牛嵴病毒单抗制备及双抗体夹心ELISA方法的建立

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中图分类号：S852.659.6 文献标志码：A 文章编号：0366-6964（2025）10-5084-11

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 VPo， VP3，and VPl in a prokaryotic expression system. Three structural proteins were purified and used for immunization in BALB/c mice respectively. After three immunizations，the spleen cells of the mice were isolated and fused with SP2/O 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 optimized，and a double antibody sandwich ELISA method for specifically detecting BKV was established. Results were as follows： Two hybridoma cell lines stably secreting monoclonal antibodies were obtained，designated 2A12 and 5El1，both of which were anti-VPl protein antibodies；The results of the superimposed ELISA and antibody variable region sequence analysis indicated 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 control of BKV.

Keywords ： bovine kobuvirus； structural protein; monoclonal antibody; double antibody sandwichELISA

∗ Corresponding author：QIAO Xinyuan，E-mail：qiaoxinyuan@126.com

牛嵴病毒（bovinekobuvirus，BKV）主要感染犊牛，常与牛冠状病毒、牛轮状病毒等其它引起特牛腹泻的病毒共同感染犊牛[]，引起犊牛腹泻和食欲减退。（剩余15833字）