利用蛋黄源免疫球蛋白（IgY）检测庆大霉素残留的间接竞争酶联免疫吸附测定法的开发。

Development of indirect competitive ELISA using egg yolk-derived immunoglobulin (IgY) for the detection of Gentamicin residues.

作者信息

He Jinxin, Hu Jianjun, Thirumalai Diraviyam, Schade Ruediger, Du Enqi, Zhang Xiaoying

机构信息

a College of Animal Science, Tarim University , Alaer, Aksu Xinjiang , China.

b College of Veterinary Medicine, Northwest A&F University , Yangling, Shaanxi , China.

出版信息

J Environ Sci Health B. 2016;51(1):8-13. doi: 10.1080/03601234.2015.1080479. Epub 2015 Oct 29.

DOI:10.1080/03601234.2015.1080479

PMID:26513166

Abstract

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.

摘要

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。