Fluorescence Luminex immunoassay of human anti-fVIII antibody calibrator. Various molar concentrations (0.01-1.0 nM) of purified human immunoglobulin in PBS/0.1% albumin/0.05% Tween 20 were added to wells containing rfVIII-coupled beads and ovalbumin-coupled beads (nonspecific binding control). Binding was detected with mouse anti–human Ig–R-PE. Each point is a mean of 8 independent determinations plus or minus standard deviation (SD). The calibrator was generated by subtraction of the MFIU signal of the nonspecific binding to ovalbumin from the MFIU of specific binding to rfVIII at each concentration of anti-fVIII Ab.