Determination of ADAMTS13 activity and detection of inhibitors by immunoblotting of VWF substrate degraded by BaCl2-activated ADAMTS13. (A) ADAMTS13 activity in 1:20 diluted patient serum or plasma. Lanes 1-6, assay calibration by normal plasma (NHP) dilutions of 1:20 (100% activity), 1:40 (50%), 1:80 (25%), 1:320 (6.25%), 1:640 (3%), and buffer control (0%). Lane 7, propositus (premortem serum); lane 8, father (plasma); lane 9, mother (plasma). (B) ADAMTS13 inhibition by purified immunoglobulin (Ig) and Ig-depleted hemolytic serum. Residual ADAMTS13 activity of NHP incubated 1:1 (vol/vol) with concentrated total Ig purified from the propositus' serum, or with concentrated Ig-depleted hemolytic serum (final NHP dilution, 1:20). Lanes 1-4, assay calibration by NHP dilutions of 1:20 (100% activity), 1:40 (50%, equivalent to an inhibitor titer of 1 BU/mL), 1:80 (25%), and buffer control (0%). Lanes 5-6, NHP 1:1 (vol/vol) with purified Ig (duplicates); lanes 7-8, NHP 1:1 (vol/vol) with Ig-depleted serum (duplicates). (C) Inhibition of ADAMTS13 activity by hemoglobin. Residual ADAMTS13 activity of NHP incubated 1:1 (vol/vol) with recombinant or erythrocyte-derived human hemoglobin (final NHP dilution, 1:20). Lanes 1-4, assay calibration by NHP dilutions of 1:20 (100% activity), 1:40 (50%, equivalent to an inhibitor titer of 1 BU/mL), 1:80 (25%), and buffer control (0%). Lanes 5-10, NHP incubated 1:1 (vol/vol) with recombinant hemoglobin; lanes 11-16, NHP incubated 1:1 (vol/vol) with purified hemoglobin from lysed erythrocytes. Final hemoglobin concentrations are 20 g/L (lanes 5, 11), 10 g/L (lanes 6, 12), 5 g/L (lanes 7, 13), 2 g/L (lanes 8, 14), 1 g/L (lanes 9, 15), and 0.5 g/L (lanes 10, 16).
