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A.S. Schelpe et al.
score,38 and the score by Benhamou et al.39 The predictive model set up by Benhamou et al. takes into account age, lactate dehydrogenase (LDH) levels, and cerebral involve- ment, and detects early death in acquired severe ADAMTS13 deficiency-associated idiopathic TTP.39 However, in iTTP, autoantibody profiling to stratify patients has not yet been fully explored.
In this study, we developed an autoantibody profiling assay for iTTP using anti-idiotypic antibodies that recog- nize particular idiotopes on anti-ADAMTS13 autoanti- bodies, idiotopes that are involved in ADAMTS13 binding (Figure 1). Since the ADAMTS13 spacer domain seems to be the main immunogenic region targeted in these patients,29 we generated an anti-idiotypic antibody against 3 available cloned human anti-spacer autoantibodies. The selected anti-idiotypic antibodies were then used to screen 151 iTTP plasmas for the presence of autoantibodies with the same idiotopes across patients, which resulted in strat- ification of iTTP patients according to these anti-spacer idiotope profiles. We next investigated in a subgroup of 95 patients whether certain anti-spacer idiotope profiles could be linked with disease severity.
Methods
Immunization strategy and characterization of anti-II-1, anti-TTP73 and anti-I-9 antibodies
Anti-II-1, anti-TTP73 and anti-I-9 antibodies were developed by immunizing BALB/c mice (Janvier Labs, Le Genest-Saint-Isle, France) with the cloned human anti-spacer autoantibodies II-1,40 TTP73, or I-9,41 respectively (see the immunization strategy in the Online Supplementary Appendix). The binding of purified anti-II-1, anti-TTP73 or anti-I-9 antibodies to II-1, TTP73, and I-9, respec- tively, and to the conserved regions (Figure 1, gray) in human immunoglobulin G (IgG) antibodies were identified using ELISA.
ELISA to identify anti-II-1, anti-TTP73 and anti-I-9 antibodies that inhibit the binding of anti-spacer autoantibodies II-1, TTP73, or I-9 to ADAMTS13, respectively
Human anti-spacer autoantibodies II-1, TTP73 or I-9 (constant final EC50: 0.04, 0.85 and 0.04 μg/mL, respectively) (see Online Supplementary Methods), were pre-incubated with a 1:2 dilution series of murine anti-II-1, anti-TTP73, or anti-I-9 antibodies (final start concentration 10 μg/mL) respectively, in a pre-blocked plate. After 30 minutes, samples were transferred to a recombinant human (rh)ADAMTS13 [2.7 μg/mL in phosphate buffered saline (PBS)] coated plate. Bound human anti-spacer autoantibodies II-1, TTP73, or I-9 were detected using a mixture of HRP-labeled anti- human IgG1-4 (IgG1: 1/20,000 and IgG2-4: 1/2,000; Sanquin, Amsterdam, the Netherlands).
ELISA to study the binding of the anti-idiotypic antibodies to the anti-spacer idiotopes of II-1, TTP73, and I-9
Murine anti-idiotypic antibodies 17H9 (anti-II-1 antibody), 9G12 (anti-TTP73 antibody) and 7D10 (anti-I-9 antibody) were coated at 5 μg/mL in carbonate/bicarbonate coating buffer (50 mM Na2CO3/NaHCO3, pH 9.6). After blocking, human anti-spacer autoantibodies II-1, TTP73, and I-9 were added at a starting con- centration of 1 μg/mL and further diluted 1:2. Bound anti-spacer autoantibodies were detected by adding a mixture of HRP-labeled anti-human IgG1-4 antibodies (Sanquin).
Patients’ samples
Detailed information about the 151 iTTP plasma samples can be found in the Online Supplementary Methods. The study protocol was approved by the Medical Ethical Committee of the University Medical Center Utrecht (Utrecht, the Netherlands), the Ethics Committee of Hospital Pitié-Salpêtrière and Hospital Saint- Antoine (Paris, France), and the Ethics Committee of Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico (Milan, Italy). The study was carried out in accordance with the Declaration of Helsinki.
ELISA to identify the presence of anti-spacer idiotope profiles in plasmas of acute iTTP patients using the newly developed anti-idiotypic antibodies
Murine anti-idiotypic antibody 17H9 (anti-II-1 antibody), 9G12 (anti-TTP73 antibody), or 7D10 (anti-I-9 antibody) were coated at 5 μg/mL. After blocking, patient plasma (starting dilution 10%, v/v) was added and diluted 1:2. Bound patient antibodies were detected with HRP-labeled anti-human IgG1-4 antibodies (Sanquin).
Statistical analysis
Graphpad Prism v.5.03 software (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. Further details of the methods used are available in the Online Supplementary Methods.
Results
Development of anti-idiotypic antibodies against idiotopes in anti-spacer autoantibodies II-1, TTP73 or I-9 involved in ADAMTS13 binding
To generate anti-idiotypic antibodies recognizing partic- ular idiotopes in anti-spacer autoantibodies involved in ADAMTS13 binding, 3 cloned human anti-spacer autoan- tibodies with different epitopes and inhibitory character- istics were available: II-1,40 TTP7342 and I-941 (see Online Supplementary Methods) and were used to immunize BALB/c mice. As the injected anti-spacer autoantibodies are full IgG antibodies in which the variable regions are grafted onto a human IgG1 constant region,40,41 the mice developed antibodies that either recognized conserved regions (e.g. constant regions: CH and CL and framework regions in VH and VL) (Figure 1, gray parts) or idiotopes in the complementarity determining regions (CDRs) of the VH and VL of II-1, TTP73 and I-9 (Figure 1, dark and light blue dots). We obtained 1 mouse monoclonal antibody that recognized anti-spacer autoantibody II-1, 2 that rec- ognized anti-spacer autoantibody TTP73 and 10 that rec- ognized anti-spacer autoantibody I-9 (Figure 2A) as the generated antibodies bound to the coated anti-spacer autoantibodies II-1, TTP73 or I-9, respectively. To identify which of the generated monoclonal antibodies recognized the conserved part of the human autoantibodies (CH, CL and framework regions in VH and VL) (Figure 1, gray parts), their binding to a pool of purified human IgG antibodies was studied. Monoclonal antibody 17H9 recognizing anti- spacer autoantibody II-1 did not recognize the conserved part of the coated human IgG antibodies (Figure 2B), while 1 of the monoclonal antibodies (20H3) recognizing anti- spacer autoantibody TTP73 and 9 of the monoclonal anti- bodies (1E6, 5C8, 6C9, 7E8, 9F9, 9G9, 9H4, 11F7, and 14G6) recognizing anti-spacer autoantibody I-9 did bind to the conserved part of the coated human IgG antibodies
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