Establishment And Application Of Competitive ELISA Based On O-Type Foot-and-Mouth Disease Virus-Like Particles

Foot-and-mouth disease(FMD)is one of the major causes of economic loss in cloven-hoofed animals worldwide.An outbreak of FMD severely decreases livestock productivity,also impacts on local and export trade of susceptible animals and their products.Currently,numerous categories of enzyme-linked immunosorbent assays(ELISA)have been developed and widely used to evaluate antibodies against FMD virus(FMDV).However,ELISA tests based on various formats and antigen types present different shortcomings,including difficulty in preparation,high risk of virus leakage,high false positive rate,low sensitivity and complicated operation.Our previous studies confirmed that O-type FMD virus-like particles(VLPs)resemble the authentic FMDV,enable to induce immune protection,and have high safety in production without limitations on biosafety facilities.Therefore,in this study,O-type FMD(VLPs)were used as a diagnostic antigen to establish the competitive ELISA(c ELISA)to evaluate antibodies against of O-type FMDV,to provide technical means for FMD diagnosis and prevention.In this study,O-type FMD VLPs were generated by prokaryotic expression,purification and self-assembly.O-type FMD VLPs were analyzed through SDS-PAGE,DLS,TEM and Western blot,followed that concentration of O-type FMD VLPs was determined.The hyperimmune sera were prepared by vaccination in rabbits using the O-type FMD VLPs as antigen.Ig G of the hyperimmune sera was purified and labeled by saturated ammonium sulfate precipitation,Protein A affinity chromatography and Na IO4 method.The HRP-Ig G was analyzed through SDS-PAGE and Western blot,and titer of the HRP-Ig G was determined by ELISA.The c ELISA method was established using O-type FMD VLPs as antigen.The optimal test conditions and cut-off value of the c ELISA were determined based on cross-checker titration,square matrix titration and ROC curve,and assembled the c ELISA kit.The characteristics of the c ELISA kit were analyzed through cross-reaction test,specificity test,sensitivity test,coincidence rate test,inter-batch and inter-batch reproducibility test and preservation test of at 37?.The test results are as follows:O-type FMD VLPs were successfully expressed,purified and assembled,the particle size ofO-type FMD VLPs basically aggregated between 20 nm and 50 nm,which were similar to the size of authentic FMDV particles.O-type FMD VLPs reacted specifically with positive serum against O-type FMDV,and the concentration of O-type FMD VLPs was 836.7 ?g/m L.The hyperimmune sera were successfully prepared by using O-type FMD VLPs as antigen,and HRP-Ig G was successfully prepared by purifying and labeling Ig G of the hyperimmune sera.HRP-Ig G titer was higher than 1:640 000,and HRP-Ig G reacted specifically with O-type FMD VLPs.The optimal test conditions and cut-off value of c ELISA method were set as follows:antigen coating concentration was set as 0.5 ?g/m L,dilution of serum and HRP-Ig G was set as1:4 and 1:18 000 respectively,condition of antigen coating was set at 4°C overnight or 37°C for2.5 h,blocking fluid was selected with 1% bovine serum albumin(BSA),condition of the blocking was set at 37°C for 30 min,reaction conditions of serum and HRP-Ig G were set at 37°C for 30 min,the substrate was kept away from light for 10 min,the cut-off value of PI was set as50%.Thus,samples with PI of < 50% were considered negative,and those with PI of ?50% were considered positive.The c ELISA has no across reaction,sensitivity was 96%,and specificity was100%.The coefficients of variation of the inter-batch and inter-batch reproducibility test of the c ELISA were less than 15%.The coincidence rate with the LPB-ELISA Kit,the VDpro?ELISA kit,and the Prio CHEK?ELISA kit for detecting antibodies against O-type FMDV were 97%,99%,and 97%,respectively.Preservation test showed that the c ELISA kit can keep its property stability after 7 day at 37?.This study successfully established a c ELISA method for the detection of antibodies against O-type FMDV and assembled the c ELISA kit.This detection method has advantages of sensitivity,specificity,simplicity and reproducibility,which can provide a better detection technology for FMD prevention and control and show a potential of further application.