Submitted August 19, 2008
Accepted December 19, 2008
Quantitation of anti-factor VIII antibodies in human plasma
Jolanta Krudysz-Amblo, Behnaz Parhami-Seren, Saulius Butenas, Kathleen E. Brummel-Ziedins, Edward D. Gomperts, Georges E. Rivard, and Kenneth G. Mann*
Department of Biochemistry, College of Medicine, University of Vermont, Burlington, VT, United States
Children's Hospital Los Angeles, Los Angeles, CA, United States
CHU Sainte-Justine, Montreal, Quebec, Canada
* Corresponding author; email: kenneth.mann{at}uvm.edu.
The presence of antibodies (Abs) in hemophilia A patients can potentially influence the therapeutic qualities of factor VIII (fVIII) administration. Much work has been focused on the presence of inhibitory antibodies, whereas the quantitation of non-inhibitory anti-fVIII antibodies has been largely undetermined. Our objective was to develop a sensitive and specific fluorescence based immunoassay (FLI) for the quantitation of anti-fVIIIAbs in human plasma. Affinity purified human anti-fVIIIAb, isolated from a hemophilia A subject, 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 (rfVIII) coupled-microspheres and probed with mouse anti-humanIg-R-Phycoerythrin. Plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were compared to 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1BU/mL (94.6±0.8nM), 11BU/mL (214.3±7.1nM), 106BU/mL (2209.4±84.9nM), 140BU/mL (2417.7±3.8nM) as measured by the Nijmegen method. We also describe the validation of a mouse anti-human fVIIIAb as a surrogate calibrator. Four healthy individuals (3%) show detectable anti-fVIIIAb in the range of 0.6-6.2nM, whereas 13 (33%) out of the 39 inhibitor-free hemophilia A subjects are positive for anti-fVIIIAb in the range of 0.5-20nM. The method may be useful for therapeutic management of hemophilia A patients.
