摘要
为建立绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)的抗原快速定量检测方法,本研究通过单克隆抗体筛选和条件优化,建立了一种检测MO抗原夹心ELISA检测方法,建立的ELISA方法的最优条件为:单克隆抗体1A11浓度为105 μg/mL,37 ℃孵育1 h后4 ℃过夜包被;用10 g/L BSA 37 ℃封闭1 h;与超声处理的样品作用1.5 h;检测抗体浓度为3.81 μg/mL,37 ℃作用2 h;酶标抗体1:4000稀释,37 ℃反应1.5h;加入底物37 ℃避光反应10min.建立的夹心ELISA方法批间和批内变异系数均低于10％,特异性、重复性、敏感性良好,本方法检测的抗原含量与MO的CCU呈正相关.本方法的建立为绵羊肺炎支原体的定量检测提供了新技术,可用于实验室MO培养物CCU含量的替代检测和MO疫苗生产过程中抗原的检测.
Abstract
In order to establish a rapid quantitative antigen detection method for Mycoplasma ovipneumoniae(MO),a monoclonal antibody-based sandwich ELISA method for detecting MO antigen was established through monoclonal antibody screening and condition optimization.The optimal conditions of the sandwich ELISA method are as follows:monoclonal antibody 1A11 was incubated at 37 ℃ for 1 h at 105 µg/mL and coated at 4 ℃ overnight.Sealed with l0g/L BSA at 37 ℃ for 1 h;act with ultrasonic treat-ed samples for 1.5 h;The antibody concentration was 3.81 μg/mL and treated at 37 ℃ for 2 h.The enzyme-la-beled antibody was diluted at 1:4 000 and reacted at 37 ℃ for 1.5 h.Add substrate and react at 37 ℃without light for 10 min.The coefficient of variation between and within batches were lower than 10％.The method showed good specificity,repeatabilityand sensitivity.And the MO antigen content detected by this method was positive correlation with its titer(CCU/mL).This study provided a new method for the quan-titative detection of MO antigen,which can be used as an alternatives to CCU(Color changing unit)test of MO cultures in the laboratory and in the production process of MO vaccine.
基金项目
国家重点研发计划项目(2022YFD1800603)
国家自然科学基金青年基金项目(32202809)
江苏省农业科学院探索性颠覆性项目(ZX211218)
江苏省第五期"333高层次人才培养工程"科研项目(BRA2019092)
国家科技期刊平台
NSTL
万方数据