title: Quantitation of anti-factor VIII antibodies in human plasma.
abstract: 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 noninhibitory 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 (+/-1.5) pM. The calibrator and the human plasma anti-fVIIIAb were captured on recombinant fVIII (rfVIII)- coupled microspheres and probed with mouse anti-human Ig-R-phycoerythrin. Plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were compared with 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1 BU/mL (94.6 +/- 0.8 nM), 11 BU/mL (214.3 +/- 7.1 nM), 106 BU/mL (2209.4 +/- 84.9 nM), 140 BU/mL (2417.7 +/- 3.8 nM) 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%) showed detectable anti-fVIIIAb in the range of 0.6 to 6.2 nM, whereas 13 (33%) of the 39 inhibitor-free hemophilia A subjects were positive for anti-fVIIIAb in the range of 0.5 to 20 nM. The method may be useful for therapeutic management of hemophilia A patients.
authors.affiliation: Department of Biochemistry, College of Medicine, University of Vermont, Burlington, VT, USA.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Krudysz-Amblo
authors.forename: Behnaz
authors.initials: B
authors.lastname: Parhami-Seren
authors.forename: Saulius
authors.initials: S
authors.lastname: Butenas
authors.forename: Kathleen E
authors.initials: KE
authors.lastname: Brummel-Ziedins
authors.forename: Edward D
authors.initials: ED
authors.lastname: Gomperts
authors.forename: Georges E
authors.initials: GE
authors.lastname: Rivard
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Mann
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D001323:Autoantibodies / Q000032:analysis* / Q000097:blood* / Q000378:metabolism; D001667:Binding, Competitive; D015415:Biomarkers / Q000032:analysis / Q000097:blood; D002138:Calibration; D005169:Factor VIII / Q000008:administration & dosage / Q000276:immunology* / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods / Q000592:standards; D006467:Hemophilia A / Q000097:blood / Q000175:diagnosis / Q000188:drug therapy / Q000276:immunology; D006801:Humans; D007260:Infusion Pumps; D007518:Isoantibodies / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D015415:Biomarkers; D007518:Isoantibodies; D011994:Recombinant Proteins; D005169:Factor VIII