2.5. Neuraminidase Assay

Neuraminidase assays were performed based on the procedures in Section 5.3 of WHO standard operating procedure 025—Fluorometric Neuraminidase Inhibition Assay (WHO, 2017) [42]. This assay measures the cleavage of 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) to 4-methylumbelliferone (4-MU), catalysed by NA [43]. The assay is performed in an assay buffer containing 33.3 mM MES pH 6.0 and 4 mM CaCl₂. To a black 96-well OptiPlate (Perkin Elmer, Roskilde, Denmark) 50 μL sample containing NA was added to a well. To start the assay 50 μL 300 μM MUNANA substrate was added, resulting in a final MUNANA concentration in the assay of 150 μM. Increasing 4-MU fluorescence was measured at excitation wavelength of 355 nm and an emission wavelength of 460 nm in a POLARstar Omega Plate Reader (BMG LABTECH, Ortenburg, Germany) for 20 min. To quantify the amount of cleaved MUNANA, a standard curve containing 4-MU at concentrations of 150, 75, 37.5, 18.75, 9.375, 4.6875, 2.34, 1.17, and 0 μM was included in the same volume as activity assays, and fluorescence measured at the same wavelengths. In this assay, 1 EU (enzyme unit) represents the amount of NA required to convert 1 µmol MUNANA to 4-MU per minute.