摘要
采用戊二醛法将半抗原伏马毒素B1分别与载体蛋白钥孔血蓝蛋白（KLH）和蛋白卵清白蛋白（OVA）偶联成为完全抗原KLH-FB1和OVA-FB1。OVA-FB1作为包被抗原建立ELISA方法;KLH-FB1作为免疫原免疫8周龄Balb/c小鼠,应用杂交瘤细胞融合技术制备单克隆抗体。经三次亚克隆后,筛选出两株（6H3,6H11）能特异、稳定分泌抗FB1单克隆抗体的杂交瘤细胞株。选择其中一株杂交瘤细胞通过体内诱生腹水的方法制备单克隆抗体,经检测表明单克隆抗体的亚型为IgG1,轻链为κ型。采用间接ELISA测得腹水纯化后效价达l∶16 000,抗体对伏马毒素的半数阻断浓度（IC50）为8.26 ng/mL,对玉米赤霉烯酮、黄曲霉素B1等结构类似物几乎不存在交叉反应,与其他结构的氯丙嗪、链霉素等无交叉反应。利用获得的抗FB1单克隆抗体,建立了间接竞争ELISA检测方法,检测灵敏度为0.44 ng/mL,检测线性范围为0.93~73.06 ng/mL,加标回收率在线性范围内达到回收率可达78.4%~102%。
To establish an indirect competitive ELISA for Fumonisin B1 detection,conjugations of KLH-FB1 and OVA-FB1 were synthetized using glutaraldhyde method and they were employed as the coating antigen and immunogen,respectively.8-week-old Balb/c mouse was immunized by the KLH-FB1 to develop the monoclonal antibody（MAb） using hybridoma technique.After three times of subcloning,two hybridoma cell lines stably secreting specific antibody against FB1 were screened by indirect competitive ELISA.The ascites MAb were purified and characterized by SDS-PAGE.The titre of the MAb reached 1∶16 000.In addition,the immunological sub-type of the MAb was identified as IgG1 and the its light chain belonged to κ type.Aflatoxin B1,Chlorpromazine,streptomycin and Zearalenone showed no cross-activity with the monoclonal antibody.Indirect competitive ELISA（IC-ELISA） was established based on the specific antibody against FB1.The sensitivity of IC-ELISA was 0.44 ng/mL and the liner range was 0.93~73.06 ng/mL.The method was used to detect FB1 in spiked samples and the recoveries were 78.4%~102% in the detection range.
出处
《上海交通大学学报（农业科学版）》
2011年第2期69-74,共6页
Journal of Shanghai Jiaotong University(Agricultural Science)
基金
国家高技术研究发展计划(863计划)项目(2007AA10Z424)
