Mabs were purified with Montage kit Prosep-G (Millipore) for IgG. Experimental serum samples Inactivated AI viruses (Table 1) were emulsified in ISA-70 (SEPPIC, France) adjuvant and injected intramuscularly
to the groups of three weeks old white leghorn chickens (n = 4). The booster was given twice at two-week intervals. Sera were prepared from the blood collected 10 days after 1st injection and 2nd injection. Antibody responses to the homologous strains were evaluated by HI as described below. Groups of mice (n = 4) were injected intramuscularly with different inactivated H7 AIVs (Table 1) individually emulsified in adjuvant (SEPPIC, France). The injections were repeated twice at two-week intervals. In addition, guinea pigs were immunized with inactivated H7N1 (A/Chicken/Malaysia/94). Blood was collected 14 days after selleck products the 2nd immunization.
Hemagglutination inhibition assay Hemagglutination inhibition (HI) assays were performed as described previously . Briefly, Mabs were serially diluted (2 fold) in V-bottom 96-well plates and mixed with 4 HA units of H7 virus. Plates were incubated for 30 min at room temperature, and 1% Trametinib concentration chicken RBCs were added to each well. The hemagglutination inhibition endpoint was the highest Mab dilution in which agglutination was not observed. Isolation and analysis of escape mutants The epitope recognized by Mab 62 was mapped by characterization of escape PSI-7977 order mutants as described previously . Briefly, H7N1 parental viruses were incubated with an excess of Mab for 1 h and then inoculated into 11 day old embryonated chicken eggs. The eggs were incubated at 37°C for 48 h. Virus was harvested and used for cloning in limiting dilution in embryonated chicken eggs and the escape mutants were plaque purified. The HA gene mutations were then identified by sequencing and comparison with the sequence of the parental virus. Microneutralization assay Neutralization activity of Mab against H7 strains was analyzed by microneutralization
assay as previously described . Briefly, Mab was serially two-fold diluted and incubated with 100 TCID50 of different clades of H7 strains for 1 h at room temperature and plated Montelukast Sodium in duplicate onto MDCK cells grown in a 96-well plate. The neutralizing titer was assessed as the highest Mab dilution in which no cytopathic effect was observed by light microscopy. H7 baculovirus production The recombinant baculovirus vector was generated as described previously . The full length HA gene was amplified from H7N7 (A/NL/219/03) reassortant virus in a standard PCR reaction. The amplified HA gene was inserted into the shuttle vector pFASTBacHT A (Invitrogen, San Diego, CA, USA) for expression under the white spot syndrome virus (WSSV) immediate early (ie1) promotor.