African swine fever virus(ASFV)infection is a big threat to the global pig industry.Because there is no effective vaccine,rapid,low-cost,and simple diagnosis methods are necessary to detect the ASFV infection in pig herds.Nanobodies,with advantages of small molecular weight and easy genetic engineering,have been universally used as reagents for developing diagnostic kits.In this study,the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel.Then,seven nanobodies against ASFV-p30 were screened using phage display technique.Subsequently,the seven nanobodies fused horseradish peroxidase(nanobody-HRP)were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA.Using the ASFV-p30-Nb75-HRP fusion protein as a probe,a competitive ELISA(cELISA)was developed for detecting anti-ASFV antibodies in pig sera.The cut-off value of cELISA was determined to be 22.7％by testing 360 negative pig sera.The detection limit of the cELISA for positive pig sera was 1:320,and there was no cross-reaction with anti-other swine virus antibodies.The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100％.More importantly,the developed cELISA showed low cost and easy production as a commercial kit candidate.Collectively,a simple nanobody-based cELISA for detecting an-tibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.

Jiakai Zhao;Jiahong Zhu;Ying Wang;Mengting Yang;Qiang Zhang;Chong Zhang;Yuchen Nan;En-Min Zhou;Yani Sun;Qin Zhao

Department of Preventive Veterinary Medicine,College of Veterinary Medicine,Northwest A&F University;Yangling Observing and Experimental Station of National Data Center of Animal Health,Ministry of Agriculture,Yangling,712100,China;Kunming Customs Technology Center,Kunming,650228,China

中国病毒学

[1]Jiakai Zhao;Jiahong Zhu;Ying Wang;Mengting Yang;Qiang Zhang;Chong Zhang;Yuchen Nan;En-Min Zhou;Yani Sun;Qin Zhao-.A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus)[J].中国病毒学,2022(06):922-933

Nanobodies,Bactrian,Nb75,tibodies

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