Antigenic characterization of secreted and intracellular wt-fVIII and N1922S-fVIII. Anti-A2 MAb 4A4 was used to capture secreted and intracellular wt-fVIII and N1922S-fVIII from BHK-M medium and cell lysates, respectively. The binding of biotinylated secondary MAbs 2-116 (anti-A1), 1D4 (anti-A2), I92 (anti-A3), G38 (anti-A3), 2-113 (anti-A3), 2A9 (anti-C1), 3E6 (anti-C2), or 2-117 (anti-C2) was measured colorimetrically, as described in “Methods.” Apparent antigen levels for wt-fVIII (A) and N1922S-fVIII (B) were calculated by means of a standard curve using purified wt-fVIII of 2 to 8 samples per construct. (C) The ratio of apparent intracellular antigen to secreted antigen was calculated from the results shown in panels A and B.