CALCIUM ION-DEPENDENT MONOCLONAL-ANTIBODY AGAINST HUMAN FIBRINOGEN - PREPARATION, CHARACTERIZATION, AND APPLICATION TO FIBRINOGEN PURIFICATION

We have produced a high-affinity monoclonal antibody classified as IgG (I) with kappa-type light chains that recognizes the calcium ion(Ca2+) -dependent conformation of the D-domain of human fibrinogen. Binding o f fibrinogen in solution to the insolubilized antibody increased in th e presence of increasing concentrations of up to 2 mM Ca2+, the half-m aximal binding being reached at 130 mu M Ca2+. The dissociation consta nt was estimated to be 1.6 x 10(-8) M at 2 mM Ca2+. The antibody was f ound also to be dependent on other divalent metal ions including Zn2+, Mn2+, Co2+ and Cu2+, but not Ba2+, Mg2+ or Sr2+. The synthetic Gly-Pr o-Arg-Pro-amide peptide, which has recently been shown to bind to clos e proximity to the calcium binding site in the D-domain, was unable to elicit the conformation for the antigen to be recognized by this anti body. This antibody was found to be a suitable ligand for the immunoaf finity chromatography of normal and abnormal fibrinogens directly from citrated plasma depleted of the vitamin K-dependent proteins or hepar inized plasma by eliminating the precipitation procedure widely adopte d in conventional techniques of fibrinogen purification. Indeed, fibri nogen Marburg I with the Act chains depleted of the carboxy-terminal A alpha(461-610) residue segment has been purified by this technique, a lthough this dysfibrinogen was difficult to purify by conventional pre cipitation techniques.