Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura by Christoph Klaus, Barbara Plaimauer, Jan-Dirk Studt, Friedrich.

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Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura by Christoph Klaus, Barbara Plaimauer, Jan-Dirk Studt, Friedrich Dorner, Bernhard Lämmle, Pier Mannuccio Mannucci, and Friedrich Scheiflinger Blood Volume 103(12):4514-4519 June 15, 2004 ©2004 by American Society of Hematology

ADAMTS13 fragments used for epitope mapping. ADAMTS13 fragments used for epitope mapping. (A) Domain organization of ADAMTS13 and a map of the ADAMTS13 fragments (I, II, III, IV, V, and VI) expressed in E coli. Indicated are the signal peptide (S), the propeptide (P), the catalytic, disintegrin, cys-rich, and spacer domains, the 2 CUB domains, and the 8 TSP type 1 repeats (1-8) (according to Zheng et al11). (B) Silver staining of purified recombinant ADAMTS13 fragments expressed in E coli (a-c). Contaminating bands derived from E coli that could not be removed during the purification process. (C) Western blot analysis of the purified recombinant ADAMTS13 fragments using an anti-histag antibody. (D) Western blot analysis of the purified recombinant ADAMTS13 fragments using a polyclonal rabbit antihuman ADAMTS13-his antiserum. Fragment I indicates propeptide; fragment II, tsp1-1; fragment III, cat/dis/tsp1-1; fragment IV, cys-rich/spacer; fragment V, tsp1/2-8; fragment VI, cub1 + 2. Christoph Klaus et al. Blood 2004;103:4514-4519 ©2004 by American Society of Hematology

Immunoblot analyses showing the domain-specific reactivity of TTP patient plasmas with different anti-ADAMTS13 inhibitor titers. Immunoblot analyses showing the domain-specific reactivity of TTP patient plasmas with different anti-ADAMTS13 inhibitor titers. Equivalent amounts of recombinant ADAMTS13 fragments were resolved on 12% SDS-PAGE and electroblotted. Plasma samples from patients 14 (32 BU/mL; panel A), 18 (6 BU/mL; panel B), 22 (10 BU/mL; panel C), and 3 (3 BU/mL; panel D) were used as sources of primary antibody and AP-conjugated antihuman immunoglobulin, and BCIP/NBT was used for visualization. Recombinant protein, lane 1: recombinant ADAMTS13 (rA-13) derived from HEK 293 cells. Purified ADAMTS13 fragments, lanes 2-7: propeptide (lane 2, I), tsp1-1 (lane 3, II), cat/dis/tsp1-1 (lane 4, III), cys-rich/spacer (lane 5, IV), tsp1/2-8 (lane 6, V), and cub1 + 2 (lane 7, VI). As a negative control, the thio/his fragment was applied (lane 8). M indicates molecular weight markers. Christoph Klaus et al. Blood 2004;103:4514-4519 ©2004 by American Society of Hematology