伏马毒素B1时间分辨免疫荧光检测试剂盒的研制

Development of TRFIA kit for quantitative analysis of fumonisin B1

目的:为快速检测食品中的伏马毒素B1(fumonisin B1,FB1),制备抗FB1的单克隆抗体并利用该抗体研制基于时间分辨免疫荧光分析方法的检测试剂盒。方法:在建立抗伏马毒素B1单克隆杂交瘤细胞株的基础上,采用小鼠腹腔注射法生产大量腹水,经Protein A亲和层析柱分离纯化、透析后得到单克隆抗体,利用所得抗体建立间接竞争性时间分辨免疫荧光分析试剂盒并优化各参数:线性范围、检出限及与黄曲霉毒素(aflatoxins,AFB1)、呕吐毒素(deoxynivalenol,DON)和载体蛋白BSA等的交叉反应性;对试剂盒稳定性采用37℃破坏实验;采用3个浓度的加标回收试验确定回收率;对19份样品及FB1玉米盲样进行检测,并同时用市售ELISA试剂盒进行验证。结果:所研制试剂盒的最低检出浓度为2ng/mL,检测线性范围为2～512ng/mL,线性方程2为Y=-0.644X+12.872(R=0.998),50%抑制浓度为10.07ng/mL,加标提取后,玉米样品3个加标水平的回收率在78.32%～116.76%之间,与AFB1、DON和BSA均无交叉反应性,试剂盒常温下可保存315d以上。结论:本研究建立了快速、敏感检测FB1的时间分辨免疫荧光检测试剂盒。

OBJECTIVE： To establish time-resolved immunofluorescence analysis （TRIFA） kit used to rapidly detect fumonisin B1 （FB1） in food. METHODS： Ascites was obtained after intraperitoneal injection of hybridoma cell lines against monoclonal antibody of FB1 into the mice, and the ascites was purified with protein A affinity chromatography so as to get a large amount of monoclonal antibody. Based on this antibody, the TRFIA kit was established and parameters including the detection limit, specificity, stability, recovery, repeatability and reproducibility were optimized. Nineteen corn samples and one blind sample of corn were detected with the established kit and the results were verified with the commercially available ELISA-kit. RESULTS： The detection limit of the established kit was 2 ng/mL, the linear range of detection was 2-512 ng/mL, the linear equation was Y=-0.644X＋12.872 （R2=0.998）, and the 50% concentration of inhibition was 10.07 ng/mL. The rate of recovery from corn samples ranged from 78.32% to 116.76%. There was no interaction with deoxynivalenol, aflatoxins A and BSA. At room temperature, the kit could be kept for more than 315 days. CONCLUSION： The fast and sensitive time-resolved immunofluorescence analysis assay was established to detect FB 1 in food. [KEY WORDS] fumonisin B1 ; monoclonal antibody; time-resolved immunofluorescence; TRFIA kit