Quantifying Influenza A/California/07/2009 Neuraminidase Activity

To determine A/California/07/2009 NA activity, high-binding flat-bottom 96-well plates (Greiner Bio-One, Monroe, NC, USA) were coated with 100 μL of 25  μg/mL fetuin (Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. Influenza virus A/California/07/2009 was diluted in sample diluent (PBS, 1% BSA, 0.5% Tween-20) to an initial dilution of 1:10 and then serially diluted two-fold for 11 dilutions. A negative-control column was included containing 100 μL of only the sample diluent. Fetuin plates were washed three times with PBS-T (PBS  +  0.05% Tween 20). Next, 50 μL of serially diluted virus was added to the fetuin-coated plate containing 50 μL of the sample diluent in duplicate. Plates were incubated for 18 h at 37 °C and 5% CO₂. After incubation, plates were washed six times in PBS-T, and 100 μL of peanut agglutinin-HRPO (Sigma-Aldrich, St. Louis, MO, USA) diluted 1000-fold in conjugate diluent (PBS, 1% BSA) was added. Plates were incubated at RT for 2 h. Plates were washed three times in PBS-T, and 100 μL (500 μg/mL) of o-phenylenediamine dihydrochloride (OPD) (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-citrate buffer (Sigma-Aldrich, St. Louis, MO, USA) was added to the plates. Plates were incubated in the dark for 10 min at RT. The reaction was stopped with 100 μL of 1 N sulfuric acid. The absorbance was read at 490 nm on a BioTek plate reader (Agilent, Santa Clara, CA, USA). The dilution of the virus needed to achieve 90 to 95% NA activity was determined and used for subsequent NA inhibition ELLAs.