Mapping of FVIII inhibitor epitopes using cellulose-bound synthetic peptide arrays.
Epitope mapping using antibodies against factor VIII (FVIII) has been performed using blotting techniques with truncated and/or digested FVIII molecules. Here, we focused on the precise mapping of affinity purified IgG from patients with an immune response against blood clotting FVIII using synthetic peptide arrays on cellulose membranes comprising the entire sequence of FVIII. The aim was to elucidate the epitope profile from different inhibitors and possibly detect new epitopes, which have not been described before. The epitope patterns from five patients showed reactivity with all domains in the FVIII molecule, but were different between various patients. These results included epitopes usually buried within the folded protein. However, in competition assays using FVIII as competitive agent in a mixture with inhibitor IgG, the most immunogenic regions were located in the FVIII light chain. Our results show that the C1 domain was the region with highest immunogenicity in all patients. Here, we demonstrate that the SPOT method is very well suited for the precise location of epitopes in the core of the protein, which usually cannot be detected by other methods.
Kopecky
Eva-Maria
EM
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Greinstetter
Sabine
S
Pabinger
Ingrid
I
Buchacher
Andrea
A
Römisch
Jürgen
J
Jungbauer
Alois
A
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D002482:Cellulose; D018604:Epitope Mapping / Q000379:methods*; D005169:Factor VIII / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology*; D006467:Hemophilia A / Q000097:blood / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D066298:In Vitro Techniques; D008297:Male; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D007074:Immunoglobulin G; D010446:Peptide Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D005169:Factor VIII; D002482:Cellulose