Preparation Of Rabbit Anti-type O Foot-and-mouth Disease VLPs IgG-HRP And Establishment Of Its Blocking ELISA Method

Foot-and-mouth disease is a highly contagious disease that invades cloven-hoofed animals and is the world’s most economically destructive livestock disease.Diseased animals have a large amount of blisters on the mouth,nose,nipples and hooves,which greatly reduces productivity.At present,the control of the disease is mainly combined with diagnostic testing and vaccine immunization.Enzyme-linked immunosorbent assay is sensitive,specific,safe,and easy to operate,and is widely used in the diagnosis of foot-and-mouth disease.Traditional ELISA method uses whole virus as coating antigen and there is a risk of virus spread during production.The structural proteins of foot-and-mouth disease virus were expressed in vitro and assembled into virus-like particles,using VLPs as antigen of ELISA method is safer and more reliable.This project carried out the preparation of polyclonal antibodies against FMD serotype O VLPs and the establishment of FMD serotype O blocking ELISA assays,laying the foundation for the development of subsequent detection kits.This research will be recombinant plasmid pSMK-VP0VP3 and pSMK-VP1 co-transformation cells BL21(DE3)-RIL expression strains,induced with IPTG the expressed fusion protein,purified with Ni-affinity chromatography method,Enzyme digestion and assembly into FMD serotype O VLPs,After immunization of rabbits with FMD serotype O VLPs and adjuvant,rabbit anti-FMD serotype O VLPs hyperimmune serum was prepared.Rabbit anti-FMD serotype O VLPs polyclonal antibodies were prepared by ammonium sulphate precipitation and affinity chromatography purification.An improved sodium periodate method was used to prepare rabbit anti-FMD serotype O VLPs enzyme antibody,and a method to detect FMD serotype O blocking ELISA was preliminarily established.The FMD serotype O VLPs were successfully expressed in this study.The protein concentration of the purified FMD serotype O VLPs was 0.687 mg/mL.Polyclonal antibodies against rabbit FMD serotype O VLPs were successfully prepared.The results of SDS-PAGE showed that the antibody heavy chain and light chain were 50 ku and 25 ku in size,respectively.By indirect ELISA detection,the titer reached 1:512000.It was demonstrated that high-purity rabbit anti-FMD serotype O VLPs enzyme-labeled antibodies can be prepared for ELISA detection of FMD serotype O.An FMD serotype O blocking ELISA method was preliminarily established,and the optimal concentration of VLPs antigen was 0.50 g /m L,the optimal dilution of enzyme-labeled antibody is 1:18000,the optimal closure time is 45 min,the optimal substrate coloring time is 15 min.The specificity test proved that the FMD serotype O positive serum was positive in colorable reaction,while others were all negative,indicating that the method has good specificity.In this study,rabbit anti-FMD serotype O VLPs polyclonal antibodies were successfully prepared,and a preliminary detection FMD serotype O blocking ELISA method was established.This can be used as a basis for the subsequent development of FMD test kits,it can pave the way for the subsequent development of FMD detection kit,and also lay the foundation for further research on FMD prevention,control and vaccine development.