title: Novel recombinant glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 and variants for assessment of anti-ADAMTS13 autoantibodies in patients with thrombotic thrombocytopenic purpura.
abstract: Immunoglobulin Gs (IgGs) against ADAMTS13 are major causes of acquired (idiopathic) thrombotic thrombocytopenic purpura (TTP). We report here a novel cell-based assay using glycosylphosphatidylinositol (GPI)-anchored ADAMTS13 or variants expressed on cell membrane for assessment of autoantibodies in patients with TTP. We showed that IgGs from all 26 patients with acquired TTP bound to cells expressing a GPI anchored full-length ADAMTS13 (gFL) and a variant truncated after the spacer domain (gS). Also, IgGs from 25/26 (96.7%) of these TTP patients bound to cells expressing a GPI-anchored C-terminal fragment, TSP1 2-8 plus CUB (gT2C). In contrast, none of the 20 healthy blood donors showed detectable binding of their IgGs to the cells expressing gFL, gS, and gT2C. A moderate, but statistically significant correlation was observed between plasma concentrations of anti-ADAMTS13 IgG and positive cells expressing gFL (r=0.65), gS (r=0.67), and gT2C (r=0.42). These results suggest that the microtiter-plate assay and the cell-based assay may detect differential antigenic epitopes. Moreover, antigens clustered on cell membranes may enhance antibody binding affinity, thereby increasing analytical sensitivity. Finally, our assay was able to determine kinetic changes of plasma levels of anti-ADAMTS13 IgGs in TTP patients during plasma therapy. Together, our findings suggest that the novel cell-based assay may be applicable for rapid identification and mapping of anti-ADAMTS13 autoantibodies in patients with acquired TTP.
authors: Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
authors: D
authors: D
authors: Li
authors: J
authors: J
authors: Xiao
authors: M
authors: M
authors: Paessler
authors: X L
authors: XL
authors: Zheng
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000071120:ADAMTS13 Protein; D000328:Adult; D000368:Aged; D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D001681:Biological Assay; D015415:Biomarkers / Q000097:blood; D016466:CHO Cells; D016022:Case-Control Studies; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D015143:Philadelphia; D011237:Predictive Value of Tests; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000628:therapy; D011994:Recombinant Proteins; D012680:Sensitivity and Specificity; D013997:Time Factors; D014162:Transfection; D016896:Treatment Outcome; D055815:Young Adult
chemical_list: D001323:Autoantibodies; D015415:Biomarkers; D017261:Glycosylphosphatidylinositols; D011994:Recombinant Proteins; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human