Development and evaluation of a novel FVIII domain-specific immunoassay for characterization of anti-FVIII antibodies

B. Pezeshkpoor, A.-C. Berkemeier, J. Müller, T. Albert, J. Oldenburg (Bonn, Germany)

Laboratory tests
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: FVIII neutralizing antibodies develop in response to replacement treatment in 30% of patients with severe hemophilia A. Such inhibitory antibodies represent a serious complication in the treatment of these patients. The aim of the present study was the development and evaluation of a microsphere-based immunoassay on the Luminex™ system for rapid detection and early characterization of anti-FVIII antibodies in patient plasma samples.

Methods: The FVIII protein was structured into several single and multi-domain fragments and introduced to a baculovirus expression system. The domains were purified (>80%) and individually coupled to colored-coded magnetic microspheres. Commercially available full length (FL-FVIII) and B-domain deleted (BDD-FVIII) FVIII proteins were immobilized accordingly. The coupled target proteins were used to establish the Luminex™ based assay, termed LumiTope.

Results: Different FVIII constructs were prepared and verified by western blotting. Later, eight constructs (A1a1, A2a2, A1a1A2, a3A3, C1, C2, C1C2 and Light chain (LC)) were chosen and purified in large scale. All prepared microspheres were tested in singleplex and multiplex assay formats on the Luminex™ system. Then, LumiTope was applied on plasma samples from healthy controls, patients with FVIII inhibitors. The first results show that LumiTope is a sensitive test for detection of anti-FVIII antibodies and can run in a routinely based multiplex-configuration covering the whole FVIII protein. LumiTope revealed positive results against FL-FVIII beads for all patients with antibody titers> 0.6 BU/ml and positive results on FVIIII-ELISA antibody test. Moreover, we were able to identify several monoclonal and polyclonal antibodies against A2a2, a3A3, LC, C1, C2 and C1C2 domains. Detected antibodies were predominantly directed against the A2a2 and C2 domains of FVIII.

Conclusion: Our new immunoassay, 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 may provide the basis for better understanding of inhibitory mechanisms and help for the eradication of FVIII inhibitors.