The development of anti-factor VIII (fVIII) antibodies is a significant complication in the management of patients with hemophilia A leading to increased morbidity and treatment cost. Physicians typically base treatment decisions on inhibitor titer as measured by the Bethesda assay. Patients with inhibitor titers greater than 10 Bethesda units (BU/ml) are unlikely to respond to fVIII and are treated with bypassing agents. Using a panel of anti-fVIII monoclonal antibodies (MAbs) to different B-Cell epitopes on fVIII, we have shown that epitope specificity, inhibitor kinetics, and time to maximum inhibition are more important than inhibitor titer in predicting response to fVIII. We have previously reported the mapping of B-Cell epitopes using competition ELISA with murine anti-fVIII MAbs. Although successful, these assays required high volumes of patient plasma. The ability to map the epitope spectrum of patient plasma using a clinically feasible assay may fundamentally change how clinicians approach treatment of high titer inhibitor patients.


B-Cell epitopes were mapped using competition ELISA and a slide based microarray. For the microarray, 15 anti-fVIII MAbs were printed on nitrocellulose slides at concentrations up to 1.0 mg/ml. Inhibitor patient or control samples were prepared at various dilutions containing 8μg/ml biotinylated fVIII (B-fVIII). Required plasma volumes ranged from 3.6-47.5μl. Following a 120' incubation at 37°C, test sample was added directly to the slide. After 60' at room temperature, capture of B-fVIII was detected with 0.5μg/ml Cy-5 conjugated streptavidin. Test samples were considered positive for the presence of antibody with a competing epitope when the average fluorescence intensity was reduced to less than 25% of the control value.

Competition ELISAs were performed using our previously published method. For each epitope tested, a single biotinylated anti-fVIII MAb was competed against antibodies present in test plasma. Samples were considered positive when a 3 fold or greater increase in antibody titer over controls was observed.


To determine if the microarray method could accurately detect anti-fVIII MAbs in plasma we chose five commercially available anti-fVIII MAbs. We created control inhibitor plasmas by adding 10μg/ml of a single MAb to fVIII deficient plasma. Antibodies selected were against 4 fVIII domains, the A1, A2, C1, and C2. Two MAbs were selected to the C2 domain as previous studies indicate overlapping epitopes. Inhibitor titer for the control MAbs ranged from <2 to 23,300 BU/mg IgG. The microarray accurately detected the presence of all control MAbs tested as measured by a signal reduction greater than or equal to 75% of control. The overlapping epitopes in the C2 domain were also resolved in the microarray as signals in the overlapping portions were significantly decreased. We also tested a patient plasma using both the microarray and ELISA against this panel of five MAbs. The assays showed agreement with detection of antibodies that overlapped C1 antibody 2A9. Neither of the C2 epitopes tested were detected by the microarray. ELISA results confirmed that our test sample was negative for competing antibodies against the C2 epitopes tested by microarray, but positive for other portions of the C2 domain.


We have shown that the B-Cell epitope mapping microarray is capable of accurately detecting anti-fVIII antibodies in patient plasma. Obtaining equivalent data by ELISA requires 37 times greater plasma volume. Although initial development testing used 5 MAbs scaling up to the full panel of 13 non-overlapping epitopes requires no larger plasma sample. The ability to map the B-cell epitopes across the fVIII protein in a single assay confers a huge advantage in terms of time, resources, and clinical feasibility. Using well-characertized murine MAbs to define B-cell epitopes may allow for functional correlations to be made. The ability to track the evolution of an individual patients' antibody epitope spectrum will allow for personalized treatment plans that could result in significantly better treatment outcomes for patients with severe Hemophilia A.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.