Comprehensive Domain-Specific Analysis and IgG Profiling of anti-FVIII Antibodies using a bead-based multiplex immunoassay.

BACKGROUND: Antibodies against factor VIII (FVIII) are a major complication in the treatment of patients with severe hemophilia A (HA). The Nijmegen Bethesda assay (NBA) is the gold standard for detection of neutralizing antibodies (inhibitors). Whereas both, inhibitors and non-neutralizing antibodies (NNAbs) can be detected by immunoassays such as enzyme-linked immunosorbent assay (ELISA) and multiplex Luminex™ bead-based assays.

AIM: Evaluation of an in-house Luminex™ bead-based assay (LumiTope) in comparison to a commercially available ELISA and NBA.

METHODS: The LumiTope method comprised full-length and B-domain deleted FVIII as well as 9 purified FVIII single or multi-domains. The respective proteins were coupled to magnetic beads to detect domain specific Immunoglobulin (IgG, IgG) anti-FVIII antibodies in a large cohort of HA patients with and without inhibitors.

RESULTS: Overall, LumiTope assay had a high sensitivity (94.9%) and specificity (91.2%), particularly in patients with low-titer inhibitors compared to ELISA (sensitivity of 72.2 vs. 27.7%). IgG was the most abundant IgG subclass in NBA positive patients. NBA positive and negative patients showed different domain profiles. Patients with genetic variants in the heavy-chain predominantly exhibited antibodies specific to this chain, while those with a light-chain variant showed a more diverse distribution of antibody specificities. Patients with an intron 22 inversion resembled those with a light-chain defect, with a majority of antibodies targeting the light-chain.

CONCLUSION: LumiTope assay provides a sensitive and specific method for detection but also domain specification of anti-FVIII-antibodies. Implementation of bead-based assays could improve antibody detection, profiling, comparability of results, and can complement NBA.

Copyright © 2024. Published by Elsevier Inc.

PMID: 38453023