Production of metal ion-dependent monoclonal antibodies against peptides in bovine prothrombin fragment 1

Abstract

Bovine prothrombin fragment 1 (F-1: the amino-terminal 156 residues of prothrombin) is used as a model to study the Ca(II) and phospholipid binding of prothrombin. The 35–46 segment in F-1 posses an α-helical region and three aromatic residues, conserved in several vitamin K-dependent blood coagulation factors. These residues are believed to have a specific function and to be important in the phospholipid binding of F-1. The 47–62 region, a disulfide loop, is believed to stabilize the γ-carboxyglutamic acid domain of the protein.

Goals of this research were to produce monoclonal antibodies against the above two sequences, for later functional studies. Antibodies S9–32.8 and S9–5.5 were produced against the 35–46 sequence; antibody S11–23.4 was raised against the 47–62 region. Both S9–32.8 and S9–5.5 bound to F-1 immobilized on ELISA plates in the presence of 10 mM Ca(II) with higher affinity than to F-1 coated in the presence of 10 mM Mg(II) or in the absence of metal ions. S11–23.4 showed greatest binding to F-1 coated in the presence of 10 mM Mg(II). Thus, the epitopes of the antibodies are metal ion-dependent and are developed by Ca(II) binding to F-1.