基于外膜蛋白的鼻气管鸟杆菌抗体间接ELISA检测方法的建立

Development of an Indirect ELISA for Antibody Detection Against Ornithobacterium rhinotracheale Based on Outer Membrane Proteins

【目的】建立鼻气管鸟杆菌(Ornithobacterium rhinotracheale,ORT)血清抗体的ELISA检测方法。【方法】利用原核表达方法克隆表达鼻气管鸟杆菌外膜蛋白OR02,经Western blotting鉴定其免疫原性。以纯化产物作为抗原包被于固相载体,采用棋盘滴定法确定抗原包被浓度和血清稀释度。同时,优化ELISA反应条件,利用鼻气管鸟杆菌阴性血清确定该检测方法的阴阳临界值判定标准。通过检测其他病原阳性血清评估该方法的特异性,检测不同比例稀释的鼻气管鸟杆菌阳性血清以评估其灵敏性,并与商品化试剂盒进行比较,评估该ELISA方法的符合率。【结果】重组蛋白OR02以包涵体形式表达,分子质量约为58 ku,与预期相符。Western blotting显示,OR02重组蛋白能与鼻气管鸟杆菌阳性血清发生特异性结合,具有良好的免疫反应性。以纯化的OR02蛋白作为包被抗原,确定最佳抗原包被浓度为2 ng/μL,血清样本的稀释度为1∶50,孵育时间为30 min,酶标二抗的稀释度为1∶5000,孵育时间为30 min,最佳底物显色条件为在避光条件下孵育15 min。阴阳临界值的判定标准为:当待检血清D_(450 nm)值≥0.266时判定为阳性,当待检血清D_(450 nm)值<0.266时判定为阴性。用该方法检测副鸡禽杆菌(Apg)、新城疫病毒(NDV)、鸡传染性法氏囊病毒(IBDV)等单因子阳性鸡血清均无交叉反应。将抗体滴度为1∶64的鼻气管鸟杆菌阳性鸡血清以1∶800稀释仍能检测到阳性结果。与商业试剂盒对相同临床样本的检测结果的符合率为91.25%。【结论】本研究利用重组OR02蛋白建立了一种检测鼻气管鸟杆菌血清抗体的间接ELISA方法,该方法具有良好的特异性和灵敏性,且与商业试剂盒的符合率较高,可作为鸡血清鼻气管鸟杆菌抗体的检测工具。

【Objective】This study was aimed to develop an ELISA detection method for serum antibodies against Ornithobacterium rhinotracheale(ORT).【Method】The outer membrane protein OR02 of ORT was cloned and expressed using prokaryotic expression methods.The immunogenicity of the purified expression product was confirmed by Western blotting.The purified product was used as an antigen coated on a solid-phase carrier,and the antigen coating concentration and serum dilution ratio were determined using checkerboard titration method.In addition,the reaction conditions for ELISA detection were optimized,and the cut-off value for the detection method was determined using ORT negative serum.The specificity of the method was evaluated by testing positive sera for other pathogens,and the sensitivity was assessed by testing ORT positive sera at different dilution ratios.Furthermore,a comparison was made with a commercial assay kit to evaluate the concordance of the ELISA method.【Result】The recombinant protein OR02 was expressed in inclusion bodies with a molecular weight of 58 ku,consistent with the expected size.Western blotting revealed specific binding of the OR02 recombinant protein with positive serum samples for,indicating good immunoreactivity.Using the purified OR02 protein as the coating antigen,the optimal antigen coating concentration was determined to be 2 ng/μL,the dilution of serum sample was 1∶50,the incubation time was 30 min,the dilution of enzyme-linked immunosorbent assay secondary antibody was 1∶5000 with the incubation time of 30 min,and the optimal substrate color development conditions were incubated for 15 min under dark conditions.The criteria for determining the critical value of positivity and negativity were:D_(450 nm) value of serum≥0.266 was considered positive,while D_(450 nm) value of serum<0.266 was considered negative.No cross-reactivity was observed when testing positive sera for other pathogens such as Avibacterium paragallinarum(Apg),Newcastle disease virus(NDV),and Infectious bursal disease virus(IBDV).Positive results was still detected when the positive serum with a titer of 1∶64 was diluted at 1∶800.The coincidence rate with commercial kits for the same clinical samples was 91.25%.【Conclusion】In this study,an indirect ELISA method was developed using recombinant OR02 protein for the detection of serum antibodies against ORT.The method demonstrated good specificity and sensitivity,with a high coincidence rate compared to a commercial kit.It could be considered a reliable tool for the detection of ORT antibodies in chicken serum.