title: Multiple B-cell clones producing antibodies directed to the spacer and disintegrin/thrombospondin type-1 repeat 1 (TSP1) of ADAMTS13 in a patient with acquired thrombotic thrombocytopenic purpura.
abstract: BACKGROUND
  The cysteine-rich/spacer domains of ADAMTS13 contain a major binding site for antibodies in patients with acquired thrombotic thrombocytopenic purpura (TTP).
  
  
  OBJECTIVE
  To study the heterogeneity of the antibody response towards these domains an immunoglobulin V-gene phage-display library was constructed to isolate monoclonal anti-ADAMTS13 antibodies from the immunoglobulin repertoire of a patient with acquired TTP.
  
  
  METHODS
  Combined variable heavy chain (VH) and variable light chain (VL) segments, expressed as single-chain Fv fragments (scFv), were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/thrombospondin type-1 repeat 1 (TSP1)/cysteine-rich/spacer domains.
  
  
  RESULTS
  Seven different scFv antibody clones were identified that were assigned to four groups based on their homology to VH germline gene segments. Epitope-mapping revealed that scFv I-9 (VH1-69), I-26 (VH1-02), and I-41 (VH3-09) bind to an overlapping binding site in the ADAMTS13 spacer domain, whereas scFv I-16 (VH3-07) binds to the disintegrin/TSP1 domains. The affinity of scFv for the disintegrin/TSP1/cysteine-rich/spacer domain was determined by surface plasmon resonance analysis and the dissociation constants ranged from 3 to 254 nM. The scFv partially inhibited ADAMTS13 activity. However, full-length IgG prepared from the variable domains of scFv I-9 inhibited ADAMTS13 activity more profoundly. Plasma of six patients with acquired TTP competed for binding of scFv I-9 to ADAMTS13.
  
  
  CONCLUSIONS
  Our data indicate that multiple B-cell clones producing antibodies directed against the spacer domain are present in the patient analyzed in this study. Our findings also suggest that antibodies with a similar epitope specificity as scFv I-9 are present in plasma of other patients with acquired TTP.
authors.affiliation: Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: B M
authors.initials: BM
authors.lastname: Luken
authors.forename: P H P
authors.initials: PH
authors.lastname: Kaijen
authors.forename: E A M
authors.initials: EA
authors.lastname: Turenhout
authors.forename: J A
authors.initials: JA
authors.lastname: Kremer Hovinga
authors.forename: J A
authors.initials: JA
authors.lastname: van Mourik
authors.forename: R
authors.initials: R
authors.lastname: Fijnheer
authors.forename: J
authors.initials: J
authors.lastname: Voorberg
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000276:immunology*; D000071120:ADAMTS13 Protein; D020816:Amino Acid Motifs / Q000235:genetics / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity / Q000235:genetics / Q000276:immunology*; D001323:Autoantibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D011697:Purpura, Thrombotic Thrombocytopenic / Q000235:genetics / Q000276:immunology*; D019700:Thrombospondin 1 / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D019700:Thrombospondin 1; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human