呋喃它酮代谢物的可视化免疫亲和凝胶柱检测分析

An Immunoaffinity Gel Column Assay for Visualizable Detection of Furaltadone Metabolites

摘要:

为了建立一种用于检测动物源性食品中呋喃它酮代谢物（AMOZ）的可视化免疫亲和凝胶柱检测方法，利用对醛基苯甲酸（CPA）对AMOZ进行衍生处理得到衍生物CPAMOZ，制备CPAMOZ完全抗原，并通过免疫小鼠获取CPAMOZ多克隆抗体，使用间接ELISA法对抗体的效价及特异性进行测定。实验采用恒温振荡法将溴化氰活化的琼脂糖凝胶分别与CPAMOZ抗体、HRP抗体偶联制得CPAMOZ抗体胶和HRP抗体胶，作为亲和凝胶柱的检测层与质控层。建立AMOZ可视化免疫亲和凝胶柱分析方法，确定了该方法在酶标抗原的稀释倍数为5000、HRP胶稀释20倍、CPAMOZ抗体胶稀释15倍的条件下，达到最佳工作状态。实验结果表明CPAMOZ多克隆抗体效价为1:64000，IC50值为2.19 μg/L，具有良好的灵敏度及特异性。建立的AMOZ可视化免疫亲和凝胶检测柱方法检测限为4 μg/L，动物源实际样品中AMOZ的检测限为2 μg/kg。该方法快速便捷，符合我国高通量筛选食品中AMOZ的要求。

Abstract:

To establish a visualizable immunoaffinity gel column detection method for analysis of the furoxone metabolites (AMOZ) in animal-derived foods, derivatization of AMOZ with aldehyde benzoic acid (CPA) was conducted to yield the derivative CPAMOZ for the preparation of AMOZ complete antigen. The CPAMOZ polyclonal antibody was obtained via immunization of the mouse, and the titer and specificity of the antibody were determined using an indirect ELISA. CPAMOZ antibody gel and HRP antibody gel were prepared by coupling the cyanogen bromide-activated agarose gel with CPAMOZ antibody and HRP antibody, respectively, as a detection layer and a quality control layer of the affinity gel column. The AMOZ visualized immunoaffinity gel column analysis method was established with the optimal working conditions as: dilution factor 5000, HRP gel dilution 20 times, and CPAMOZ antibody gel dilution 15 times. The experimental results showed that the CPAMOZ polyclonal antibody had a titer of 1:64000 and an IC50 value of 2.19 μg/L, with good sensitivity and specificity. The detection limit of the established AMOZ visualizable immunogel column method was 4 μg/L, and the detection limit of AMOZ in the actual sample of animal origin was 2 μg/kg. The method is fast and convenient and meets the requirements for high-throughput screening of AMOZ in foods in China.