Calcium Ion-Dependent Monoclonal Antibody Against Human Fibrinogen: Preparation, Characterization, and Application to Fibrinogen Purification

Abstract

SummaryWe have produced a high-affinity monoclonal antibody classified as IgG1 with K-type light chains that recognizes the calcium ion(Ca2+)- dependent conformation of the D-domain of human fibrinogen. Binding of fibrinogen in solution to the insolubilized antibody increased in the presence of increasing concentrations of up to 2 mM Ca2+, the half-maximal binding being reached at 130 μM Ca2+. The dissociation constant was estimated to be 1.6 × 10-8 M at 2 mM Ca2+. The antibody was found also to be dependent on other divalent metal ions including Zn2+, Mn2+, Co2+ and Cu2+, but not Ba2+, Mg2+ or Sr2+. The synthetic Gly-Pro-Arg-Pro-amide peptide, which has recently been shown to bind to close proximity to the calcium binding site in the D-domain, was unable to elicit the conformation for the antigen to be recognized by this antibody. This antibody was found to be a suitable ligand for the immunoaffinity chromatography of normal and abnormal fibrinogens directly from citrated plasma depleted of the vitamin K-dependent proteins or heparinized plasma by eliminating the precipitation procedure widely adopted in conventional techniques of fibrinogen purification. Indeed, fibrinogen Marburg I with the Aa chains depleted of the carboxy-terminal Aα(461-610) residue segment has been purified by this technique, although this dysfibrinogen was difficult to purify by conventional precipitation techniques.

The activation peptide cleft exposed by thrombin cleavage of FXIII-A2 contains a recognition site for the fibrinogen α chain

“…Recombinant fibrinogen (a,b,g) 2 was obtained from stably transfected Chinese hamster ovary (CHO) cells (a generous gift from Susan Lord) and purified by ammonium sulfate precipitation and IF-1 affinity chromatography as previously described. [29][30][31] The purified fibrinogen was dialyzed against 100 mM NaCl, 50 mM tris(hydroxymethyl)aminomethane (Tris) at pH 7.4 and stored at 280°C. All proteins were analyzed for purity using 4% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).…”

The influence of type 2 diabetes on fibrin structure and function

“…Fibrinogen purification Fibrinogen was purified from the plasma of each subject using IF-1 affinity chromatography as described previously [24], and was standardised at a concentration of 1 mg/ml for the analysis of clot structure.…”

Mural thrombus generation in type 2A and 2B von Willebrand disease under flow conditions

“…12,14,15 Antifibrinogen monoclonal antibody IF-1 recognizes the fibrinogen conformation in the presence of calcium ions. 16 Both monoclonal antibodies were used as F(abЈ) 2 fragments prepared by pepsin digestion of purified immunoglobulin G (IgG) at low pH and the collection of flow-through fractions in a protein A-Sepharose (Pharmacia-LKB Japan, Tokyo, Japan) column. 12,15 …”