4.4. Enzyme-Linked Immunosorbent Assay (ELISA)

Indirect ELISA was performed as described previously [23]. Briefly, the viruses were diluted with 50 mM bicarbonate/carbonate coating buffer (pH 9.6) at 1000 HAU/mL (Hemagglutination unit), coated on a 96-well microtiter plate (Greiner, Germany), and incubated at 37 °C for 2 h. The plate was washed with 200 μL PBS, 0.1% Tween 20 (PBS-T, pH 7.4), and then blocked with 5% non-fat dry milk at 37 °C for 2 h. To react the antibody, first McAbs (2 μg/100 μL/well) were added to each well and positive control (anti-influenza A NP, 2 μg/100 μL/well) following which the subtype virus was added to each well and incubated at 37 °C. After 1 h, horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Abcam, Cambridge, UK) was added to each well according to the manufacture’s protocol. Stringent washing with PBS-T was performed five times to remove nonspecific binding, and 100 μL of 3,3′,5,5′-tetramethyl benzidine (Sigma-Aldrich, St. Louis, MO, USA) substrate solution was added.