Differentiation of Antibodies Against H5 and H6 Influenza A Viruses by Blocking ELISA Using Anti-HA Monoclonal Antibodies
Date Issued
2015
Date
2015
Author(s)
Lee, Kuang-Hsiu
Abstract
H6N1 and H5N2 AIV have been circulated in Taiwan since 1972 and 2003 respectively. H6N1 strains are low pathogenic avian influenza viruses (LPAIV). However, some of the H5N2 strains isolated currently become highly pathogenic avian influenza viruses (HPAIV). Developing an effective detection tool to differentiate whether the serum antibodies are against H5 or H6 subtypes is essential for AIV surveillance and management. In this study, BALB/c mice were immunized with inactivated H5N2 or H6N1 AIVs. After evaluated the antibody titer of the mice sera by immunofluorescent assay (IFA), we fused mice splenocytes with myelomas to generate hybridomas. Anti-HA Monoclonal antibodies (αHA MAbs) were screened by IFA, hemagglutination inhibition (HI) assay, western blotting (WB) and indirect ELISA (iELISA) to confirm the specificity to HA and no interaction between H5 and H6 AIVs. On the other hand, we established two sandwich blocking ELISAs (1209/H5N2 bELISA；rHAΔTM/H6N1 bELISA) with αHA MAbs and formalin-inactivated 1209/H5N2 viruses or recombinant HA protein of A/chicken/Taiwan/2838V/00 (H6N1) by baculovirus-insect cell expression system. Hemagglutination inhibition test was taken as the golden standard method of detecting anti-H5 or anti-H6 antibodies in the chicken sera. 702 chicken sera were tested by 1209/H5N2 bELISA and rHAΔTM/H6N1 bELISA. The cut-off value of 1209/H5N2 bELISA was 27%. The sensitivity and specificity were 93.31% (265/284) and 90.91% (380/418). In addition, the cut-off value of rHAΔTM/H6N1 bELISA was 28%. The sensitivity and specificity were 95.11% (214/225) and 96.44% (460/477). Also, both of the two bELISAs can detect the antibodies in chicken sera with low HI titer. The results show that these bELISAs could be used to differentiate H5 and H6 infection in the field.
Subjects
avian influenza virus (AIV)
hemagglutinin (HA)
monoclonal antibody (MAb)
baculovirus
protein expression
blocking ELISA (bELISA)
Type
thesis
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