Fig. 7: NASPA is compatible with a bulky synthetic NA inhibitor.
a Diagram showing the novel chemoenzymatically synthesized biotinylated NA inhibitor (NAi: 4-guanidino-Neu5Acα2–3 Galβ1–4GlcNAc–βProNH-PEG4-Biotin) that was combined with streptavidin (SA) for use in NASPA. b NASPA schematic showing how the synthetic NA inhibitor bound to streptavidin (NAi + SA) can detect steric (purple) or active site (red) NAI antibodies. c IC50 values are displayed for the NAi, NAi + SA, NAi-MAb (FNI-9), and zanamivir after the indicated incubation times with TX100-treated H1N1/BR18 virus. d Streptavidin binding to the NAi is required for NASPA. NA activity data is displayed from NASPA performed using TX100-treated H1N1/BR18 virus with the NAi, NAi + SA, or SA and the indicated ferret antisera. Assays were performed in duplicate using amounts approximating the IC65 and are displayed as the mean ± SD. Gray region indicates the mean (dashed line) ± 3 SDs (dotted line) of the mock control wells. e IC50 values of the NAi, NAi + SA, and the NAi-MAbs (FNI-9 and FNI-9 R27D) for the indicated H3N2 vaccine strains are displayed. Each assay was run twice using either a 30 min incubation (NAi and NAi + SA) or 3 h incubation (NAi-MAbs) at 37 °C. f Representative NA activity data set is shown from NASPA that was performed using TX100-treated H3N2/Dar21 virus with the NAi + SA mixture and the indicated purified N2 MAbs. Purified N2 MAbs were diluted as indicated from a starting concentration of ~20 μg/ml. The assay was performed using NAi + SA amounts approximating the IC80. Gray region indicates the mean (dashed line) ± 3 SDs (dotted line) of the mock control wells.
