基于异源策略的黄曲霉毒素B_1酶联免疫(ELISA)分析方法的建立  

Heterology strategy-based competive indirect enzyme-linked immunosorbent assay for aflatoxin B_1

作　　者：李敏[1,2] 马飞[1,3] 李培武[1,4,2,5,3] 张奇[1,2,3] 张文[1,3] 周海燕[1,3] 印南日[1,3] 王恒玲[1,2] 吴慧[1,2] 

机构地区：[1]中国农业科学院油料作物研究所,湖北武汉430062 [2]农业部生物毒素检测重点实验室,湖北武汉430062 [3]农业部油料及制品质量监督检验测试中心,湖北武汉430062 [4]农业部油料作物生物学与遗传育种重点实验室,湖北武汉430062 [5]农业部油料产品质量安全风险评估实验室(武汉),湖北武汉430062

出　　处：《中国油料作物学报》2014年第6期802-807,共6页Chinese Journal of Oil Crop Sciences

基　　金：农业行业科技专项(201303088);国家科技支撑计划(2012BAB19B09);国家农产品质量风险评估重大项目(GJFP2014006)

摘　　要：为提高黄曲霉毒素B1(aflatoxin B1,AFB1)酶联免疫分析灵敏度,建立了基于异源策略的间接竞争酶联免疫分析(IC-ELISA)方法。首先采用黄曲霉毒素B1与羟基乙酸(glycolic acid,GA)进行衍生化反应,生成缩醛(AFB2-GA),即AFB1半抗原,质谱鉴定结果为ESI-MS:388.07[M+H]+,表明半抗原合成成功。然后采用活泼酯法,将AFB1半抗原羧基与牛血清白蛋白(BSA)的氨基经脱水反应实现化学共价偶联,得到AFB1人工抗原AFB2-GA-BSA。紫外扫描结果表明人工抗原AFB1特征吸收峰与BSA和AFB1的特征吸收峰有明显差异,BSA与黄曲霉毒素B1成功偶联,摩尔结合比分别为8.45∶1。最后将黄曲霉毒素B1人工抗原AFB2-GA-BSA作为包被原,利用黄曲霉毒素B1高特异性3G1抗体,建立了基于异源策略的IC-ELISA方法。利用该方法测定了3G1抗体的灵敏度,检测灵敏度达到0.3ng/m L,比同源ELISA方法检测灵敏度(1.6ng/m L)提高了81.25%,并将方法应用于花生中黄曲霉毒素的实际检测。测定结果与高效液相色谱法结果进行比对,结果符合曲线y=1.044 4x+0.090 2,R2=0.975 1,说明该方法能满足花生中黄曲霉毒素快速筛查的测定准确度的要求。To improve the sensitivity of enzyme -linked immunosorbent assay for aflatoxin BI, indirect competitive enzyme - linked immunosorbent assay （ IC - ELISA） was developed with selective antibody 3GI. The procedure was modified by working preoperatively under dry conditions and using glycolic acid as a spacer providing a nucleophilie hydroxyl group for formation of aflatoxin acetal AFB2 - GA in 8 - position on one side. The structure was successfully identified by MS. The ESI - MS identification demonstrated that [ M ＋ H ]＋ was 388.07. Active ester method （AEM） was used to couple the acetal of aflatoxin B1 to cartier protein （BSA） to obtain artificial antigen for aflatoxin B1. Hapten was then linked to BSA through active ester,and this was confirmed by UV spectrum. A result of UV spectra of AFB2 - GA - BSA was obviously different from BSA and aflatoxin B~, showing that the preparation of artificial antigen for aflatoxin B~ was successful. The coupling ratio of AFB1 to BSA was 8.45：1 in the conjugate. Consequently, using novel AFB2 - GA - BSA as envelope antigen, indirect competitive enzyme - linked immunosorbent assay （IC -ELISA） method was developed with selective antibody 3G1 for aflatoxin B~. Sensitivity of 0.3ng/mL for 3G1 was achieved by heterologous ELISA with working range of 0.03 - 1.08ng/mL, which was 81.25% higher than that obtained by homologous detection. 12 peanut samples were analyzed by this method, fitted well with standard curve of y = 1. 044 4x ＋ 0.090 2 and corresponding coefficient of determination R2 = 0.975 1, comparing with the result by high -performance liquid chromatography （HPLC）.

关 键 词：黄曲霉毒素B1 人工抗原 合成鉴定 ELISA 异源策略 

分 类 号：S379.7[农业科学—农产品加工]