河南农业大学学报2025，Vol.59Issue(6)：1028-1036,9.DOI:10.16445/j.cnki.1000-2340.20250214.001
高效区分胸膜肺炎放线杆菌自然感染和疫苗免疫猪的ELISA检测方法构建
Establishment of ELISA assay to efficiently distinguish natural infection of Actinobacillus pleuropneumoniae from vaccine-immunized pigs
摘要
Abstract
[Objective]To establish a method capable of distinguishing between Actinobacterium pleuro-pneumoniae(APP)natural infection and vaccine-immunized pigs,thus facilitating an accurate assess-ment of the prevalence of APP natural infection.[Method]In this study,soluble protein APP ApxⅣwas expressed in a low temperature induction way and used as the coated antigen.Reaction conditions were optimized through matrix titration.An indirect ELISA antibody detection method for APP ApxⅣwas initially established,and its specificity,sensitivity,reproducibility,and compliance rate of the commercialized reagent kit were subsequently determined.[Result]The results of screening and optimi-zation of reaction conditions showed that the optimal coating concentration of antigen was 0.75 mg·L-1 and the optimal coating condition was 37℃for 1 h+4℃for 14 h.The best sealing solution is trehalose with a mass fraction equal to 2%,and the best sealing condition was 37℃for 1.5 h.The optimal dilu-tion ratio of serum was 1∶50,and the optimal incubation condition was 37℃for 1 h.The optimum dilution ratio was 1∶10 000 and the optimum incubation condition was 37℃for 30 min.The optimal color development condition was 25℃for 10 min.The specific evaluation results showed that the method had no cross-reaction with the positive sera of bacteria such as Escherichia coli,Streptococcus suis,and Haemophilus parahaemolyticus,with the positive sera of porcine circovirus type 2,classical swine fever virus,porcine reproductive and respiratory syndrome virus,pseudorabies virus,and the positive sera of pigs immunized with APP vaccine.Sensitivity evaluation results showed that APP posi-tive serum was still positive after 200 times dilution.The results of repeatability evaluation showed that the coefficient of variation of intra-batch and inter-batch repeatability tests were 1.31%-6.27%and 0.73%-4.05%,respectively.The coincidence rate evaluation results showed that 200 clinical serum samples were randomly selected for detection,and the overall coincidence rate with commercial kits reached 86.50%.[Conclusion]In summary,the indirect ELISA antibody detection method estab-lished based on APP ApxⅣ protein in this study exhibited high specificity,sensitivity,good repeat-ability,and high coincidence rate,making it suitable for distinguishing between APP naturally infected pigs and vaccine-immune pigs.关键词
胸膜肺炎放线杆菌/ApxⅣ蛋白/间接ELISA/抗体检测/自然感染/疫苗免疫Key words
Actinobacterium pleuropneumoniae(APP)/ApxⅣ protein/indirect ELISA/antibody testing/natural infection/vaccine immunity分类
农业科技引用本文复制引用
李建铭,侯晋辉,徐广鑫,高俊龙,史郑婷,赵睿,袁晋..高效区分胸膜肺炎放线杆菌自然感染和疫苗免疫猪的ELISA检测方法构建[J].河南农业大学学报,2025,59(6):1028-1036,9.基金项目
国家自然科学基金青年基金项目(32402924) （32402924）
国家重点研发计划青年科学家项目(2023YFD1801800) （2023YFD1801800）
河南省重点研发专项(231111113100) （231111113100）
