摘要
目的 建立灵敏、特异的布鲁氏菌双抗体夹心ELISA检测方法。方法 筛选用于布鲁氏菌检测的单克隆捕获抗体和检测抗体,优化并确定最佳抗体包被时间和浓度,最佳封闭液、封闭时间和阴阳性临界值;通过检验其他易与布鲁氏菌发生交叉反应的细菌来验证该方法的特异性;通过对灭活布鲁氏菌梯度稀释液的检测,验证该方法的灵敏性;将该夹心ELISA检测结果与试管凝集和qPCR检测结果进行比较。结果 筛选出的捕获抗体为4A12,检测抗体为6C12。试验分析显示,捕获抗体4A12的最佳包被浓度为5μg/mL,检测抗体6C12的最佳稀释比为1∶2 000。该研究的最佳包被条件为4℃过夜、5%脱脂奶粉封闭、封闭时间2 h。该研究建立的双抗体夹心ELISA方法只与布鲁氏菌发生反应,与其他细菌未发生反应;布鲁氏菌灭活菌液浓度稀释到1×10^(5) CFU/mL时仍能检测出。批内、批间变异系数均小于10%。结论 该研究成功建立了布鲁氏菌双抗夹心ELISA检测方法,该方法具有良好的特异性与灵敏性,为布鲁氏菌病的精确诊断和有效防控提供有效途径。
This study was aimed at establishing a sensitive and specific sandwich ELISA detection method for Brucella.We screened monoclonal capture antibodies and detection antibodies for Brucella detection,and optimized and determined the optimal antibody coating time and concentration,as well as the optimal blocking solution,blocking time,and yin-yang critical value.The specificity of this method was verified by examination of other bacteria prone to cross-reacting with Brucella.The sensitivity of the method was verified by detection of a gradient dilution of inactivated Brucella.Moreover,the sandwich ELISA detection results were compared with test tube agglutination and qPCR results.The selected capture antibody was 4A12,and the selected detection antibody was 6C12.Experimental analysis indicated that the optimal coating concentration for the 4A12 capture antibody was 5μg/mL,and the optimal dilution ratio for the 6C12 detection antibody was 1:2000.The optimal coating conditions were overnight at 4℃,and blocking with 5%skim milk powder for 2 hours.The established double antibody sandwich ELISA method reacted with only Brucella but not other bacteria,thus demonstrating the method’s good specificity.Inactivated Brucella solution was still detectable after dilution to 1×10^(5) CFU/mL,thus demonstrating the method’s good sensitivity.The intra-and inter batch coefficients of variation were both below 10%,thus indicating the method’s good repeatability.Thus,this study successfully established a dual antibody sandwich ELISA method for Brucella detection,which has good specificity and sensitivity,and might provide an effective approach for the precise diagnosis and effective prevention and control of brucellosis.
作者
姚梦欣
彭泽宇
任文浩
徐艺玫
郭伟
陈创夫
马忠臣
王勇
YAO Meng-xin;PENG Ze-yu;REN Wen-hao;XU Yi-mei;GUO Wei;CHEN Chuang-fu;MA Zhong-chen;WANG Yong(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;International Research Center for Animal Health and Breeding,Shihezi 832003,China;Collaborative Innovation Center for Healthy Sheep Breeding and Zoonose Prevention and Control,Shihezi 832003,China;Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention,Urumqi 830000,China;Fujian Shengwei Biotechnology Co.,Ltd.,Nanping 353000,China)
出处
《中国人兽共患病学报》
北大核心
2025年第3期255-262,共8页
Chinese Journal of Zoonoses
基金
国家自然科学基金项目(No.32060789)
2023/2024人才发展专项(No.CZ003315,No.CZ004317)
兵团重大科技项目(No.2017AA003)。
关键词
单克隆抗体
布鲁氏菌
双抗夹心ELISA法
诊断
monoclonal antibody
Brucella
double antibody sandwich ELISA method
diagnosis
