A soluble recombinant factor VIII fragment containing the A2 domain binds to some human anti-factor VIII antibodies that are not detected by immunoblotting.
Human factor VIII (fVIII) inhibitors are pathologic antibodies that inactivate fVIII. A cDNA clone was modified to encode fVIII amino acid residues 373-740 for expression in a baculovirus vector in insect cells. The encoded protein fragment H2 was produced as a soluble, secreted protein, and it was used to test inhibitor plasmas for the presence of antibodies that were not detected by immunoblotting. Seven of 13 inhibitors that bound only to the fVIII light chain by immunoblotting also bound to fragment H2 in an immunoprecipitation assay. Thus multi-chain inhibitor reactivity of inhibitors is more frequent than previously reported. One of these inhibitors was shown to share the epitope for other inhibitors that bind to H2 within amino acid residues 373-541 in immunoblotting assays. The sensitive immunoprecipitation assay described allows determination of relative H2 binding capacity of the total IgG and epitope localization of inhibitors that cannot be similarly characterized by immunoblotting.
Scandella
D
D
Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross Blood Services, Rockville, MD.
Timmons
L
L
Mattingly
M
M
Trabold
N
N
Hoyer
L W
LW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D015592:Immunoradiometric Assay; D007313:Insecta / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D012995:Solubility
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005169:Factor VIII