伏马菌素B_1单克隆抗体的制备及免疫学检测方法初步应用  

Development and Preliminary Application of the Monoclonal Antibody against Fumonisin B_1

作　　者：姚静静[1] 胡骁飞[1] 韩俊岭[2] 李青梅[1] 王方雨[1] 邓瑞广[1] 张改平[1,3] 

机构地区：[1]河南省农业科学院动物免疫学重点实验室农业部动物免疫学重点实验室,郑州450002 [2]新乡医学院第一附属医院,新乡453100 [3]河南农业大学,郑州450002

出　　处：《畜牧兽医学报》2016年第5期1009-1017,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技支撑计划课题(2014BAD13B05);河南省农业科学院优秀青年基金项目(2013YQ29);河南省基础与前沿项目(162300410135)

摘　　要：拟制备灵敏高、特异性强的抗伏马菌素B_1(FB_1)单克隆抗体,并初步应用于FB_1的检测,以期为FB_1的免疫学快速检测提供技术支持。采用碳二亚胺法(EDC)制备人工抗原FB_1-BSA和FB_1-OVA。以免疫原FB_1-BSA免疫BALB/c小鼠,每四周免疫一次。四免后,小鼠脾B细胞与SP2/0骨髓瘤细胞融合后,采用ELISA筛选,建立分泌抗FB_1抗体的单克隆杂交瘤细胞株;采用动物体内诱生腹水方法制备出抗FB_1单抗,并鉴定单抗的各种性能;初步建立FB_1免疫学检测方法。结果得到1株稳定分泌抗FB_1单抗的杂交瘤细胞株2E11-H3,该细胞株分泌的mAbs的效价高达1∶1.02×10~6,亲和力常数平均值为7.98×10^(10) L·mol^(-1),亚型为IgG_3。FB_1mAbs的工作浓度为1∶6.0×10~4,与FB_1发生特异性反应,间接竞争ELISA测得IC_(50)=43.65ng·mL^(-1);与FB_1结构类似物FB_2和FB_3的交叉反应率分别为385%和72.4%,与其他霉菌毒素及载体蛋白BSA和OVA均无交叉反应。样品回收率在85.85%~114.07%,平均值为98.81%;变异系数为10.11%。本研究制备出了灵敏度高、特异性强、亲和力高的FB_1单抗,并初步建立FB_1免疫学检测方法。A monoclonal antibody against fumonisin B1 （FB1） was developed and applied to estab- lish rapid immunology detection method of FB1. FB1 was successfully conjugated with BSA and OVA using EDC,respectively. Hybridoma cell line secreting monoclona antibodys （mAbs） against FB1 was produced by fusing murine SP2/0 cells with the spleen B cells of BALB/c mice which was immunized with FB1-BSA for four times,every four weeks at a time. The mAbs against FB1 were obtained by inducing ascites in the mice body. The affinity, titer, substype and sensibility of the mAbs were assayed with ELISA. Furthermore, the mAbs were preliminarily applied in immunolo- gy detection of FB1. The mAbs against FB1 whose substype was tested as IgG3 were secreted byhybridoma cell line （2Ell-H3）. The working concentration of constant was 7.98〉〈 10^10L · mo1-1 ,ICso was 43.65 ng · mL the mAbs was 1：6.03〈104 ,affinity 1. The cross reaction ratios with Fu- monisin Bz and B3 were accordingly 385% and 72.4% ,while none with other analogues and carrier protein. The recovery rate of sample was among 85.85%-114.07% ,the average was 98.81%and the coefficient of variation was 10.11%. The monoclonal antibody against FB1 with high affinity,spe- cificity and sensibility was produced and preliminarily applied in immunology determination of FB1.

关 键 词：伏马菌素B1 单克隆抗体 杂交瘤 ELISA 

分 类 号：S859.8[农业科学—临床兽医学]