摘要
采用活泼酯法将玉米赤霉烯酮（Zaralenone，ZEN）分别与牛血清白蛋白（BSA）和卵清蛋白（OVA）偶联合成免疫原ZEN—BSA和包被原ZEN—OVA，经紫外扫描和聚丙烯酰胺凝胶电泳鉴定后免疫BALB/c小鼠；挑选产生高效价和高敏感性抗体的小鼠进行抗原超强免疫．取脾细胞与SP2／0细胞融合获得1株稳定分泌抗玉米赤霉烯酮单克隆抗体的杂交瘤细胞（3D8）。经鉴定，该抗体亚类为IgG1，轻链为k型。经体内诱生腹水并纯化获得效价为l：2．048x106的单克隆抗体。建立了基于单克隆抗体的ZEN间接竞争ELISA法（ic—ELISA），该方法ic50和检出限分别为22．89pg／mL和10．07pg/mL。与a-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮、a-玉米赤霉醇和β-玉米赤霉醇的交叉反应率分别为95％、8％、12％、6％和5％，而与黄曲霉毒素B1、呕吐毒素、伏马毒素B1和赭曲霉毒素A未见有交叉反应。在玉米、大麦、小麦和燕麦中加标的回收率在86．4％～104．8％之间。该方法灵敏特异。可作为快速检测谷物中玉米赤霉烯酮的初筛方法。
Zearalenone （ZEN） was coupled with bovin serum albumin（BSA） and ovalbumin（OVA） to prepare immunogen ZEN-BSA and coating antigen ZEN-OVA by ester activation method. The two antigens were identified by UV Scan and SDS-PAGE. The spleen cells of BALB/c mice immunized by ZEN-BSA were fused with SP2/O myeloma cells, and hybridomas secreting antibodies against ZEN were selected and cloned. A stable hybridoma cell line 3D8 that secret MAb of subclasses IgG1, kappa light chain were established. The titer of the MAb determined by indirect ELISA was 1：2.048x1@ in ascites. Based on the ascites MAb, an indirect competitive ELISA （ic-ELISA） method was developed for the quantitative detection of ZEN. The ic-ELISA had a good sensitivity with an IC50 of 22.89 pg/mL, a detection limit of 10.07 pg/mL, and 95%, 8%, 12%, 6%, 5% cross-reactivitives to a-zearalenol, β-zearalenol, zearalanone, a-zearalanol, 13-zearalanol respectively, and scarcely showed cross-reactivity to others. The spiked recovery of this method ranged from 86.4% to 104.8% in maize, barley, wheat and oat samples. It is indicated that the developed method in this paper was sensitive and convenient in preliminary screening of ZEN.
出处
《中国食品学报》
EI
CAS
CSCD
北大核心
2013年第1期145-152,共8页
Journal of Chinese Institute Of Food Science and Technology
基金
浙江省重大科技专项(2006C12102)
杭州市重大科技创新专项(20083212A25)
浙江省重点科技创新团队(2010R50028)
浙江省科技成果转化项目(2011E61018)
浙江省重大科技专项重点项目(2012C12013-3)
