中国兽医科学2025，Vol.55Issue(8)：1018-1025,8.DOI:10.16656/j.issn.1673-4696.2025.0137
布鲁菌抗体阻断ELISA检测方法的建立与应用
Establishment and application of a blocking ELISA for detection of antibodies against Brucella
摘要
Abstract
This study aimed to develop a blocking ELISA for detection of antibodies against Brucella using lipopolysaccharides(LPS)and horseradish peroxidase(HRP)-labeled monoclonal antibodies(HRP-mAb),and to evaluate its diagnostic performance and preliminary application.Results indicated that the optimal reaction system was achieved when the coating amount of Brucella LPS was 400 ng per well and the well was blocked with 10 g/L gum arabic solution for 2 h.1∶4 diluted serum was incubated at 37 ℃ for 20 min,and HRP-mAb diluted with 1∶6 000 was incubated at 37 ℃ for 20 min,following color development for 10 min.The cut-off value for this method was determined to be PI=46.7％.The minimum detection limit was 3.125 IU/mL,and cross-reactivity with positive sera from other bacteria was not detected.The coefficient of variations within and between batches were less than 10％.The method demonstrated good sensitivity,specificity and reproducibility.This method could detect Brucella positive sera in cattle,sheep,deer,dogs and other hosts.The blocking ELISA was used to test 284 clinical serum samples.The overall coincidence rates with the rose bengal test,competitive ELISA and indirect ELISA kits reached 97.18％(276/284),98.59％(280/284)and 96.13％(273/284),respectively.In conclusion,a highly sensitive,specific and repeatable blocking ELISA for detecting anti-Brucella serum antibodies was estab lished,with a total reaction time not exceeding 1 h.Moreover,this method could detect anti-Brucella antibodies from different animals.In China,this assay addresses the gap in blocking ELISA technique and provides a support for rapid diagnosis and monitoring immune level of brucellosis.关键词
布鲁菌/脂多糖/单克隆抗体/阻断ELISAKey words
Brucella/lipopolysaccharide/monoclonal antibody/blocking ELISA分类
农业科技引用本文复制引用
宋国栋,郑福英,葛家振,Weldeab Solomon,刘一健,高鹏程,王梓晴,郭双双,谢莹影,储岳峰..布鲁菌抗体阻断ELISA检测方法的建立与应用[J].中国兽医科学,2025,55(8):1018-1025,8.基金项目
甘肃省科技重大专项(23ZDNA007) （23ZDNA007）
中国农业科学院重大科技任务项目(CAAS-ZDRW-202410) （CAAS-ZDRW-202410）
甘肃省农业农村厅农业科技支撑项目(KJZC-2024-13) （KJZC-2024-13）
