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Anti-spacer idiotope profiles in iTTP patients
(Figure 2B). Therefore, monoclonal antibodies 17H9, 9G12 and 7D10 are anti-idiotypic antibodies that target idiotopes in the CDRs of VH and VL of anti-spacer autoan- tibody II-1, TTP73, or I-9, respectively (Figure 2B).
We next aimed to identify if the anti-idiotypic antibod- ies recognizing particular idiotopes in the anti-spacer autoantibodies II-1, TTP73, and I-9 are anti-idiotypic anti- bodies that are involved in ADAMTS13 binding (Figure 1, dark blue antibody). To do so, we used a competition ELISA where we studied if the binding of anti-spacer autoantibodies II-1, TTP73 and I-9 could be inhibited by their respective anti-idiotypic antibody. The 3 developed anti-idiotypic antibodies (17H9, 9G12 and 7D10) inhibited the binding of their respective anti-spacer autoantibodies
A
(II-1, TTP73 and I-9) to rhADAMTS13 (Figure 2C).
In conclusion, we developed 3 anti-idiotypic antibodies that recognize particular idiotopes in the anti-spacer autoantibodies II-1, TTP73 and I-9 that are involved in ADAMTS13 binding, as they strongly inhibit the binding of anti-spacer autoantibodies II-1, TTP73 or I-9, respec-
tively, to rhADAMTS13.
Anti-idiotypic antibodies and their binding to idiotopes in II-1, TTP73 and I-9
Since anti-spacer autoantibodies II-1 and I-9 have over- lapping but different epitopes (see Online Supplementary Methods),43 they will have both shared and unique idiotopes. We, therefore, investigated whether anti-idio-
B
C
Figure 2. Development and characterization of anti-idiotypic antibodies that inhibit the binding of respectively anti-spacer autoantibody II-1, TTP73 or I-9 to rhADAMTS13. (A) Binding of purified murine anti-II-1 (red), anti-TTP73 (green) and anti-I-9 (orange) antibodies to coated human anti-spacer autoantibodies II-1, TTP73 or I-9. Bound murine anti-II-1, anti-TTP73 and anti-I-9 antibodies were detected using GAM-HRP. Murine anti-II-1, anti-TTP73 or anti-I-9 antibody binding was expressed as relative absorbance values [mean ± Standard Deviation (SD), n=3] with absorbance of the respective positive controls (sera of mice immunized with either II-1, TTP73 or I-9) set as 1. (B) Binding of purified murine anti-II-1 (red), anti-TTP73 (green) and anti-I-9 (orange) antibodies to a coated human IgG pool. Bound murine anti-II-1, anti-TTP73 and anti-I-9 antibodies were detected using GAM-HRP. Murine anti-II-1, anti-TTP73 or anti-I-9 antibody binding was expressed as relative absorbance values (mean±SD, n=3) with absorbance of the respective positive controls (sera of mice immunized with either II-1, TTP73 or I-9) set as 1. Binding to coated human IgG pool indicates that the murine antibodies bind to the conserved regions of antibodies. (C) Inhibition of anti-spacer autoantibody binding to rhADAMTS13 by anti-idiotypic antibodies. A 1:2 dilution of murine anti-II-1 (red), anti-TTP73 (green) or anti-I-9 antibodies (orange) were pre-incubated with constant amounts of anti-spacer autoantibody II-1, TTP73 and I-9, respectively, before addition to a rhADAMTS13 coated 96-well microtiter plate. Bound II-1 (red), TTP73 (green) and I-9 (orange) antibodies were detected using a mixture of anti-human IgG1-4-HRP. Data are expressed as % binding (mean±SD, n=3) of anti-spacer autoan- tibodies II-1 (red), TTP73 (green) or I-9 (orange) to rhADAMTS13 in the presence of the competing murine anti-II-1 (17H9), anti-TTP73 (9G12) or anti-I-9 (7D10) anti- body relative to the binding of II-1, TTP73 or I-9 in the absence of anti-idiotypic antibodies (dotted lines). OD: optical density.
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