Abstract
Background
ADAMTS13-binding immunoglobulin G (IgG)-type autoantibodies are present in patients with acquired thrombotic thrombocytopenic purpura (TTP), thereby causing severe deficiency of plasma ADAMTS13 activity. Some specific autoantibodies directly inhibit the enzymatic activity by interfering with the access of its substrate, von Willebrand factor (VWF), particularly in the spacer domain, although others bind to almost all of the domains in the molecule, possibly leading to the acceleration of the clearance from blood stream. Not only the inhibitor titer, but the importance of ADAMTS13 autoantibody titer was highlighted by many previous clinical studies, associating with the prognosis such as recurrence rate.
Objective
We targeted to establish a novel high-sensitive assay to measure ADAMTS13-binding IgG autoantibody titer using three kinds of radioisotope-labeled antigens, ADAMTS13 whole molecule, MDTCS and T2-8/CUB, and aimed to analyze the association between the autoantibody titer and the clinical characteristics of TTP patients.
Materials and Methods
Human Cell-Free Protein Expression System (Takara #3281, Shiga, Japan) was used to synthesize radioisotope-labeled antigens. ADAMTS13 cDNA corresponding to whole molecule (A13), metalloprotease to spacer domains (MDTCS) and TSP1-2 to CUB2 domains (T2-8/CUB) were cloned into an expression vector pT7-IRES and mixed with Cell Lysate containing T7 RNA polymerase, methionine-free amino acids, ATP, translation enhancement factor and 35S-methionine. The correct synthesis and molecular size of the radiolabeled antigens were checked with SDS-PAGE. To assess the utility as a quantitative assay, each of the antigens was mixed with mouse anti-ADAMTS13 monoclonal antibodies, whose epitopes were determined in our previous study (Thromb Res. 2012; 130(3):e79-83), and the immune complex was precipitated with protein G beads, washed and measured in a liquid scintillation counter. Plasma samples from acquired TTP patients were tested to quantify the autoantibody titers using radiolabeled A13, MDTCS and T2-8/CUB antigens, respectively. As a control, plasma samples from healthy subjects with no histories of autoimmune disease were also tested.
Results
Each of the radiolabeled antigens was detected as a single band at the correct molecular weight size and successfully immnoprecipitated with several mouse anti-ADAMTS13 monoclonal antibodies, indicating the intact molecular conformation of the synthesized proteins using the cell-free protein synthesis system. Moreover, the appropriate dose-dependent escalation curves in accordance with the addition of the monoclonal antibodies were observed, thereby confirming the utility of the assay as a quantitative analysis. We tested TTP patient plasma at onset (n=5) and were able to detect ADAMTS13 autoantibody titers with each of the radiolabeled A13, MDTCS and T2-8/CUB antigens. We next applied this assay for monitoring ADAMTS13 autoantibody titer in a clinical course of TTP patient. The patient developed the first episode of TTP at the age of 2 month and treated with steroid pulse and plasma exchange therapy for six consecutive days. Remission was once achieved but 6 months later from the onset, the second episode of TTP occurred and the patient was treated with 11 plasma exchange and rituximab at the dose of 375 mg/m2 once a week for 4 weeks. Plasma samples at onset, after the first 6 consecutive plasma exchange and after rituximab administration were examined about autoantibody titer using this assay. Interestingly, the titers remained high even after the plasma exchange but declined clearly after the rituximab treatment, whereas no reduction of total IgG level was observed. These findings suggest that the autoantibody titration using this assay might be useful to assess the effect of treatment and associate with the prognosis related to the recurrence.
Conclusion
We developed a novel quantitative radioimmunoprecipitation assay to measure ADAMTS13 autoantibody titer related to three antigens, ADAMTS13 whole molecule, MDTCS and TSP2-8/CUB. This assay may serve not only as a diagnostic test but as a monitoring index to evaluate the prognosis of TTP.
Disclosures:
No relevant conflicts of interest to declare.
Antibodies in human sera to oncorna virus-like proteins from normal or leukemia marrow cell cultures.
