绵羊肺炎支原体夹心ELISA检测方法的建立及初步应用  

Establishment and preliminary application of antibody-sandwich ELISA for detection of Mycoplasma ovipneumoniae antigen

作　　者：王晓楠 张纹纹[2,3] 陈思宇 单依依 陈益 李文良 程子龙[2,3] 杨蕾蕾 王金泉 刘茂军[2,3] WANG Xiaonan;ZHANG Wenwen;CHEN Siyu;SHAN Yiyi;CHEN Yi;LI Wenliang;CHENG Zilong;YANG Leilei;WANG Jinquan;LIU Maojun(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biologic al Engineering and Technology,Ministry of Agriculture and Rural Affairs,Nanjing 210014,China;Guotai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou,225300,China)

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052 [2]江苏省农业科学院兽医研究所农业农村部兽用生物制品工程技术重点实验室,南京210014 [3]兽用生物制品(泰州)国泰技术创新中心,江苏泰州225300

出　　处：《中国兽医科学》2024年第9期1202-1208,共7页Chinese Veterinary Science

基　　金：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ZX(21)1218;江苏省第五期“333高层次人才培养工程”科研项目(BRA2019092)。

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105μg/m L,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3.81μg/m L,37℃作用2 h;酶标抗体1∶4000稀释,37℃反应1.5 h;加入底物37℃避光反应10 min。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。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 105μg/mL and coated at 4℃overnight.Sealed with 10 g/L BSA at 37℃for 1 h;Act with ultrasonic treated samples for 1.5 h;The antibody concentration was 3.81μg/mL and treated at 37℃for 2 h.The enzyme-labeled antibody was diluted at 1:4000 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,repeatability and 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 quantitative 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.

关 键 词：绵羊肺炎支原体 夹心ELISA 建立 

分 类 号：S852.62[农业科学—基础兽医学]