Phenotypes of Allo- and Autoimmune Antibody Responses to FVIII Characterized by Surface Plasmon Resonance

Kenneth B. Lewis, Richard J. Hughes, Melinda S. Epstein, Neil C. Josephson, Christine L. Kempton, Craig M. Kessler, Nigel S. Key, Tom E. Howard, Rebecca Kruse-Jarres, Jeanne M. Lusher, Christopher E. Walsh, Raymond G. Watts, Ruth A. Ettinger, Kathleen P. Pratt

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17 Scopus citations


Evidence of antibody isotype/subtype switching may provide prognostic value regarding the state of immune responses to therapeutic proteins, e.g. anti-factor VIII (FVIII) antibodies that develop in many hemophilia A patients, clinically termed "inhibitors". A sensitive, high- information-content surface plasmon resonance (SPR) assay has been developed to quantify IgG subtype distributions and the domain specificity of anti-drug antibodies. Plasma samples from 22 subjects with an allo- or auto-immune reaction to FVIII were analyzed. Pre-analytical treatment protocols were developed to minimize non-specific binding and specific matrix interference due to von Willebrand factor-FVIII interactions. The dynamic range for IgG quantification was 0.2-5 μg/ml (∼1-33 nM), allowing characterization of inhibitor-positive samples. Subtype-specific monoclonal antibodies were used to quantify the IgG subtype distribution of FVIII-specific antibodies. Most samples obtained from multiply-infused inhibitor subjects contained IgG4 antibodies. Several distinct phenotypes were assigned based on the IgG subtype distribution: IgG1, IgG4, IgG1 & IgG4, and IgG1, IgG2 & IgG4. An IgG1-only response was found in mild/moderate HA subjects during early FVIII infusions, and analysis of serial samples followed antibody class switching as several subjects' immune responses developed. Competition studies utilizing a recombinant FVIII-C2 domain indicated 40-80% of FVIII-specific antibodies in most samples were directed against this domain.

Original languageEnglish
Article numbere61120
JournalPLoS ONE
Issue number5
StatePublished - 10 May 2013
Externally publishedYes


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