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Thromb Haemost 1995; 73(04): 662-667
DOI: 10.1055/s-0038-1653837
Original Articles
Coagulation
Schattauer GmbH Stuttgart

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

Mikihiro Takebe
1   The Division of Hemostasis and Thrombosis Research, Institute of Hematology, Jichi Medical School, Tochigi, Japan
,
Gilbu Soe
2   Central Research Laboratories, latron Laboratories Inc., Mitodai, Mito, Takomachi, Katori-Gun, Chiba, Japan
,
Isao Kohno
2   Central Research Laboratories, latron Laboratories Inc., Mitodai, Mito, Takomachi, Katori-Gun, Chiba, Japan
,
Teruko Sugo
1   The Division of Hemostasis and Thrombosis Research, Institute of Hematology, Jichi Medical School, Tochigi, Japan
,
Michio Matsuda
1   The Division of Hemostasis and Thrombosis Research, Institute of Hematology, Jichi Medical School, Tochigi, Japan
› Author Affiliations
Further Information

Publication History

Received 08 September 1994

Accepted after resubmission 04 January 1995

Publication Date:
26 July 2018 (online)

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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.

 

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