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）.
出处
《中国油料作物学报》
CAS
CSCD
北大核心
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)
