Epitope mapping / IgG isotyping
J. Oldenburg and B. Pezeshkpoor will map inhibitor B-cell epitopes with the following three strategies:
- In a first round samples will be tested for binding to FVIII domains. For this FVIII chains will be dissociated by incubation. Plasma samples will be added to FVIII domains and analysed for binding
- Based on the x-MAP technology, three different anti-FVIII antibody tests will be performed. After incubation and washing, the IgG subclasses (IgG1 to IgG4) of the FVIII inhibitor will be identifed by a specifc IgG antibody.
- Additionally in a second multiplexed assay certain antibodies will be immobilized and incubated. Finally the C2 domain will be directly coupled to beads for detection of anti-C2 domain antibodies. The date will be expressed as the mean ﬂuorescence intensity (MFI) read by the BioPlexTM instrument. Results will be expressed in terms of relative antigenic reactivity (RAR), corresponding to the patient’s MFI value / negative control mean MFI value + 3 Standard Deviations ratio. Plasma samples will be tested for IgG-subclasses specifc for FVIII. Most inhibitors belong to the subclasses IgG1 and IgG4. There are some indications that the ratio of IgG1 and IgG4 might inﬂuence ITI outcome.
- Lapalud P et al. Anti-A2 and anti-A1 domain antibodies are potential predictors of immune tolerance induction outcome in children with hemophilia A. J Thromb Haemost 2015; 13: 540-547