An indirect ELISA method based on recombinant nucleoprotein(NP) for detection of antibodies to swine influenza virus(SIV)

The soluble SIV recombinant nucleoprotein(NP) was expressed by Escherichia.coli and purified through native conditions purification method. Using purified soluble nucleoprotein,an indirect ELISA for detection of anti-SIV antibodies was developed and its optimal reaction conditions were determined: coating antigen concentration was 12.5 μg/ml,at 37℃ for 1 h and 4℃ overnight,serum samples were diluted to 1∶40,Peroxidase-Labeled SPA(1∶10000) was incubated at 37 ℃ for 40 min,the substrate for showing color was incubated at 37℃ for 15 min.The ELISA assay was confirmed to have a good,specificity and sensitivity by repeatability,Cross test and competitive-inhibition test.The critical OD values for the ELISA was 0.20,which was determined by using the Two Graph-ROC functions of the CMDT software package,and it was found that the specificity and sensitivity of the ELISA were 95.83% and 91.43%,respectively.And compared with the IDEXX Test Kit,the accordant is about 94.00%,which showed no significant difference between the two assays.In addition,356 serum samples collected from different swine farms were detected by the developed assay,it showed that the positive rate of antibody against SIV was 35.40%.