Development And Preliminary Application Of A Sandwich ELISA For Capture Of Emerging Virus Aichivirus D Antigen Based On Monoclonal Antibody

Kobuvirus（Ko V）belongs to the family members of the genus Picornavirus,which causes emerging infectious diseases characterized by diarrhea and poses a risk to human and animal health.At present,there is a lack of research regarding the diagnosis,prevention,and treatment of the disease and the viral pathogenic mechanism.In this study,the primers for the Aichivirus L gene were designed based on the nucleotide sequence of the first domestic Aichivirus D（Ai V-D）revealed in our laboratory from cattle,and used to amplify the Leader protein gene fragment using RT-PCR.The PCR-amplified fragment was cloned into the prokaryotic expression vector to construct the recombinant plasmid p GEX-4T-1-Ko V-L.After the transformation in BL21,the recombinant protein was expressed by induction with IPTG in the E.coli expression system.The purified recombinant protein used as an immunogen was emulsified with Freund’s adjuvant and injected into healthy experimental animals to produce the corresponding antibodies.Splenocytes collected from mice with higher serum titers were fused with Sp2/0 cells.Three hybridoma cell clones that stably secrete anti-Leader protein monoclonal antibodies（m Abs）were obtained through screening and subcloning four times.Two clones designated as 3F3and 4G10 and one clone named 4F5 were determined as Ig G1 and Ig G2a subclasses,separately.The specificity and stability assay showed that all three m Abs showed specificity to Ai V-D antigens with good stability.Determinations of the antigenic epitopes for the m Abs were performed by Western blot using a segment truncation approach followed by peptide scanning.The antigenic epitopes recognized by m Abs3F3,4F5,and 4G10 were ⁹⁷RICAKTLPGPWHS¹⁰⁷,¹⁰⁵GPWHSKLTKAERIF¹¹⁸,¹³ERPFHYSLPKPSS²⁵,respectively,in the Leader protein.Sandwich ELISA methods for the detection of Ai V-D antigens were initially established by combining individual monoclonal antibodies of three with rabbit-derived polyclonal antibodies.The detection effect was best when mouse monoclonal antibody 4G10 was used as the capture antibody and polyclonal antibody was used as the detection antibody.The optimized conditions for sandwich ELISA methods were as follows:the capture antibody coating amount was 200 ng/well,the optimal dilution of detection antibody was 1:4000,and the enzyme-linked secondary antibody dilution concentration was 1:5000.The specimen was judged positive by the criteria when the OD₄₉₀ was≥0.191.Specificity test showed the assay were positive for kobuvirus and negative for bovine viral diarrhea virus and bovine enterovirus.The sensitivity assay demonstrated that after 160-fold dilution of positive feces,the test result was still positive by the sandwich ELISA method,which was consistent with the RT-PCR method,indicating that the method had high sensitivity.The intra-plate coefficients of variation ranged from 3.4%to 6.2%,and the interplate coefficients of variation ranged from 2.7%to 7.5%,indicating that the sandwich ELISA method was reproducible well.Detection of 457 cattle feces samples from certain regions of Xinjiang and Jilin provinces using the established ELISA method showed that Ai V-D infections existed in the above regions with the infection rate of Ai V-D ranging from 8.24%to 32.50%.In summary,we have generated and obtained three m Abs against Ai V-D for the first time and characterized the antigenic epitopes,which will provide the basis for the studies on diagnostic method development,pathogenic mechanisms,and the function of the Leader protein for the emerging virus.Using the prepared antibody,we established a specific and sensitive sandwich ELISA method for detecting the Ai V-D antigen and revealed different degrees of Ai V-D infection in certain regions of Xinjiang and Jilin provinces.These results provide an effective method for the diagnosis of Ko V infection and epidemiological theoretical basis for the prevention and control of Ai V-D infection in the future.