Direct antibody-inhibition of GpA binding by RII.

<p>(A) RII binds to GpA (lane 1), but not to neuraminidase treated GpA (GpA-NA – lane 2). Addition of R217 IgG or Fab prevents RII from binding GpA (lane 3 and 4). Neither R218 IgG (lane 5), R218 Fab (lane 6), control IgG (lane 7) nor control Fab (lane 8), block RII/GpA receptor binding. Binding is specific as neuraminidase treatment prevents binding in all cases (lanes 2 and 9–14). Note that an increased signal is observed with R218 IgG over R218 Fab (compare lanes 5 and 6) suggesting bivalent binding. This assay was performed with RII at 3 µM and antibody or Fab fragments at 6 µM. (B) Titration of R217 demonstrates the specificity of interaction as all concentrations around and above the available RII concentration (3 µM) prevents binding (lanes 6–10). At concentrations below the available RII binding occurs as not all the RII is bound by R217 (lanes 3–5). (C) Titration of R218 demonstrates that R218 is unable to directly prevent GpA binding at any concentration.