呋喃唑酮代谢物间接竞争酶联免疫检测方法的研究

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  • 1(山西大学 生命科学学院, 山西 太原, 030006) 2(山西大学,食品药品快速检测中心, 山西 太原,030006)
    3(山西省食品药品监督管理局, 山西 太原,030006)
硕士

网络出版日期: 2018-03-26

Indirect competitive enzyme linked immunosorbent assay for detection of furazolidone metaboliteod

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  • 1(College of Life Science, Shanxi University, Taiyuan 030006, China) 2(The Food and Drug Safety Rapid Inspection Center,
    Shanxi University, Taiyuan 030006, China) 3(Shanxi Food and DrugAdministration, Taiyuan 030006, China)

Online published: 2018-03-26

摘要

建立了呋喃唑酮代谢物间接竞争酶联免疫检测法。用活化酯法将卵清蛋白和半抗原连接成为包被原,对包被原和单克隆抗体的稀释倍数,封闭液浓度、竞争时间、酶标二抗孵育时间和显色反应时间进行优化,同时通过灵敏度、特异性、精密度和准确度等指标进行方法学评价。结果显示,最佳条件为:包被原和单克隆抗体稀释倍数分别为800倍和6 400倍,封闭液是1%脱脂乳,竞争时间是60 min,辣根过氧化物酶标记羊抗鼠二抗孵育时间是45 min,显色时间是15 min。通过建立的标准曲线计算得到线性方程是Y=-0.253X+1.336(R2=0.995),IC50是2.015 ng/mL,线性范围是0.131~30.911 ng/mL,空白猪肉添加回收率是84.8%~90.3%,批内和批间变异系数分别为3.3%~4.1%和4.6%~6.7%。此方法特异性强,准确性、精密度和重现性均良好,可用于动物源性食品中呋喃唑酮代谢物的快速筛查。

本文引用格式

史晓亚 , 高丽霞 , 黄登宇 . 呋喃唑酮代谢物间接竞争酶联免疫检测方法的研究[J]. 食品与发酵工业, 2018 , 44(3) : 260 . DOI: 10.13995/j.cnki.11-1802/ts.015791

Abstract

An indirect competitive enzyme linked immunosorbent assay (ic-ELISA) using monoclonal antibody was developed to determine furazolidone metabolite. Made 3-{[(4-carboxyphenyl) methylene]amino}-2-oxazolidinone (CPAOZ) was conjugated with Ovalbumin (OVA) using N-hyduoxysuccinimide ester method to obtain CPAOZ-OVA. Then the influences of several physicochemical factors such as dilution ratios of CPAOZ-OVA and monoclonal antibody, blocking solution formulation, competitive reaction time, incubation time of HRP-IgG, chromogenic reaction time were optimized and the sensitivity, specificity, accuracy and precision of the method were evaluated. In the optimized system, the dilution ratios of CPAOZ-OVA and monoclonal antibody were 800-fold and 6400-fold respectively; blocking solution contained 1% skimmed milk; competitive reaction time was 60 min; incubation time for HRP-IgG was 45 min; chromogenic reaction time was 15 min. Based on the established standard curve, the linear equation for ELISA was set as Y=-0.253X+1.336(R2=0.995). IC50 was 2.015 ng/mL and linearity range was 0.131-30.911 ng/mL. The recovery rate of negative pork samples was 84.8%-90.3%. The coefficients of variation in intra-assay and inter-assay were 3.3%-4.1% and 4.6%-6.7% respectively. The method was proved to have excellent specificity, accuracy, precision and reproducibility, thus could be applied to rapid detect AOZ content in animal-derived food.
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