Identification of novel factor VIII inhibitor epitopes using synthetic peptide arrays.
OBJECTIVE
Mapping the antibody-binding sites on the factor VIII (FVIII) protein opens the prospect of studying the development of FVIII inhibitors and the alteration of inhibitor specificities over time. This paper describes a novel approach to the mapping of FVIII antibody-binding sites.


METHODS
Immobilized synthetic peptide arrays covering 80% of the complete 2351 amino acid sequence of factor VIII (FVIII) were used to determine epitope specificity of 6 alloantibodies and 3 autoantibodies inhibitory to FVIII activity. This detailed assessment was carried out using a modified enzyme-linked immunosorbent assay with plasma from normal persons or hemophilia A patients without inhibitors as negative controls.


RESULTS
Antibody-combining sites could be differentiated in both a qualitative and quantitative manner and were patient-specific. Highly reactive peptides were restricted to specific sites in the A1-A3 and C1-C2 domains and were not proximal to known proteolytic cleavage sites. Free peptides incubated in vitro with the plasmas of 3 patients significantly reduced residual inhibitor titers in a dose-dependent manner.


CONCLUSIONS
This technique permits the study of the development and specificity of FVIII inhibitors, can detect and differentiate between inhibitory and noninhibitory antibodies using immobilized or free peptides respectively, permits correlation of antibody-combining sites with inhibition of FVIII activity and provides a basis for the development of inhibitor adsorption or neutralization technology.
Palmer
D S
DS
Ottawa Centre, Canadian Red Cross Society, Canada.
Dudani
A K
AK
Drouin
J
J
Ganz
P R
PR
D000595:Amino Acid Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping / Q000379:methods; D005169:Factor VIII / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000627:therapeutic use; D006467:Hemophilia A / Q000188:drug therapy / Q000276:immunology; D006801:Humans; D007518:Isoantibodies / Q000276:immunology*; D019151:Peptide Library
D007518:Isoantibodies; D019151:Peptide Library; D005169:Factor VIII