Journal: PLoS ONE
Article Title: Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin
Figure Lengend Snippet: Inhibition of H2 rHA low pH induced conformational change by mAb C179. A. 1:50 unlabeled goat anti-mouse IgG (UNLB, Southern Biotech, AL) completely masked C179 that bound to H2 rHA. Because both C179 and 1/87 are mouse mAbs, a blocking step was required for detection specificity. H2 rHA coated nickel plates were incubated with 100 ng/well of C179 for 1 hour followed by incubation with diluent only, 1:5000, 1:500, or 1:50 diluted UNLB for 1 hour. The effects of this blocking step was confirmed by ELISA using HRP-conjugated goat anti mouse IgG (SouthernBiotech, AL). B. H2 rHA was bound to a nickel-coated 96-well plate, treated with 200 ng/ml trypsin, then incubated with or without mAb C179 (100 ng/well), and treated with pH adjusted buffer in 0.2 unit increments ranging from pH 4.6–5.8 and pH 7.0. The plate was washed once with PBS followed by blocking with UNLB (1:50), H2 rHA was fixed with 0.05% glutaraldehyde/PBS and washed. To ascertain the conformation of H2 rHA, the plates were incubated with the pH specific mAb 1/87 followed by incubation with an HRP-conjugated goat anti-mouse IgG. Reactions were terminated and the OD 450 nm was measured in ELISA. C. To confirm the results in Fig 3B , the protease susceptibility assay was performed without the blocking step. H2 rHA was bound to a nickel-coated 96-well plate, cleaved with 200 ng/ml of trypsin, incubated with or without mAb C179 (100 ng/well), treated with neutral (7.0) or low pH (4.8) buffer, and subsequently treated with trypsin at 0 or 10 μg/ml. Samples including both the released digested rHA and rHA remaining on the plate, were eluted from the nickel-coated plate by adding an equal volume of 2X non-reducing SLB supplemented with 1M imidazole, and were separated by SDS-PAGE under non-reducing conditions. PAGE-separated proteins were transferred to a nitrocellulose membrane and probed with an anti H2 HA mAb (2/9). HA proteins were detected by chemiluminescence with an HRP-conjugated secondary antibody.
Article Snippet: The plate was washed with 0.05% Tween 20/PBS followed by incubation with an HRP-conjugated secondary antibody (SouthernBiotech, AL) for 1 hour.
Techniques: Inhibition, Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay, Drug Susceptibility Assay, SDS Page, Polyacrylamide Gel Electrophoresis