Preparation Of Monoclonal Antibodies Against Foot-and-Mouth Disease Vrius And Screening Of Mimotope

Foot-and-mouth disease(FMD) is a fever,acute and high contacted contagious disease in cloven-hoofed animals infected by foot-and-mouth disease virus(FMDV).FMDV is a member of the picornavirus family.Non-enveloped FMDV has a single stranded positive-sense RNA genome.The virus exists as one of seven different serotypes:O,A,C, Asia1,SAT1,SAT2 and SAT3.However,based on serological tests,extensive antigenic variation among FMDV isolates was recognized in the seven serotypes,resulting in a large number of subtypes that have evolved within each serotype.In developing countries,the key to control FMD is rapid and accurate diagnosis and effective use of the vaccine. Therefore,the development of rapid diagnostic reagents and effective vaccine is of great practical significance to prevention and control of FMD.The monoclonal antibody(mAb) has strong specific and repeat characters,and it can be make into productions of detection and be used in research fields.A panel of three specific monoclonal antibodies(mAbs) against FMDV seretype O,named as 2B9,5F7 and 8E8,were produced by fusing between SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with purified virus preparations.These mAbs were IgG1 isotype and contained kappa light chains.The titers of antibody in supematant of the cell culture and ascites of the inoculated mice were 1:256,1:512,1:512 and 1:8×10~3,1:1.6×10~4,1:1.6×10~4, respectively.Detection using ELISA and IFA showed that these mAbs bound to FMDV strains of serotype O,but not to FMDV strains of serotype Asia1,speculating that they are specific to FMDV serotype O.Western blot analysis suggested that the mAbs 2B9 and 5F7 recognized conformational epitopes since they didn't show any specific band,whereas mAb 8E8 recognized linear epitope.The additive ELISA showed that antigen epitopes recognized by 2B9 and 5F7 were close to each other,and these epitopes were different from the epitope recognized by 8E8.Neutralization test showed that mAb 8E8 revealed a strongly neutralizing activity(1:1024).Relative affinity index(RAI) 2B9,5F7 and 8E8 were 1.5 mol/L,1.5 mol/L and 2.5 mol/L,which were indicated by thiocyanate elution measurement.Epitopes,or antigenic determinants,are antigen domains with affinity to antibody. Epitopes play a very important role in the structure and function of protein antigens. Precise epitope mapping may help design more reasonable vaccines and diagnostic reagents.In order to study antigenic property of FMDV seretype Asia1,we had produced neutralizing mAb 3E11,which recognizes a conformation-dependent epitope in seretype Asia1 FMDV.Here we investigated structural features of this epitope by screening random 12-peptide phage-displayed libraries with four rounds of biopanning.A solid-phase biopanning was used in the previous three rounds,and the last panning round used solution binding with protein G capture,and eight phage clones were positive to react specificly with mAb 3E11.The results of sequence alignment showed that five of them shared a motif GSLxxL which is mimotope recegnised by mAb 3E11.Peptide-ELISA showed that mAb 3E11 could react with dodecapeptide containing the motif,but not scrambled dodecapeptide.Site-directed mutagenesis analysis identificated critical residues in the motif required for mAb 3E11 binding.Competition tests using soluble peptides showed that this mimotope inhibit mAb 3E11 binding to viral protein.In this study,three mAbs against FMDV seretype O were produced and a mimotope in FMDV seretype Asia1 were identified which is helpful to develop novel vaccine and diagnosis reagent for control of FMD.