinxin;Li, Xinping
鸡是简化重组抗体库生成的理想模型。将鸡单链可变片段（scFv）应用于免疫分析中检测动物食品中抗生素和化学污染物的报道很少。在本研究中，scFv（S-1 和 S-5）是从源自超免疫鸡的噬菌体展示文库中分离出来的。棋盘滴定结果表明，S-1和S-5的最佳浓度分别为0.78μg/mL和0.44μg/mL，在5μg/mL的Gent-OVA包被浓度下获得约1.0的OD450。 S-1和S-5与卡那霉素和阿米卡星的交叉反应性可以忽略不计。 S-1和S-5的50%抑制浓度(IC50)分别为12.418μg/mL和14.674ng/mL。在间接竞争ELISA (ic-ELISA)中，S-1和S-5的检测限分别为0.147 ng/mL和0.219 ng/mL。 Gent 的平均回收率范围为 60.91% 至 118.09%，测定内和测定间的相对标准偏差 (RSD) 不超过 10.35%。这些结果表明基于鸡scFv的ic-ELISA方法适用于动物源性可食用组织和牛奶中Gent残留的检测。
Chicken is an ideal model for simplified recombinant antibody library generation. It has been rarely been reported to apply chicken single-chain variable fragments (scFvs) in immunoassays for the detection of antibiotic and chemical contaminants in animal food products. In this study, the scFvs (S-1 and S-5) were isolated from a phage display library derived from a hyperimmunized chicken. The checker board titration revealed that the optimum concentrations of S-1 and S-5 were 0.78 mu g/mL and 0.44 mu g/mL respectively, to obtain OD450 around 1.0 at 5 mu g/mL of Gent-OVA coating concentration. Both S-1 and S-5 exhibited negligible cross reactivity with kanamycin and amikacin. The 50% inhibitory concentration (IC50) of S-1 and S-5 were 12.418 mu g/mL and 14.674 ng/mL respectively. In the indirect competitive ELISA (ic-ELISA), the limits of detection for S-1 and S-5 were 0.147 ng/mL and 0.219 ng/mL respectively. The mean recovery for Gent ranged from 60.91% to 118.09% with no more than 10.35% relative standard deviation (RSD) between the intra-assay and the inter-assay. These results indicate the chicken scFv based ic-ELISA method is suitable for the detection of Gent residue in animal derived edible tissues and milk.