Fig 3. Effect of different stress treatments and group 2 stalk on assay performance.

Detergent treatment is necessary for the assay for optimal access antigenic sites on the HA trimers. In addition, conditions during the vaccine production process also require detergent and high salt concentrations. To test if these conditions influence the stalk conformation we treated recombinantly expressed HA protein (which is used as standard in the assay) with (A) 0.05% zwittergent and (B) 2% Triton X-100+340mM NaCl and compared the readout to the signal obtained with untreated HA. In addition, we tested the influence of different treatments on the concentrations of properly folded HA in purified viral preparations. We exposed the viral preparations to (C) low pH (pH 4 for 60 minutes) or (D) heat (100°C for 10 minutes) treatment. Post treatment HA concentrations were normalized to pre-treatment concentrations. (E) A capture ELISA was performed to measure the HA content of a group 2 cHA expressing virus, cH4/3N2, with a recombinant cH4/3 protein as standard.