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. 2022 Aug 28;23(17):9765. doi: 10.3390/ijms23179765

Figure 4.

Figure 4

238D2 binds A/SOLOMON ISLANDS/3/2006 hemagglutin. (A) ELISA on H1N1. H1N1 was coated on MaxiSorp plates and incubated with the myc-tagged Nb at 1 µM. Nb was revealed with an HRP-coupled anti-c-myc antibody. Here are shown luminescence signals in mean +/− sem obtained from 5 independent experiments. Identical letters show no statistical difference, and different letters show statistical differences at p < 0.05. (B) Nb-directed immuno-precipitation of H1N1. Magnetic beads were coupled to an anti-c-myc antibody and exposed to a mixture of H1N1 and the biotinylated c-myc-tagged 238D2 (AviTag-238D2, lane 1), or the biotinylated c-myc-tagged 3SM5 (AviTag-3SM5, lane 2) or to H1N1 alone (lane 3). In lanes 4, 5, and 6 are shown the inputs. The beads eluate, and the input samples were analyzed by Western blot. H1N1 was revealed by an anti-His tag primary antibody (upper panel), and the Nb by an AlexaFluor680-coupled streptavidin (lower panel). (C) BLI streptavidin sensors were loaded with biotinylated AviTag-238D2 (top) or AviTag-3SM5 (bottom). Association of H1N1 on the 238D2 or 3SM5 was measured at 84.75 (bottom curve), 127.1, 169.5, 254.2, 339, 508.5 and 847.5 nM (upper curve). The dissociation occurred in the running buffer. The KD indicated on the graph was obtained from the simultaneous fit of the 5 curves.