Characterization of domain-specific anti-A3 MAbs. (A) Overlapping epitopes recognized by anti-A3 MAbs were identified by competition ELISA. Human fVIII was bound to MAbs I143, 2-113, I92, I130, F147, or G38 that were immobilized to microtiter wells. Corresponding biotinylated MAbs were added and binding was detected using streptavidin-alkaline phosphatase conjugate. The rows and columns correspond to unmodified, immobilized MAbs, and solution-phase, biotinylated MAbs, respectively. Epitopes recognized by MAbs were mapped as described in supplemental Figure 1. (B) Native wt-fVIII or wt-fVIII were denatured by heating in SDS and adsorbed to nitrocellulose membranes, followed by the addition of the indicated domain-specific murine anti-fVIII MAb and goat anti–mouse IR conjugate. The ratio of fluorescence of goat anti–mouse IR conjugate bound to denatured fVIII versus wt-fVIII was determined using a LI-COR Odyssey infrared imaging system. Results are reported as mean and sample standard deviation of triplicate samples for each MAb.