Production of metal ion-dependent monoclonal antibodies against peptides in bovine prothrombin fragment 1.
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 alpha-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 gamma-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.
Mahassni
Sawsan H
SH
Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia. sawsanmahassni@hotmail.com
Klapper
David G
DG
Hiskey
Richard G
RG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002118:Calcium / Q000276:immunology*; D002413:Cations, Divalent / Q000276:immunology; D002417:Cattle; D005260:Female; D006825:Hybridomas / Q000276:immunology; D008274:Magnesium / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000276:immunology*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology*; D011498:Protein Precursors / Q000276:immunology*; D011516:Prothrombin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D010446:Peptide Fragments; D010455:Peptides; D011498:Protein Precursors; C029733:prothrombin fragment 1; D011516:Prothrombin; D008274:Magnesium; D002118:Calcium