细交链孢菌酮酸酶联免疫吸附分析方法研究

采用水合肼和乙醛酸依次对细交链孢菌酮酸(Tenuazonic acid,TeA)进行衍生化,设计合成了含有氮杂共轭双键偶联手臂,可增强免疫效果的半抗原TeAHGA.通过偶联载体蛋白BSA后的免疫原TeAHGABSA免疫新西兰大白兔,成功制备了特异性识别TeA水合肼衍生物TeAH的多克隆抗体；优化确立了ELISA最佳反应条件(TeAH-OVA为异源包被原、包被浓度0.156 μg/L、药物稀释及反应缓冲液为PBS、一抗反应时间40 min、二抗反应时间20 min),建立了TeA间接竞争ELISA(icELISA)检测方法,其抑制中浓度(IC50)为1.61 μg/L,检出限(LOD)为0.08 μg/L,定量线性检测范围为0.19～12.89 μg/L (IC20～IC80).番茄、面粉样品平均添加回收率分别为67.2％～89.8％和74.8％～93.7％.

Development of an Enzyme-linked Immunosorbent Assay Method for Detection of Tenuazonic Acid

To develop an immunoassay method for tenuazonic acid(TeA),two haptens,2-(2-(1-(5-(sec-butyl)-2-oxo-2,5-dihydro-1H-pyrrol-3-yl)ethylidene)-hydrazinyl)acetic acid(TeAHGA) and 5-(sec-butyl)-3-(1-hydrazonoethyl-4-hydroxy-1H-pyrrol-2(5H)-one(TeAH) derived from TeA by hydrazine hydrate and glyoxylic acid were synthesized.Then TeAHGA-BSA conjugate was used as immunogen and the specific polyclonal antibody against TeAH was prepared.Furthermore,an indirect competitive ELISA(icELISA) method for TeA with TeAH as target analyte was established.The optimized assay conditions were 0.156 μg/L of heterologous coating antigen(TeAH-OVA,ovalbumin),with PBS as assay buffer,the incubation times for the primary antibody and the secondary antibody were 40 and 20 min,respectively.The icELISA results showed IC50 value,limit of detection(LOD) and linear range as 1.61 ng/mL,0.08 μg/L and 0.19-12.89 μg/L,respectively.The average recovery rates for standard addition of TeA from tomato and flour samples were 67.2%-89.8% and 74.8%-93.7%,respectively.