摘要
为了对产气荚膜梭菌α毒素进行快速、有效的检测,本研究以单克隆抗体(monoclonal antibody,mAb)为基础,建立了双抗体夹心ELISA(double antibody sandwich ELISA,DAS-ELISA)方法。纯化原核表达的α毒素蛋白为免疫原,制备腹水型mAb并分离纯化作为检测抗体,免疫新西兰大白兔制备抗α毒素多克隆抗体纯化后作为捕获抗体,通过试验优化反应条件,建立标准曲线;并对建立的DAS-ELISA方法进行性能评价及初步应用。DAS-ELISA方法对α毒素的有效检测范围为5.86~375μg/L,最低检测线为4.57μg/L,比多抗-多抗双夹心ELISA具有更高的特异性和敏感性。初步应用结果显示,相同培养条件下,A^E型产气荚膜梭菌标准菌株的培养上清中,α毒素含量差异较大,A型菌(NCTC528)中α毒素含量显著高于其他菌型。本研究成功制备了高特异性的mAb,并建立了特异、灵敏、稳定的DAS-ELISA方法,为高效检测α毒素提供了有效工具。
Based on the monoclonal antibody(mAb),a double antibody sandwich ELISA(DASELISA)was established in this study with the aim to effective detection for Clostridium perfringens(C.perfringens)α-toxin.With prokaryotic express alpha toxin as antigen,the ascitic mAb and polyclonal antibody were prepared as detection antibody and the capture antibody respectively,optimizing reaction conditions and estiblishing a standard curve,the performance of DAS-ELISA was evaluated and was also preliminarily applied in culture samples.The results showed that the detection range and the sensitivity of DAS-ELISA were respectively 5.86-375μg/L and 4.57μg/L,with higher sensitivity than polyclonal-polyclonal sandwich ELISA.The content ofα-toxin in culture filtrates fromC.perfingens standard strains biotypes A^E with the same culture condition has high difference,and the type A(NCTC528)produces significantly higher quantities ofα-toxin than other C.perfingens strains biotypes.This study,therefore,successfully developed a specific and sensitive DAS-ELISA method,providing an effective method for the efficient detection ofα-toxin of C.perfringens.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2014年第6期930-935,941,共7页
Chinese Journal of Veterinary Science
基金
山东农业大学博士后基金资助项目(23476)
关键词
产气荚膜梭菌
Α毒素
单克隆抗体
多克隆抗体
双抗体夹心ELISA
Clostridium perfringens
α-toxin
monoclonal antibody(mAb)
polyclonal antibody
double antibody sandwich ELISA(DAS-ELISA)
