A型肉毒毒素内肽酶免疫检测方法的建立

Development of endopeptidase immunoassay for botulinum toxin type A

目的建立一种A型肉毒毒素内肽酶生物活性定量检测方法。方法首先构建参考底物蛋白SNAP251-197质粒并表达纯化,合成检测目标8肽序列且于兔体内制备并纯化其抗体;96孔板包被底物蛋白,利用ELISA方法建立A型肉毒毒素生物活性定量检测体系,并对检测方法的检测限、重复性及精密度等因素进行评价。结果成功表达纯化了SNAP251-197底物蛋白,制备并纯化了8肽兔多抗,建立了BoNT/A ELISA检测体系。结论成功建立了一种简便、灵敏、快速、稳定、可靠的A型肉毒毒素内肽酶生物活性定量检测方法。

Objective To develop an endopeptidase immunoassay for botulinum toxin type A.Method Firstly,the substrate protein SNAP251-197 was constructed and expressed and purified.The target octapeptide sequence was synthesized and prepared,and the antibody was prepared,purified in rabbits.Then,the substrate protein was coated in a 96-well plate,and botulinumneurotoxin type A(BoNT/A)was established by ELISA.Results We successfully expressed and purified the protein of SNAP251-197.Its purity was up to 95%after purification.The antibody of octapeptide was successfully raised in New Zealand white rabbits before it was purified by protein A column.Then,we developed the endopeptidase immunoassay for BoNT/A by ELISA method.The detection limit,repeatability and precision of the test method were evaluated.Conclusion We have successfully developed an endopeptidase immunoassay for BoNT/A by ELISA method,which is simple,sensitive,rapid,stable and reproducible.