Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display.
Clotting factor VIII (fVIII)-inhibitory antibodies represent a major problem in the treatment of haemophilia A. To understand the inactivation mechanisms and to pave the way towards modifications of recombinant clotting factors that reduce their immunogenicity, the exact localization of immunodominant epitopes is required. Here, a random peptide phage display library was employed to identify epitopes of polyclonal fVIII antibodies isolated from patient's plasma by affinity chromatography. FVIII-binding specificity and inhibitory activity of the isolated fVIII antibodies were confirmed by ELISA and Bethesda assays. Phage selection on the individual samples yielded several phages which were displaced from binding to the respective antibody preparation by fVIII. Their homology with amino acid motifs of human fVIII and immunoprecipitation results with radioactively labelled fVIII fragments suggested putative epitopes in the A1, A2 and C1 domains of fVIII for one and in the C2 domain for another patient. Synthetic peptides corresponding to the A2, C1 and C2 domain epitopes blocked antibody binding to fVIII and partially neutralized the inhibitory activity of the respective plasma in Bethesda assays. These results provide the proof of principle that random peptide libraries can be used for the mapping of epitopes in a polyclonal antibody preparation.
Children's Hospital, Nikolaus Fiebiger Center of Molecular Medicine, University of Erlangen-Nuernberg, Glückstr. 6, 91054 Erlangen, Germany.
Christiane
C
Mühle
Stefan
S
Schulz-Drost
Alexey V
AV
Khrenov
Evgueni L
EL
Saenko
Jens
J
Klinge
Holm
H
Schneider
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000906:Antibodies / Q000737:chemistry*; D002675:Child, Preschool; D002846:Chromatography, Affinity; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry; D005169:Factor VIII / Q000737:chemistry*; D006467:Hemophilia A / Q000628:therapy; D006801:Humans; D007223:Infant; D008875:Middle Aged; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D000906:Antibodies; D000939:Epitopes; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D005169:Factor VIII