Development Of Nanobodies Against Goose Astrovirus And Establishment Of Competitive ELISA

Goose Astrovirus(GAstV)is one of the main pathogens causing gout disease in goslings.At present,there is a lack of efficient epidemiological investigation methods and commercially available GAstV serological testing kits,which often leads to delayed clinical treatment and expanding economic losses.The purpose of this study is to prepare GAstV nanobody(Nb)for the establishment of competitive ELISA detection methods,providing a reliable technical means for monitoring the health status of geese and preventing the spread of gout disease in goslings.In this study,GAstV was used as the research object to conduct the following experiments:(1)GAstV was used to immunize alpacas,and the IgG antibody titer in alpaca serum was detected by indirect ELISA.(2)Total RNA from alpaca peripheral blood lymphocytes(PBL)was extracted and a phage display library of GAstV Nb was constructed using Nest PCR combined with phage display technology.(3)Using GAstV as the target antigen,a recombinant phage Nb positive clone was obtained through enrichment and elutriation,and cloned into pcDNA3.1-Fc eukaryotic expression vector for sequencing analysis and expression.(4)Using GAstV ORF2 protein as the target antigen,the specificity,binding activity,and affinity of Nb were detected using indirect ELISA and Western blotting tests.(5)A competitive ELISA method was established by selecting one strain of Nb with the best biological activity.The optimal antigen coating amount and the optimal Nb dilution were determined by chessboard method,and the ELISA reaction conditions were optimized.(6)Evaluate the specificity,sensitivity,and repeatability of competitive ELISA methods.The results showed that:(1)The titer of IgG antibody against GAstV in alpaca serum reached 1:64000.(2)Obtain a storage capacity of 3.8×10~8PFU/mL GAstV Nb phage display library.(3)After three rounds of enrichment and elutriation,39 recombinant phage Nb positive clones were initially obtained,and only 10 of them were successfully expressed in HEK-293-F cells.(4)ELISA and Western blotting confirmed that 8 strains of Nb specifically reacted with ORF2 protein were obtained,and one strain had the best biological activity.(5)The optimal antigen coating amount for this method is 4μg/mL,the optimal dilution of Nb is 1:400;The optimal serum dilution is 1:20;The best sealing condition is 5%skimmed milk,sealed at 37℃for 60 minutes;The optimal competition time is 60 min and the optimal dilution of enzyme labeled secondary antibody is 1:6000.(6)This method does not cross react with the positive sera of avian influenza virus(AIV),goose plague(GPV),duck hepatitis virus(DHV),and muscovy duck reovirus(MDRV);Sensitivity is 1:320;The coefficient of variation within and between plates is≤10%.The results showed that in this study,a phage display library of GAstV Nb was successfully constructed,and a strain of Nb with the best biological activity was obtained;A competitive ELISA method for detecting antibodies to GAstV based on Nb has been established.This method has good specificity,sensitivity,and repeatability,laying a foundation for the monitoring and epidemiological investigation of antibodies to GAstV.