Heat-inactivated immune sera from chicken blood samples were tested for hemagglutination inhibition (HI) activity with 1% chicken red blood cells and 4 hemagglutinating units (HAU) of H5N1 (A/Viet Nam/1194/04 NIBRG-14). In addition, the chicken sera were tested for HI activity using 8 HAU (67 ng) of recombinant soluble trimeric HA protein. To this end the recombinant proteins were precomplexed with the anti-Strep-tag antibody as described above, mixed with limiting dilutions of the chicken sera and incubated with 0.5% chicken red blood cells. Red button formation was scored as evidence of hemagglutination. Antibody titers were expressed as the reciprocal of the highest serum dilution showing HI. Immune sera prepared from mouse blood samples were treated with VCNA, heat-inactivated at 56uC for 30 min and tested by HI assay using 8 HAU of sH5 3 as described above.