Detection of antibodies against avian influenza virus by anti-NS1 monoclonal antibodies-based ELISA

The non-structural protein 1 (NS1) of AIV is an important indictor Ag of AIV infection because it''s not essential for formation of the viral particle but produced during viral replication. Detection of anti-NS1 antibodies in the serum can differentiate AIV infection from vaccination. Therefore, we intended to establish a blocking ELISA which enable to detect antibodies against avian influenza virus. In this study, BALB/c mice were immunized with H6N1 AIVs for generating anti-NS1 monoclonal antibodies (αNS1 MAbs). αNS1 MAbs were screened by immunofluorescence assay (IFA) and characterized by IFA, eukaryotic expression system (EES), western blot (WB), and indirect ELISA (iELISA) to identify the specificity to NS1. On the other hand, recombinant NS1 proteins (rNS1 and brNS1) from A/chicken/Taiwan/2838V/00 (H6N1) were produced by E. coli and baculovirus-insect cell expression system respectively. After optimizing the condition, we established three ELISA systems with αNS1 MAbs and rNS1, they are rNS1 blocking ELISA, rNS1 sandwich ELISA and rNS1 indirect ELISA. The IFA test of EES were taken as the golden standard method of detecting anti-NS1 antibodies in the chicken sera. 115 chicken sera were tested by three ELISA systems. The cut-off value, sensitivity and specificity of rNS1 bELISA were 10%, 77.8% (35/45) and 74.3% (52/70). The cut-off value, sensitivity and specificity of rNS1 sELISA were 0.59, 93.3% (42/45) and 92.9% (65/70). The cut-off value, sensitivity and specificity of rNS1 iELISA were 0.76, 91.1% (41/45) and 97.1% (68/70). These results show that rNS1 sELISA and rNS1 iELISA have potential to be applied on detecting serum anti-NS1 antibodies.