LumiTope: Toward Improving the Performance of Identification of Antifactor VIII Antibodies in Congenital and Acquired Hemophilia A

Introduction Development of anti-FVIII antibodies in congenital hemophilia A (HA) and acquired HA (AHA) is a great burden and serious complication concerning the treatment and morbidity/mortality, respectively. The aim of the present study was to evaluate the performance of an in-house microsphere-based immunoassay on the Luminex (LumiTope) for early detection and characterization of anti-FVIII antibodies.

Materials and Methods Plasma samples were tested in parallel using the Bethesda assay, an indirect FVIII ELISA kit (Immucor) and the results were compared with LumiTope. A full-length (FLFVIII) and a BDD-deleted (BDD-FVIII) FVIII recombinant product as well as nine single or multidomain FVIII proteins (A1, A2a2, A2, B, a3A3, C1, C2, C1C2, a3A3C1C2 (light chain, LC)) were covalently coupled on magnetic microsphere beads and tested after dilution in assay buffer and incubation with bead mix. Anti-FVIII antibodies were detected using a PEphycoerythrin (PE) antihuman IgG/IgG1-4 or IgA antibody.

Results LumiTope testing showed several advantages over the ELISA and for the evaluation of the patient’s treatment course in combination with the Bethesda assay. (1) In comparison with conventional ELISA, a smaller volume of sample is needed (13× less) for performing the test against 11 proteins using several detecting antibodies. Here we need ~100 µL of plasma vs. ~1,300 µL of ELISA (20 µL per domain and detecting antibody). (2) Using LumiTope, we were able to detect IgM antibodies in the very early phase of treatment (<10 EDs) in a HA patient. (3) Due to the domain-specific detection of the antibodies, in a HA patient we were able to detect noninhibitory antibodies against the B-domain, which were present against FL-FVIII but absent in BDD-FVIII. (4) Several noninhibitory antibodies against single domains of FVIII were detected which were detectable in ELISA. These antibodies decreased the FVIII half-life/clearance depicted by low trough levels in HA patients. (5) Using LumiTope, persistent antibodies against the A1 domain of IgA type were detected in an AHA patient where despite negative ELISA and Bethesda results, the remission was not achieved.

Conclusion LumiTope provides a sensitive and fast method for early characterization of inhibitory anti-FVIII antibodies in hemophilia A patients. The characterization of the binding regions of these antibodies provides the basis for better understanding of inhibitory mechanisms and help for the eradication of FVIII inhibitors.

Publikationsverlauf

Artikel online veröffentlicht:
13. November 2020