Quantitation of Non-Inhibitory Anti-Factor VIII Antibodies in human Plasma

Abstract

Hemophilia A is a clinically heterogeneous bleeding disorder characterized by the absence of functional factor VIII (FVIII). The most severe complication from exposure to exogenous factor VIII therapy is the development of alloantibodies that neutralize the effect of the therapeutic agent. The standard method for quantitation of FVIII inhibitors in human plasma is by the Nijmegen method, a modification of the Bethesda assay. Our objective was to develop a sensitive and specific fluorescence based immunoassay for the quantitation of anti-FVIII antibodies (FVIIIAb) in human plasma. An affinity purified human anti-FVIIIAb isolated from a hemophilia A patient was used as a calibrator, with a detectability limit of 40±1.5pM. The calibrator and the human plasma anti-FVIIIAb were captured on recombinant FVIII coupled microspheres and probed with mouse anti-human Ig conjugated to R-Phycoerythrin. Citrated human plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were studied and compared to 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1BU (94.6±0.08nM), 11BU (214.3±7.1nM), 106BU (2209.4±84.9nM) and 140BU (2417.7±3.8nM) as measured by the Nijmegen method. Among the 150 normal control plasmas, 4 subjects (3%) were determined as positive (anti-FVIIIAb ≥ 40pM) with concentrations in the range of 0.6nM to 1.9nM, well below that of 1BU (94.6±0.08nM) inhibitor-positive sample. Anti-FVIIIAb was also evaluated in seven commercial inhibitor-free plasma samples reported to contain <1% FVIII activity. Two of the seven plasmas were found to contain anti-FVIIIAb with concentrations of 112.9±1.9nM and 71.1±8.5nM. The concentration of the antibody in both samples was within the range of the measured molar concentration of an inhibitor-positive 1BU titer plasma sample. Thirty two inhibitor-negative hemophilia A subjects were also quantitated for anti-FVIIIAb. Eleven of the 32 samples tested positive for anti-FVIIIAb with molar concentrations in the range of 0.6–20nM. The overall prevalence of the non-inhibitory anti-FVIIIAbs in hemophilia A subjects evaluated by our immunoassay (n=39) is approximately 33% relative to healthy donor population in which 3% were determined as positive (≥40pM) for FVIIIAb.