Iga antibodies

The levels of antigen-specific serum antibodies (total IgG, IgG1 and IgG2a) and of sIgA antibodies in mucosal washes (nasal, tracheal and lung washes) were measured via indirect ELISA as described previously. The ELISA plates were coated overnight at 4 °C with H9N2 WIV and then blocked by incubation in 1% (w/v) BSA in PBS containing 0.05% Tween (PBST) for 1 h at 37 °C. Thereafter, the plates were washed 5 times with PBST (0.01 M, pH 7.4). Two-fold serial dilutions of serum or lavage fluid samples from mice were applied to the plates and incubated for 1.5 h at 37 °C. After washing 5 times with PBST (0.01 M, pH 7.4), the plates were incubated in HRP-conjugated anti-mouse IgG (total IgG, IgG1 or IgG2a) (Santa Cruz, CA) or IgA antibodies (Southern Biotech, Birmingham, AL, USA) for 1 h. The plates were washed 5 times and incubated in 3,3′, 5,5′- tetramethylbenzidine (TMB). After 20 min, the reaction was stopped using sulfuric acid, and the absorbance was measured at 450 nm using a microplate reader. The results were expressed as the ratio of OD₄₅₀ produced by the serum or mucosal wash samples relative to the produced by the negative control serum or mucosal wash sample (P/N). Samples with an OD₄₅₀ ratio higher than 2.1 were considered to be positive. The titer was expressed as the highest dilution of antibody that produced a P/N ratio ≥2.1.

Blood and BAL of mice infected with recombinant vNA-Δ or PR8 virus were collected at pre-determined time points after inoculation. Serial dilutions of serum samples were used to determine flu-specific antibodies titers by ELISA using PR8 virus as antigen. Briefly, 96-well ELISA plates (NUNC Maxisorp) were coated with 0.5 µg of detergent-disrupted purified PR8 virus per well in 0.2 M Na-carbonate buffer, pH 9.6 (overnight at 4°C). Bound antibody was detected with anti-mouse total IgG (H+L; Amersham) and IgA antibodies (SouthernBiotech) carrying the Horseradish Peroxidase and revealed by the addition of TMB peroxidase substrate (KPL) as indicated by the supplier.
Serum was used for hemagglutinin inhibition (HI) assay, and to this aim, serial dilutions (2 fold) of mice sera were incubated with 4 hemagglutinin units (HU)/25 µl of PR8 virus and 1% turkey red blood cells. HI titers were determined as the highest serum dilution able to completely inhibit hemagglutination.