title: Immunoblot cross-reactivity of factor VIII inhibitors with porcine factor VIII.
abstract: Porcine factor VIII has been used successfully to treat factor VIII inhibitor patients whose plasmas have minimal cross-reactivity to porcine factor VIII. However, some inhibitor plasmas do inhibit porcine factor VIII, and the extent of procoagulant inhibition often increases after treatment with porcine factor VIII. Because there is no information about the porcine factor VIII epitopes with which these antibodies react, we have compared the immunoblot and enzyme-linked immunosorbent assay (ELISA) reactivities with porcine and human factor VIII for 20 inhibitor plasmas (11 from hemophilia A patients and 9 autoantibodies). Immunoblots identified binding to porcine factor VIII for only 2 of the 12 plasmas from patients who had not received porcine factor VIII, but this reactivity could not be predicted from the inhibitor titer to porcine factor VIII. Immunoblot reactivity with porcine factor VIII was detected for 7 of 8 inhibitor plasmas from patients who had been previously treated with porcine factor VIII, and the strength of this reactivity was generally related to the inhibitor titer. Of the 5 plasmas that were immunoblot positive with the porcine factor VIII A2 domain, 4 had inhibitor titers greater than 45 Bethesda units when tested with porcine factor VIII, whereas only 1 of 15 of the other plasmas had this level of inhibitor activity with porcine factor VIII. In contrast, immunoblot reactivity to the porcine factor VIII A1 domain did not correlate with the antiporcine VIII inhibitor titer. We also determined the effect of preincubation with human or porcine factor VIII on immunoblot reactivity. In one case, immunoblot reactivity with porcine factor VIII was absorbed with porcine, but not human, factor VIII, which is consistent with antibody formation after treatment with porcine factor VIII. In no cases did human factor VIII reduce the reactivity of inhibitor plasmas with the porcine A1 domain, suggesting that these antibodies are directed at unique porcine factor VIII determinants. The reactivity to porcine A2 in 2 plasmas probably represented cross-reactivity of similar A2 determinants, because it was absorbed by both human and porcine factor VIII. Although the ELISA assays with porcine factor VIII detected antibodies in some plasmas that could not be identified by inhibitor assay or immunoblot, the level of ELISA reactivity was generally consistent with the titers of the other assays.
authors.affiliation: Holland Laboratory, American Red Cross, Rockville, MD 20855, USA.
authors.forename: K
authors.initials: K
authors.lastname: Koshihara
authors.forename: J
authors.initials: J
authors.lastname: Qian
authors.forename: P
authors.initials: P
authors.lastname: Lollar
authors.forename: L W
authors.initials: LW
authors.lastname: Hoyer
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000276:immunology*; D015153:Blotting, Western; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology*; D006467:Hemophilia A / Q000097:blood / Q000276:immunology*; D006801:Humans; D007518:Isoantibodies / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D013045:Species Specificity; D013552:Swine / Q000276:immunology*; D013917:Thrombin / Q000378:metabolism
chemical_list: D001323:Autoantibodies; D007518:Isoantibodies; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005169:Factor VIII; D013917:Thrombin