title: A recombinant factor VIII A2 domain polypeptide quantitatively neutralizes human inhibitor antibodies that bind to A2.
abstract: Human antibodies that inactivate coagulation factor VIII (fVIII), known as inhibitors, have been shown by immunoblotting or immunoprecipitation assays to bind predominantly to epitopes within the A2 and/or C2 domains of the fVIII protein. Because these assays simply measure antibody binding, a soluble recombinant polypeptide containing the fVIII A2 domain was used to develop a quantitative inhibitor neutralization assay for antibodies that bound only to A2 by immunoblotting assay. Antibodies from six of eight inhibitor plasmas were fully neutralized by A2 (> or = 90%), whereas two were only partially neutralized. These results established the fVIII inhibitor properties of anti-A2 antibodies. In immunoprecipitation assays, five of the eight inhibitors also had significant levels of anti-light-chain antibody. In one case, this light-chain antibody was shown to have inhibitor activity. Because it did not bind to the C2 domain, this antibody appears to define a new inhibitor epitope within the fVIII light chain. Another inhibitor, which was partially neutralized by A2, was not neutralized by the light chain, even though it contained anti-light-chain antibodies by immunoprecipitation assay. Our results show additional complexities of the immune response to fVIII.
authors.affiliation: Holland Laboratory, American Red Cross, Rockville, MD 20855.
authors.forename: D
authors.initials: D
authors.lastname: Scandella
authors.forename: M
authors.initials: M
authors.lastname: Mattingly
authors.forename: R
authors.initials: R
authors.lastname: Prescott
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D000939:Epitopes / Q000378:metabolism; D005169:Factor VIII / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D010957:Plasmids; D021382:Protein Sorting Signals / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism*; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005169:Factor VIII