基于单克隆抗体的玉米赤霉烯酮检测方法研究  

Study on Immunoassay for the Detection of Zearaleone Based on Monoclonal Antibody

作　　者：刘琦 生威[1] 李志 李诗洁[1] 张燕[1] 王硕[1] 

机构地区：[1]天津科技大学食品工程与生物技术学院,教育部食品营养与安全重点实验室,天津300457

出　　处：《食品研究与开发》2017年第21期111-115,共5页Food Research and Development

基　　金：“十二五”国家科技支撑计划(2013BAD18B11);国家国际科技合作专项项目(2014DFR30350)

摘　　要：建立一种检测谷物样品中玉米赤霉烯酮的酶联免疫分析方法。将玉米赤霉烯酮修饰羧基制得半抗原,再与钥孔嘁血蓝蛋白偶联,免疫BALB/c小鼠,经过细胞融合与筛选,得到3株具有高亲和力和特异性的杂交瘤细胞,分别为2E8、2C7、6E11,重链类型均为IgG_1,轻链类型均为Kappa。细胞株2E8分泌的抗体特异性较好,制备腹水得到单克隆抗体用于后续实验。建立检测玉米赤霉烯酮的间接竞争酶联免疫吸附(Enzyme-Linked Immunosorbent Assay,ELISA)方法,方法的灵敏度(IC_(50))为(0.13±0.02)ng/mL,检测限(IC_(15))为(0.02±0.01)ng/m L,在玉米和燕麦中的检出限分别为2.4μg/kg,4.0μg/kg。加标回收率为82.80%~109.20%,变异系数为3.27%~15.38%。经高效液相色谱串联质谱仪验证(R^2=0.996 3)二者具有良好的相关性。The aim of this research was to develop an enzyme linked immunosorbent assay to detect zearalenone in grain samples. Three hybridoma cells named 2E8,2C7,6E11 were obtained after cell fusion and filtering by immunizing Balb/c mouses with conjugate of ZEN and keyhole limpet hemocyanin. The subtypes of three monoclonal antibody were identified with IgG1 for heavy chain and Kappa for light chain. The cell 2E8 was chosen for subsequent experiments. An indirect-competitive Enzyme-Linked Immunosorbent Assay（ELISA） was developed for the detection of zearalenone with high specificity and affinity after optimizing. The half maximal inhibitory concentration （IC50） was（0.13±0.02） ng/mL, the limit of detection （IC15） was（0.02±0.01） ng/mL while the the limits of detection are 2.4 μg/kg, 4.0 μg/kg for corn and oat with the recoveries from 82.80 % to 109.20 % and the coefficients of variation was between 3.27 % and 15.38 %. The method was confirmed by high performance liquid chromatography tandem mass spectrometry with a good correlation.

关 键 词：玉米赤霉烯酮 单克隆抗体 间接竞争酶联免疫吸附法 高效液相色谱-串联质谱法 

分 类 号：O657.63[理学—分析化学] TS210.7[理学—化学]