Western blot analysis of antibody responses to influenza virion proteins

An immunoblotting procedure was developed to detect antibody responses in mice and humans to influenza virion proteins. The technique was capable of detecting 1.5 micrograms of haemagglutinin (HA) on nitrocellulose strips at a 1:5000 dilution of a mouse serum with an initial haemagglutination inhibition titre of 20. The effects of the use of the blocking agent Tween-20 on virion proteins were also studied. The commonly used concentration of 0.05% (v/v) Tween-20, when included in blocking and incubation buffers, greatly reduced the amount of detectable matrix protein but caused no detectable loss of HA and neuraminidase/nucleoprotein proteins. If virion proteins were separated by polyacrylamide gel electrophoresis under reducing conditions, antibody bound to HA2 more strongly than to HA1. Under non-reducing conditions, more antibody bound to the uncleaved HA protein than to other proteins. IgG1 and IgG2a antibody responses in mice to each protein were stronger than IgG2b and IgG3 responses.