Immunoblot for detecting autoantibodies against ADAMTS13 in plasma samples. (A) Comparison of different plasma samples with a monoclonal anti–human ADAMTS13 antibody. Lane 1: molecular mass marker (full-range rainbow marker, Amersham Pharmacia). Lanes 2-4: plasma samples from a patient⁶  with antibody-induced TTP taken at different stages during her disease course. Lane 5: plasma sample from a patient with antibody-induced TTP. Lane 6: plasma sample from a patient with hereditary TTP. Lane 7: pooled normal plasma. Lane 8: monoclonal anti–human ADAMTS13 antibody applied in 1/1000 dilution. The plasma samples used for staining lanes 2-7 were diluted 1/200 with 20 mM Tris (tris(hydroxymethyl)aminomethane), 130 mM NaCl, pH 7.2 buffer, containing 0.05% (wt/vol) Tween 20 and 10% (vol/vol) blocker casein in Tris-buffered saline (TBS; Pierce, Rockford, IL). The bottom panel shows the ADAMTS13 activities and the inhibitor concentrations measured by the collagen-binding assay. (B) Quantitative evaluation of the immunoblots. The intensity of the ADAMTS13 bands in the Western blots developed with serial dilutions of a plasma sample from patient A were plotted against the applied inhibitor concentration. The inset shows the ADAMTS13 bands as they appeared on the immunoblots.