摘要
为制备B族黄曲霉毒素(BGAFs)特异性强、广谱性好的抗体,本研究根据黄曲霉毒素B1(AFB1)的分子结构和活性位点,采用肟化活泼酯法(OAE)、氨甲基化法(MOA)、混合酸酐法(MA)、半缩醛法(SA)、环氧化物法(EP)和烯醇醚衍生物法(EED)6种方法合成BGAFs人工抗原AFB1-BSA,并通过UV和SDS-PAGE进行鉴定;采用AFB1-BSA免疫新西兰白兔制备多克隆抗体(AFB1 pAb),间接ELISA检测AFB1 pAb效价,间接竞争ELISA(icELISA)分析其敏感性,交叉反应试验(CR)分析其特异性和广谱性。结果表明,AFB1-BSA合成成功,在BGAFs抗原合成的6种方法中OAE效果最好,AFB1与BSA的分子结合比为8.46∶1;AFB1 pAb的间接ELISA效价为1∶(1.28×104),IC50为10.32μg·L-1,与AFB2、AFG1、AFG2、AFM1、AFM2的CR分别为75.21%、44.13%、14.72%、16.36%、1.44%。本研究制备出了高效价、敏感、特异、广谱的AFB1 pAb,为BGAFs免疫检测方法的建立奠定了物质和技术基础。
The aim of this study was to prepare B-group aflatoxins(BGAFs)antibody with strong specificity and goodeurytopicity.According to the molecular structure and active site of aflatoxin B1(AFB1),the BGAFs artificial antigen AFB1-BSA was prepared by 6 methods including oxime active ester(OAE),methylation of ammonia(MOA),mixed anhydride(MA),semi acetal(SA),epoxide(EP)and enol ether derivative(EED)and identified by UV and SDS-PAGE.Polyclonal antibodies against AFB1(AFB1 pAb)were prepared by immunizing New Zealand rabbits with AFB1-BSA,and the titers of AFB1 pAb was detected by indirect ELISA,the sensitivity of AFB1 pAb was analyzed by indirect competitive ELISA(icELISA)and the specificity and eurytopicity of AFB1 pAb was analyzed by cross-reactivity(CR)test.The results showed that AFB1-BSA was synthesized successfully and the best one was OAE method among the six synthesis methods of BGAFs artificial antigen and its conjugation ratio of AFB1 to BSA was 8.46∶1.The immune efficacy of OAE method was the best,that its AFB1 pAb had the highest titers of 1∶1.28×104 by indirect ELISA,a good sensitivity with the 50%inhibition concentration(IC50)of 10.32μg·L-1 to AFB1 by icELISA and showed a high CR to AFB2 of 75.21%,AFG1 of 44.13%,AFG2 of 14.72%,AFM1 of 16.36%and AFM2of 1.44%,respectively.In this study,AFB1 pAbs with high titer,sensitivity,specificity and eurytopicity were prepared,proving a technical support for the establishment of BGAFs immunoassay.
作者
张海棠
王晓斐
职爱民
王亚楠
王自良
ZHANG Haitang;WANG Xiaofei;ZHI Aimin;WANG Yanan;WANG Ziliang(The Animal Science and Technology College,Henan Institute of Science and Technology,Xinxiang,Henan 453003;Henan Xu Rui Food Co.,Ltd.,Jiaozuo,Henan 454150)
出处
《核农学报》
CAS
CSCD
北大核心
2019年第12期2414-2420,共7页
Journal of Nuclear Agricultural Sciences
基金
“十二五”国家科技支撑计划项目(2014BAD13B05)
河南省产学研项目(162107000056)
关键词
B族黄曲霉毒素
抗原合成设计
多克隆抗体
特性分析
B-group aflatoxins
antigen synthesis design
polyclonal antibody
characteristics analysis
