Patients with hereditary or acquired
haemophilia A may develop inhibitory
factor VIII (FVIII)
antibodies. These disrupt FVIII activity predominantly by preventing the formation of the
tenase complex, leading to a serious
bleeding disorder.
Antibodies without inhibiting activity, however, can also be found when screening patients with
haemophilia A under FVIII supplementation. Therefore, the detection of only these allo- or
autoantibodies from plasma is not sufficient. Rather, the characterization of the antibody-induced effects on the coagulation cascade should be considered due to its great diagnostic importance. Currently, inhibitory activities are detected by the functional Bethesda assay, which directly measures the delay in clotting time by the patient plasma. However, this assay does not provide information on the cause of the inhibition. Here, we report the development of a surface plasmon resonance (SPR) biosensor that has the potential to integrate both quantitative and functional information on patient antibody characteristics in one measurement. Recombinant FVIII
protein was immobilized on the sensor surface to detect
antibodies from patient plasma. The interaction of the FIX- and FXa-clotting
proteins with the formed anti-FVIII/FVIII complex could be detected subsequently within the same SPR measurement cycle. Inhibitory
antibodies led to the prevention of these interactions. Thus, discrimination between the clinically relevant inhibitory and non-inhibitory
antibodies was enabled. In a group of 16 patients with inhibitory
antibodies (both ELISA- and Bethesda-positive), 5 patients with non-inhibitory
antibodies (ELISA-positive but Bethesda-negative) and 12 healthy controls, diagnostic sensitivity and specificity data of 100% for the FIX interaction were achieved using this biomimetic biosensor approach. The new method allows for detection and quantification, as well as for evaluation of inhibitory activity of allo- and
autoantibodies, using small sample volume and short analysis time.