He Jinxin, Hu Jianjun, Thirumalai Diraviyam, Schade Ruediger, Du Enqi, Zhang Xiaoying
a College of Animal Science, Tarim University , Alaer, Aksu Xinjiang , China.
b College of Veterinary Medicine, Northwest A&F University , Yangling, Shaanxi , China.
J Environ Sci Health B. 2016;51(1):8-13. doi: 10.1080/03601234.2015.1080479. Epub 2015 Oct 29.
Gentamicin (Gent) is an aminoglycoside antibiotic being used in livestock sector. Gent residues could cause some genetic disorders by nonsense mutations. This study aimed to develop IgY-based ELISA for the detection of Gent in animal products. Gent was conjugated with Bovine serum albumin (BSA) by carbodiimide method for further immunization in the laying chickens. PEG-6000 extraction method was employed to extract IgY from the egg yolk. The titer of anti-Gent-IgY attained the peak of 1:256,000 after the 5(th) booster immunization. Checkerboard titration confirmed that, anti-Gent IgY in 1:2,000 dilution could give an Optical Density (OD) 1.0 at 2 µg mL(-1) of Gent-OVA coating concentration. IgY-based indirect competitive ELISA (Ic-ELISA) showed that, the IC50 value of anti-Gent IgY was 2.69 ng mL(-1) and regression curve equation was y = -16.27x + 56.97 (R(2) = 0.95, n = 3), confirming that, the detection limit (LOD, IC10 value) was 0.01 ng mL(-1). Recoveries from fresh milk, pork and chicken samples were ranged from 69.82% to 94.32%, with relative standard deviation lower than 10.88%. Our results suggested that generated anti-Gent IgY antibodies can be used in routine screening analysis of Gent residues in food samples.
庆大霉素（Gent）是一种用于畜牧业的氨基糖苷类抗生素。庆大霉素残留可通过无义突变导致一些基因紊乱。本研究旨在开发基于卵黄抗体（IgY）的酶联免疫吸附测定（ELISA）法用于检测动物产品中的庆大霉素。采用碳二亚胺法将庆大霉素与牛血清白蛋白（BSA）偶联，用于进一步免疫蛋鸡。采用聚乙二醇-6000（PEG-6000）提取法从蛋黄中提取IgY。在第5次加强免疫后，抗庆大霉素-IgY的效价达到峰值1:256,000。棋盘滴定法证实，在庆大霉素-卵清蛋白（Gent-OVA）包被浓度为2 μg mL⁻¹时，1:2,000稀释的抗庆大霉素IgY的光密度（OD）值为1.0。基于IgY的间接竞争ELISA（Ic-ELISA）表明，抗庆大霉素IgY的半数抑制浓度（IC50）值为2.69 ng mL⁻¹，回归曲线方程为y = -16.27x + 56.97（R² = 0.95，n = 3），证实检测限（LOD，IC10值）为0.01 ng mL⁻¹。鲜奶、猪肉和鸡肉样品的回收率在69.82%至94.32%之间，相对标准偏差低于10.88%。我们的结果表明，所产生的抗庆大霉素IgY抗体可用于食品样品中庆大霉素残留的常规筛查分析。
