2.7. Neuraminidase inhibition (NAI) assay

The neuraminidase inhibition activity was determined using the NA-Fluor Influenza Neuraminidase Assay kit (Life technologies) according to the manufactures protocol, where serum and virus was allowed to incubate 30 min at 37 °C before adding the NA-Fluor™ Substrate. The two virus strains, A/California/07/09 (H1N1)pdm09 and A/swine/DK/10496/2008 (H1N1), were used at 1:10 dilution, determined to give a relative fluorescence unit (RFU) within the linear range. Serum from day −36, day 0 and day 13 pc were tested at 4-fold dilutions starting at 1:10 up to 1:1040. Non-linear regression, with log-transformed serum dilutions as the independent variable, was performed to calculate the serum dilution that would inhibit 50% NA activity (IC₅₀) (Graph-Pad Prism software).