Monoclonal antibody (VII-M31) to bovine factor VII: a specific epitope in the gamma-carboxyglutamic acid domain.
A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized. Antibody VII-M31 inhibited the activations of both factors IX and X catalyzed by factor VIIa in the presence of tissue factor, phospholipids, and Ca2+. It possessed a strong affinity for factor VII in the presence of 5 mM Ca2+ (Kd = 1.12 x 10(-10)M). The immunoblotting test of other bovine proteins with the antibody, such as prothrombin, factor X, factor IX, protein C, protein S, and protein Z, in addition to human factor VII, revealed that it recognizes only a Ca2(+)-dependent epitope in bovine factor VII. Furthermore, this antibody VII-M31 covalently coupled with Affi-Gel allowed a simple and rapid purification of bovine factor VII. To localize the antigenic site in factor VII, various segments including a gamma-carboxyglutamic acid (Gla)-domainless protein, a Gla-domain peptide and the fragments isolated from the lysyl endopeptidase digest, were prepared. Among them, the isolated Gla-domain peptide and Gla-domainless factor VII were no longer recognized by antibody VII-M31, indicating that the sequence around the cleavage site by a-chymotrypsin is required for the interaction between the antibody and factor VII. In accordance with this result, the antibody bound specifically to a Gla-containing peptide corresponding to the NH2-terminal 23-50 residues of factor VII, which contains the chymotryptic cleavage site. These results suggest that the specific epitope of this antibody is localized in the carboxy-terminal 28 residues of the Gla-domain constituting the amino-terminal portion of bovine factor VII.
Higashi
S
S
Department of Biology, Faculty of Science, Kyushu University, Fukuoka.
Kawabata
S
S
Nishimura
H
H
Funasaki
H
H
Ohyama
S
S
Miyamoto
S
S
Funatsu
A
A
Iwanaga
S
S
D015055:1-Carboxyglutamic Acid / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005167:Factor VII / Q000276:immunology*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D000939:Epitopes; D015055:1-Carboxyglutamic Acid; D005167:Factor VII; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase