Development Of The Chemiluminiscence Diagnositic Kit For Rapidly Detection Of The Antibodies To Non-structure Proteins Of Foot-and-mouth Diseases

Foot-and-mouth disease(FMD)is a severely infectious illness caused by foot-and-mouth disease virus(FMDV)for cloven-hoofed animals such as pigs,bovines and sheeps.In clinical diagnosis,detection of FMDV infection in the early infection phase at a rapid and accurate way is crucially important to control the outbreak of FMD.Chemiluminescence immunoassay(CLIA)as a new detection technique has showed perfect performances for diagnosis of some human diseases such as AIDS,tumor and hepatitis due to the merits of ultra sensitivity,high signal to noise ratio and wider linear range.However,there has been not a report about CLIA for diagnosis of FMD.In this research,two full-length non-structure proteins 3ABC,2C and five recombinant proteins that contain single epitope located in 3A and 3B respectively named by 3Aepit1,3Aepit2,3Bepit1,3Bepit2,3Bepit3 were expressed in bacteria.Among of them,the recombinant 3ABC and 2C proteins were expressed in the form of inclusion body,while the five epitope-based proteins were expressed in the soluble form.All of these proteins were purified using affinity chromatography.Western-blotting showed that all the proteins had good reactions with FMDV positive serum samples.And indirect ELISA showed that the proteins of 3ABC,2C,3Bepit1 and 3Bepit2 had a strong discrimination power between FMDV positive sera and negative sera.The 3ABC-CLIA and 3ABC-2C-CLIA for diagnosis of FMDV in pigs were developed by using the single 3ABC protein and both the 3ABC and 2C proteins as the coating antigens respectively.The diagnosis sensitivity(100%)and diagnosis specificity(96.47%)of 3ABC-2C-CLIA were better than the diagnosis sensitivity(96.73%)and diagnosis specificity(95.87)of 3ABC-CLIA by detection of 712 of known status FMD sera.By detecting the 962 sera from the field,the concordance rate of3ABC-2C-CLIA with the home-made ELISA kit was 96.15%and with the ProCHECK~@NSP ELISA kit was 97.81%.By surveillance of the serum samples that have infected by FMDV,the 3ABC-2C-CLIA can make an accurate detection during 1 days post infection(DPI)-4DPI,compared with 6DPI-9DPI of the commercial ELISA kits in most conditions.In addtion,the detection time of the CLIA was 15 min and it also had a good repeatability and stability.Based on above,a 3ABC-2C-CLIA for diagnosis of FMD in bovines has been developed.The diagnosis sensitivity and diagnosis specificity of the method were 96.86%and 93.38%respectively by detecting 460 known statuses bovine sera.After detection of 810 sera from the field,it showed that the CLIA method has a concordance rate at 91.85%and at 92.83%with home-made ELISA kit and ProCHECK~@NSP ELISA kit correspondingly.In addition,the detection time of this method was 25min.In conclusion,the rapid,sensitive and accurate CLIA kit for detection of antibodies to both 3ABC and 2C has been developed firstly.