猪支原体重组MSG1蛋白间接ELISA检测方法的建立

为确定猪支原体在临床的感染情况和进行血清流行病学调查,本研究以原核表达纯化的猪支原体MSG1重组蛋白为包被抗原,建立了检测猪支原体的间接ELISA诊断方法。通过对ELISA反应一系列条件的优化,确定了最佳抗原包被浓度为0.5μg/mL;最佳封闭条件为用1%BSA37℃温箱内封闭2 h;血清最适稀释度为1∶200,作用时间为1.5 h;酶标二抗最适稀释度为1∶15 000,最适作用时间为1 h;最后37℃显色15 m in。判定标准为OD450≥0.239时判为阳性,OD450<0.198时判为阴性,0.198≤OD450<0.239时判为可疑。敏感性、特异性和可重复性试验证明,该检测方法敏感性高、特异性强和可重复性好。该研究表明建立的间接ELISA方法为猪支原体的检测和区域流行病学调查提供了一种快速简便的血清学诊断方法。

Development of an indirect-ELISA for detection of Mycoplasma suis with recombinant MSG1 protein

An indirect-ELISA for detection of antibody against Mycoplasma suis was developed using the recombinant MSG1 protein.The optimization of ELISA reactions was as followings: the optimal concentration of coated antigen was 0.5 μg/mL;the blocking buffer was 1% BSA and incubated at 37 ℃ for 2 h;positive and negative sera dilutions were 1∶200 and incubated for 1.5 h;SPA-HRP dilution was 1∶15000 and incubated for 1 h;the substrate incubation condition was 37 ℃ for 15 min.The cutoff values of ELISA were defined as OD450≥0.239 for positive reaction and OD450<0.198 for negative.This method had high sensitivity and specificity and good repeatability.This study demonstrated the established ELISA method will provide a good serological tool for epidemiological investigation.