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本研究旨在研制一种以紧密素为检测靶标的间接ELISA技术,检测血清中产志贺毒素大肠杆菌(Shiga toxigenic Escherichia coli,STEC)的抗体水平。根据紧密素27个亚型的N端保守序列,体外表达390~543 aa片段制备包被抗原,通过敏感性、特异性、重复性和稳定性实验,建立检测STEC的抗体的间接EIISA方法。经优化筛选的间接ELISA最佳反应条件:抗原包被浓度3 ng/孔,待检血清稀释比例1∶200,HRP-兔抗羊Ig G、HRP-兔抗牛Ig G的工作浓度均为1∶15000,5%脱脂奶封闭l h,底物作用时间10min,用于牛、羊疫苗抗体检测或者STEC大肠杆菌感染的监测。
The aim of this study was to develop an indirect ELISA technique to detect the level of antibodies against Shiga toxigenic Escherichia coli (STEC) in sera. According to the N-terminal conserved sequence of 27 subtypes of compactin, 390 ~ 543 aa fragments were expressed in vitro to prepare coated antigen. The indirect ELISA method for detecting antibodies to STEC was established by experiments of sensitivity, specificity, repeatability and stability. The optimal reaction conditions for optimized indirect ELISA were as follows: the concentration of antigen coating was 3 ng / well, the dilution of serum to be tested was 1: 200, the working concentration of HRP-rabbit anti-goat IgG, HRP- : 15000, 5% skimmed milk closed lh, substrate action time 10min, for cattle and sheep vaccine antibody test or STEC E. coli infection monitoring.