Epitope localization of monoclonal antibodies against factor VIII light chain which inhibit complex formation by factor VIII with von Willebrand factor.
We obtained three clones of monoclonal antibodies against factor VIII by immunization with purified human factor VIII. The anti-factor VIII procoagulant activity of these antibodies ranged from 2 to 53 Bethesda units/mg of IgG. According to an immunoblotting study, all antibodies reacted with the 80 kDa light chain but not with 72 kDa peptides derived from thrombin digestion of factor VIII. We attempted to localize the antigenic epitopes of these antibodies by a competitive blocking assay using synthetic peptides and recombinant fragments of the amino-terminal region of factor VIII light chain. In the former assay, a 50 microM peptide containing the fifteen amino acid residues from the Val1670-Glu1684 completely inhibited the binding of the three monoclonal antibodies to immobilized factor VIII. In the latter experiment, 13 reactive recombinant peptides were obtained. Sequences of these peptides revealed fourteen overlapped amino acid residues from Glu1675 to Pro1688. All three antibodies at a final concentration of around 10 micrograms/ml completely inhibited the binding of 125I-labelled factor VIII to immobilized von Willebrand factor (vWF). We conclude that ten amino acid residues, 1675EDFDIYDEDE1684 in the factor VIII light chain are important for complex formation with vWF.
Shima
M
M
Department of Pediatrics, Nara Medical College, Japan.
Yoshioka
A
A
Nakai
H
H
Tanaka
I
I
Sawamoto
Y
Y
Kamisue
S
S
Terada
S
S
Fukui
H
H
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D005169:Factor VIII / Q000276:immunology*; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology*; D011485:Protein Binding; D014841:von Willebrand Factor / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D014841:von Willebrand Factor; D005169:Factor VIII