Summary
We 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.
