The presence of anti-CS autoantibodies that open ADAMTS13 is a common feature of the autoimmune response in patients with iTTP. (A-B) IgG fractions from 13 patients with acute iTTP were purified against CS (A) and CUB (B) using affinity chromatography. The specificity of the purified autoantibody fractions against CS (A) or CUB (B) was tested, using ELISA, on coated CS (light green) and MDT (orange) (A) and CUB (lavender) and T2-T8 (red) (B). Bound autoantibodies were detected using HRP-labeled polyclonal goat anti-human (Fc-specific) IgGs. Data are shown as the residual OD value that was calculated by subtracting the OD value of the sample on the fragment of interest (CS, MDT, CUB, or T2-T8), with the mean OD + 3 SD value of the binding of purified IgGs of 9 healthy donors to the corresponding fragment. Anti-CS (A) or anti-CUB (B) autoantibody fractions were used for further analysis if the residual OD value of the sample on CS(A, light green) or CUB (B, lavender), respectively, was positive, whereas the residual OD of binding to the other ADAMTS13 domains (MDT (A, orange) or T2-T8 (B, red)) was <50% of the binding to CS (A, light green) or CUB (B, lavender). Hence, only the anti-CUB autoantibody fraction from patient F was not used further. The anti-CS autoantibody fractions isolated from 10 patients (C) and the anti-CUB autoantibody fractions isolated from 4 patients (D) were individually preincubated with NHP containing closed ADAMTS13 and tested, using 1C4-ELISA, to investigate which autoantibodies could open ADAMTS13. Data are expressed as the mean CI ± SD (n = 3). A mean CI ≤ 0.50 (filled circle) represents closed ADAMTS13, whereas a mean CI > 0.50 (open circle) represents open ADAMTS13.^15,16