Anti-ADAMTS13 autoantibodies from acute iTTP patients induce an open ADAMTS13 conformation in healthy donor plasma. (A) ADAMTS13 antigen levels in acute iTTP patients (n = 19) and healthy donors (n = 10). Plasma was added to coated antibody 3H9. Bound ADAMTS13 was detected using biotinylated antibodies 19H4 and 17G2 and horseradish peroxidase–labeled streptavidin. NHP was used as a reference and set as 1 µg/mL ADAMTS13.⁵  Only samples containing ≥0.03 µg/mL (dotted line) could be tested for ADAMTS13 conformation. (B) ADAMTS13 conformation was tested in plasma samples of acute iTTP patients (n = 18) and healthy donors (n = 10) using our 1C4-ELISA; open ADAMTS13 (Conformation Index >0.5; open circles), but not closed ADAMTS13 (Conformation Index ≤0.5; filled circles), is specifically captured on the coated antibody 1C4. (C) Anti-ADAMTS13 autoantibody levels in acute iTTP patients (n = 18) and healthy donors (n = 10). Plasma was added to coated rhADAMTS13, and bound anti-ADAMTS13 autoantibodies were detected by horseradish peroxidase–labeled polyclonal goat anti-human IgG (Fc-specific) antibodies. To calculate anti-ADAMTS13 autoantibody levels, the iTTP plasma sample TTP07-A was used as a reference and set as 100%. The threshold of positivity was determined by the 97.5th percentile of anti-ADAMTS13 autoantibody levels measured in healthy donors (n = 404) and set as 6.7% (supplemental Figure 1). Filled circles, closed ADAMTS13; open circles, open ADAMTS13. (D) Purified IgGs from acute iTTP patients (n = 18) or healthy donors (n = 10) were individually preincubated with closed ADAMTS13 present in NHP. Next, the preincubated samples were tested using the 1C4-ELISA to determine whether the purified IgGs had opened the closed ADAMTS13 conformation in NHP. Murine monoclonal antibody 17G2, which induces an open ADAMTS13 conformation, was used as a positive control (n = 5). Filled circles, closed ADAMTS13; open circles, open ADAMTS13.