Abstract Number: PB0206
Meeting: ISTH 2020 Congress
Background: Development of FVIII-neutralising antibodies (‘inhibitors’) occurs in around one third of severe haemophilia A (HA) patients receiving therapeutic factor VIII (FVIII). Inhibitors limit treatment options and negatively impact patient quality of life. Detecting and measuring inhibitors is notoriously error-prone, with high inter- and intra-laboratory variability. We have developed a candidate reference reagent using patient-derived FVIII-neutralising antibodies and tested this reagent against patient plasmas and a variety of commercially available FVIII activity assays.
Aims: To develop a Bethesda assay reference reagent from patient-derived monoclonal antibodies.
Methods: Recombinant immunoglobulin (IgG)4 was expressed in mammalian cells and purified using protein A. FVIII-neutralising activity was assessed using one-stage clotting assays (OSCAs) and chromogenic assays.
Results: We have generated a panel of neutralising and non-neutralising antibodies that bind the A2, A3, C1 and C2 domains of FVIII. The panel contains both type-1 and type-2 inhibitors, one of which is an immediate acting inhibitor. Due to differences in the biochemical characteristics of the antibodies (Bethesda titres, time-dependency, epitopes) we made type I antibody mixtures to have more confidence of capturing the variety in patient responses. One mixture included the fast-acting antibody (‘4-mix’) and one did not (3-mix). Both mixtures compared well with patient plasmas (Figure 1A). Antibody titre (potency, BU/ml) for 4-mix was more consistent across the different FVIII assay platforms compared to 3-mix (Figure 1B).
Conclusions: We have a candidate reference reagent that will be further developed as a reference reagent for the Bethesda assay. A mixture of type I antibodies that included an immediate-acting antibody is compatible with, and shows consistency in, a range of commercially available FVIII activity assay kits.
To cite this abstract in AMA style:Coxon C, Beavis J, Yu X, Diamond S, Riches A, Raut S. Characterisation and Evaluation of a Candidate Bethesda Assay Reference Reagent Developed from Recombinant, Patient-Derived FVIII Inhibitors [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/characterisation-and-evaluation-of-a-candidate-bethesda-assay-reference-reagent-developed-from-recombinant-patient-derived-fviii-inhibitors/. Accessed October 1, 2023.
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