Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

• Published in: PloS one Vol. 8; no. 4; p. e60688
• Main Authors: Hausammann, Stefanie, Vogel, Monique, Kremer Hovinga, Johanna A, Lacroix-Desmazes, Sebastien, Stadler, Beda M, Horn, Michael P
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 23.04.2013; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Analysis; Ankyrin; Ankyrin Repeat; Ankyrins; Antibodies; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antigenic determinants; Antigens; Antigens - genetics; Antigens - immunology; Assaying; Autoantibodies - blood; Autoantibodies - immunology; Binding Sites; Biology; Blood plasma; Coagulation; Coagulation factor VIII; Coagulation factors; Design; Diagnostic systems; Enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; Epitopes; Factor VIII; Factor VIII - immunology; Factor VIII - metabolism; Factor VIII deficiency; Hematology; Hemophilia; Hemophilia A - blood; Hemophilia A - diagnosis; Hemophilia A - immunology; Human health and pathology; Humans; Immunoglobulins; Immunology; Inhibition; Laboratories; Life Sciences; Medical diagnosis; Medicine; Mimicry; Molecular biology; Molecular Mimicry; Molecular Sequence Data; Mutation; Patients; Peptide Library; Peptides; Peptides - genetics; Peptides - immunology; Protein Binding; Proteins; Target recognition
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0060688

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.