Aurintricarboxylic Acid Is a Potent Inhibitor of Influenza A and B Virus Neuraminidases

Hashem, Anwar M.; Flaman, Anathea S.; Farnsworth, Aaron; Brown, Earl G.; Van Domselaar, Gary; He, Runtao; Li, Xuguang

Extracellular influenza A antigens were detected in cell culture supernatants using a commercial ELISA kit (Takara Bio Inc., Otsu, Shiga, Japan). Culture media were removed 48 h after cells were infected with virus and treated with ATA, AH or both. Supernatants were clarified by centrifugation at 50006g for 5 min. Samples, positive control and standards diluted in diluent/ lysis buffer were added to individual wells (100 ml/well), and incubated at 37uC for 1 h with immobilized monoclonal antibody directed against influenza A nucleoprotein (NP), then were washed three times with PBS containing 0.1% Tween-20. Samples were incubated with biotinylated rabbit polyclonal anti-influenza virus antibodies for 1 h at 37uC. Following three washes, streptavidin-ophenylenediamine dihydrochloride conjugates were added to wells and the plate incubated for 30 min at 37uC. Wells were washed four times and incubated with the substrate solution consisting of hydrogen peroxide and tetramethylbenzidine at room temperature for 15 min. The reaction was stopped by the addition of 1 N H 2 SO 4 prior to absorbance reading at 450 nm using a Synergy TM 2 Multi-Mode Microplate Reader. Viral abundance was calculated as hemagglutination (HA) units from a standard curve using a positive control with known HA content. One HA unit is equal to the quantity of virus required to completely aggregate erythrocytes in an HA assay (100 ml of 0.25% v/v). The commercial ELISA kit has certain limitations as it employs the anti-nucleoprotein as the capture antibodies coated on the plates and anti-influenza viral proteins (total influenza viral proteins including hemagglutinins) as the detecting antibodies in solution for sandwich ELISA. Because hemagglutinins vary from strain to strain in terms of amino acid sequences or the ratio of viral proteins, one cannot directly compare the HA units between two different strains. Therefore, we present the results as percentage of HA units from treated infected samples relative to that from untreated infected control for the same strain.