Establishment and preliminary testing of a double antibody sandwich ELISA method for Brucella detection
10.3969/j.issn.1002-2694.2025.00.039
- VernacularTitle:布鲁氏菌双抗夹心ELISA检测方法的建立与初步验证
- Author:
Meng-xin YAO
1
;
Ze-yu PENG
;
Wen-hao REN
;
Yi-mei XU
;
Wei GUO
;
Chuang-fu CHEN
;
Zhong-chen MA
;
Yong WANG
Author Information
1. 石河子大学动物科技学院,石河子 832003;动物健康养殖国际联合研究中心,石河子 832003;绵羊健康养殖与人兽共患病防控协同创新中心,石河子 832003
- Publication Type:Journal Article
- Keywords:
monoclonal antibody;
Brucella;
double antibody sandwich ELISA method;
diagnosis
- From:
Chinese Journal of Zoonoses
2025;41(3):255-262
- CountryChina
- Language:Chinese
-
Abstract:
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 opti-mal antibody coating time and concentration,as well as the optimal blocking solution,blocking time,and yin-yang critical val-ue.The specificity of this method was verified by examination of other bacteria prone to cross-reacting with Brucella.The sen-sitivity 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 5 μg/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 sand-wich ELISA method reacted with only Brucella but not other bacteria,thus demonstrating the method's good specificity.Inac-tivated Brucella solution was still detectable after dilution to 1 × 105 CFU/mL,thus demonstrating the method's good sensitiv-ity.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 spe-cificity and sensitivity,and might provide an effective approach for the precise diagnosis and effective prevention and control of brucellosis.
