细交链孢菌酮酸酶联免疫吸附分析方法研究  

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

作　　者：杨星星[1] 刘细霞[1] 王弘[1] 徐振林[1] 沈玉栋[1] 孙远明[1] 

机构地区：[1]广东省食品质量安全重点实验室,华南农业大学食品学院,广州510642

出　　处：《分析化学》2012年第9期1347-1352,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(Nos.30871755,30901005)资助项目

摘　　要：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TeAHGA-BSA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"g/L、药物稀释及反应缓冲液为PBS、一抗反应时间40min、二抗反应时间20min),建立了TeA间接竞争ELISA(icELISA)检测方法,其抑制中浓度(IC50)为1.61"g/L,检出限(LOD)为0.08"g/L,定量线性检测范围为0.19～12.89"g/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%。To develop an immunoassay method for tenuazonic acid （TEA）, two haptens, 2-（2-（1-（5- （sec-butyl）-2-oxo-2,5-dihydro-lH-pyrrol-3-yl） ethylidene）-hydrazinyl） acetic acid （TeAHGA） and 5- （sec-butyl）-3-（ 1-hydrazonoethyl-4-hydroxy-lH-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 rain, 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.

关 键 词：细交链孢菌酮酸 异源包被 半抗原 多克隆抗体 免疫分析 

分 类 号：S482.2[农业科学—农药学]