The aim of this study is to identify the linear Fc-binding epitope for IgG1 on bovine IgG Fc receptor Ⅱ(boFcγRⅡ)to understand the molecular basis of IgG-Fcγ interaction.The boFcγRⅡ molecules were expressed on cell surface of the boFcγR Ⅱ-transfected COS-7 cells.The extracel-lular domain of boFcγRⅡ was expressed in NS0 cells,and the boFcγRⅡ recombinant protein was purified from ascites by Ni-chelation chromatography.Peptides derived from the membrane-distal extracellular domain(EC2)of boFcγR Ⅱ were synthesized and conjugated to a carrier protein of IgG-free bovine serum albumin(BSA).Binding of bovine IgG1 to the different peptides was tested by dot-blot assay,and the IgG-binding peptide was further modified by truncation and mutation to identify the Fc-binding epitope as well as its key amino acids for Fc-binding.The inhibition effect of the Fc-binding peptide was determined by competitive ELISA and Fc-rosetting inhibition assay,re-spectively.The results showed that boFcγR Ⅱ molecules were stably expressed on surface of the transfected COS-7 cells,which showed about 90％rosetting with IgG1-RBCs.The soluble boFcγRⅡ recombinant protein specifically bound to bovine IgG1.The minimal effective peptide of 122FYQDRKSKIF131 of boFcγRⅡ was able to bind bovine IgG1 specifically,suggesting it repre-sents a linear Fc-binding epitope located in the putative C-C'loop of the EC2 domain on the recep-tor.The Ala-substitution of Phe122,Tyr123,Arg126,Lys127,Ser128,Lys129 or Phe131 within the linear epitope led to a complete loss of its IgG1-binding capability,indicating those residues are critical for IgG1-binding on boFcγRⅡ.The Fc-binding peptide inhibited bovine IgG1 binding to the soluble recombinant protein of boFcγRⅡ with IC50 of 20.05 μmol/L,and inhibited the rosette formation of bovine IgG1-sensitized RBCs on the boFcγRⅡ transfected cells with IC50 of 80.15 μmol/L.The re-sults indicate that boFcγRⅡ possesses the linear epitope for Fc-binding,and the Fc-binding pep-tide showed well capability of regulating boFcγR Ⅱ-IgG1 interaction on cell surface,thereby provi-ding a research foundation for understanding the IgG-Fcγ interaction.

Objective:To establish a highly sensitive indirect competitive ELISA(icELISA)method for detecting ribavirin in chicken.Methods:Based on the obtained monoclonal antibodies against ribavirin,a chessboard test was employed to determine the optimal working concentration of artificial antigen and antibody,and then established an icELISA method.Furthermore,performance of the detection method was evaluated.Results:The established icELISA method has a linear range of 0.44～32.71 ng/ml,IC50 of which was 3.78 ng/ml,and the limit of detection(LOD)was 0.20 ng/ml.Except for specific reaction with ribavirin,there were no cross reactions with other antiviral drugs.Recovery rate of sample spiking was between 91.60%and 100.76%,and coefficient of variation was between 7.29%and 10.63%.Conclusion:A highly sensitive and specific icELISA method for detection of ribavirin has been estab-lished,which can be used to determine the residue of ribavirin in chicken.

Objective:To establish a highly sensitive indirect competitive ELISA(icELISA)method for detecting ribavirin in chicken.Methods:Based on the obtained monoclonal antibodies against ribavirin,a chessboard test was employed to determine the optimal working concentration of artificial antigen and antibody,and then established an icELISA method.Furthermore,performance of the detection method was evaluated.Results:The established icELISA method has a linear range of 0.44～32.71 ng/ml,IC50 of which was 3.78 ng/ml,and the limit of detection(LOD)was 0.20 ng/ml.Except for specific reaction with ribavirin,there were no cross reactions with other antiviral drugs.Recovery rate of sample spiking was between 91.60%and 100.76%,and coefficient of variation was between 7.29%and 10.63%.Conclusion:A highly sensitive and specific icELISA method for detection of ribavirin has been estab-lished,which can be used to determine the residue of ribavirin in chicken.

The aim of this study is to identify the linear Fc-binding epitope for IgG1 on bovine IgG Fc receptor Ⅱ(boFcγRⅡ)to understand the molecular basis of IgG-Fcγ interaction.The boFcγRⅡ molecules were expressed on cell surface of the boFcγR Ⅱ-transfected COS-7 cells.The extracel-lular domain of boFcγRⅡ was expressed in NS0 cells,and the boFcγRⅡ recombinant protein was purified from ascites by Ni-chelation chromatography.Peptides derived from the membrane-distal extracellular domain(EC2)of boFcγR Ⅱ were synthesized and conjugated to a carrier protein of IgG-free bovine serum albumin(BSA).Binding of bovine IgG1 to the different peptides was tested by dot-blot assay,and the IgG-binding peptide was further modified by truncation and mutation to identify the Fc-binding epitope as well as its key amino acids for Fc-binding.The inhibition effect of the Fc-binding peptide was determined by competitive ELISA and Fc-rosetting inhibition assay,re-spectively.The results showed that boFcγR Ⅱ molecules were stably expressed on surface of the transfected COS-7 cells,which showed about 90％rosetting with IgG1-RBCs.The soluble boFcγRⅡ recombinant protein specifically bound to bovine IgG1.The minimal effective peptide of 122FYQDRKSKIF131 of boFcγRⅡ was able to bind bovine IgG1 specifically,suggesting it repre-sents a linear Fc-binding epitope located in the putative C-C'loop of the EC2 domain on the recep-tor.The Ala-substitution of Phe122,Tyr123,Arg126,Lys127,Ser128,Lys129 or Phe131 within the linear epitope led to a complete loss of its IgG1-binding capability,indicating those residues are critical for IgG1-binding on boFcγRⅡ.The Fc-binding peptide inhibited bovine IgG1 binding to the soluble recombinant protein of boFcγRⅡ with IC50 of 20.05 μmol/L,and inhibited the rosette formation of bovine IgG1-sensitized RBCs on the boFcγRⅡ transfected cells with IC50 of 80.15 μmol/L.The re-sults indicate that boFcγRⅡ possesses the linear epitope for Fc-binding,and the Fc-binding pep-tide showed well capability of regulating boFcγR Ⅱ-IgG1 interaction on cell surface,thereby provi-ding a research foundation for understanding the IgG-Fcγ interaction.

This study aims to obtain highly sensitive and specific monoclonal antibodies to ribavirin.Ribavirin was coupled with carrier proteins to synthesize artificial complete antigen by glutaralde-hyde method.UV scanning,gel electrophoresis and animal immunization were employed to identify quality of the synthesized artificial complete antigens.The monoclonal antibody was prepared by cell fusion,positive hybridoma screening and induction of ascites in vivo,and the immunological characteristics of monoclonal antibodies were identified.The results showed that RBV was coupled to carrier proteins,and the artificial antigens were prepared successfully.One cell line that can stably secrete monoclonal antibodies against ribavirin was obtained,and the titers of the mono-clonal antibodies secreted by cell line was above 1∶512 000,with IC50 values of 4.74 μg/L.Fur-thermore,there were no cross-reactivity with other antiviral drugs such as amantadine and carrier proteins.In this study,monoclonal antibodies of ribavirin with high sensitivity and specificity was synthesized successfully,which lays a solid foundation for the establishment of immunoassay methods of ribavirin.