CHARACTERIZATION OF MONOCLONAL ANTI-HUMAN FACTOR-VII ANTIBODY (B101 B1) THAT RECOGNIZED 3-DIMENSIONAL STRUCTURES NEAR ARG AT POSITION-79 INTHE FIRST EGF-LIKE DOMAIN/

Murine monoclonal antibodies (designated hVII-B101/B1, hVII-DC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B 1 that did not bind reduced FVIIa. The immunoglobulin of the three mon oclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation fact ors except FVII and did not cross-react with proteins in the immunoblo tting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVII-DC6/3D8 the heavy chain. hVII-B 101/B1 bound FVII without Ca2+, and possessed stronger affinity for FV II in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10(-10) M in the presence of 5 mM CaCl2. The antibody inhibited the b inding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced gamma-carboxyglutaminic aci d (Gla)-domainless-FVII and/or FVIIa, hVII-B101/B1 showed a similar pa ttern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test, hVII-B101/B1 reacted differen tly with the FVII from the dysfunctional FVII variant, FVII Shinjo, wh ich has a substitution of Gin for Arg at residue 79 in the first epide rmal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97, 1995) compared with normal FVII when used as a solid phase- antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. Thes e results suggested that the specific epitope of the antibody, hVII-B1 01/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.