Nov 5, 1989

Conformational changes in an epitope localized to the NH2-terminal region of protein C. Evidence for interaction of protein C domains

The Journal of Biological Chemistry
C L OrthnerD K Strickland

Abstract

Murine monoclonal antibodies, developed following immunization with human protein C, were characterized for their ability to bind antigen in the presence of either CaCl2 or excess EDTA. Three stable clones were obtained which produced antibodies that bound to protein C only in the presence of EDTA. All three antibodies bound to the light chain of protein C on immunoblots and also bound to the homologous proteins factor X and prothrombin in solid-phase radioimmunoassays. One antibody, 7D7B10 was purified and studied further. The binding of 7D7B10 to human protein C was characterized by a KD of 1.4 nM. In competition studies, it was found that the relative affinity of the antibody for protein C was 20-40-fold higher than for prothrombin, fragment 1 of prothrombin, or factor X. In contrast, 7D7B10 was unable to bind to factor IX or bovine protein C. The effect of varying Ca2+ concentration on the interaction of the antibody with protein C was complex. Low concentrations of Ca2+ enhanced the formation of the protein C-antibody complex with half-maximal effect occurring at approximately 60 microM metal ion. However, higher concentrations of Ca2+ completely inhibited 7D7B10 binding to protein C with a K0.5 of 1.1 mM. Furthermore, mil...Continue Reading

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Mentioned in this Paper

Monoclonal Antibodies
Monoclonal antibodies, antineoplastic
Immunoblotting, Reverse
Clotting Factor II Assay
Mice, Inbred BALB C
Epidermal Growth Factor
F9 gene
Protein C, human
Murine
Antigenic Specificity

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