畜牧兽医学报2025，Vol.56Issue(10)：5084-5094,11.DOI:10.11843/j.issn.0366-6964.2025.10.028
牛嵴病毒单抗制备及双抗体夹心ELISA方法的建立
Preparation of Monoclonal Antibody against Bovine Kobuvirus and Establishment of Double Antibody Sandwich ELISA Method
摘要
Abstract
This study aimed to prepare monoclonal antibody against bovine kobuvirus(BKV)and establish a double antibody sandwich ELISA method for detection.The pET-30a(+)vector was utilized to express the BKV structural proteins VP0,VP3,and VP1 in a prokaryotic expression system.Three structural proteins were purified and used for immunization in BALB/c mice re-spectively.After three immunizations,the spleen cells of the mice were isolated and fused with SP2/0 cells to prepare hybridoma cells.Then the hybridoma cells were screened by the indirect ELISA method,the prepared monoclonal antibodies were used as capture antibody and detection antibody respectively,the conditions such as antibody concentration and incubation time were op-timized,and a double antibody sandwich ELISA method for specifically detecting BKV was es-tablished.Results were as follows:Two hybridoma cell lines stably secreting monoclonal anti-bodies were obtained,designated 2A12 and 5E11,both of which were anti-VP1 protein antibod-ies;The results of the superimposed ELISA and antibody variable region sequence analysis indi-cated that they recognized different antigen sites.The results of monoclonal antibody specificity detection showed that the two monoclonal antibodies were specifically bound to BKV and were not bound to other bovine diarrhea viruses.One hundred and seven clinical samples were detected by RT-PCR method and the double antibody sandwich ELISA method established in this study.The results demonstrated that the positive coincidence rate between the two methods was 92.2％,the negative coincidence rate was 95.3％,and the overall coincidence rate was 93.5％.In conclusion,two monoclonal antibodies against BKV were prepared in this study,and a double antibody sandwich ELISA method for BKV was established.This method has good specificity,repeatability and sensitivity,which lays a foundation for the rapid diagnosis,prevention and con-trol of BKV.关键词
牛嵴病毒/结构蛋白/单克隆抗体/双抗体夹心ELISAKey words
bovine kobuvirus/structural protein/monoclonal antibody/double antibody sandwich ELISA分类
畜牧业引用本文复制引用
曹恒志,马芊玥,姜艳平,崔文,李佳璇,乔薪瑗..牛嵴病毒单抗制备及双抗体夹心ELISA方法的建立[J].畜牧兽医学报,2025,56(10):5084-5094,11.基金项目
黑龙江省重点研发计划项目(GA21B004) （GA21B004）
