Conformation-specific monoclonal antibodies to the calcium-induced structure of protein C.
Monoclonal antibodies to various domains of human protein C were characterized, and the cross-reactivity of these antibodies with other vitamin K-dependent proteins was explored. Three antibodies, JTC-1, -2, and -3 reacted with protein C only in the presence of Ca2+ and were shown to bind to the light chain of protein C. It is suggested that these antibodies recognize a gamma-carboxyglutamic acid domain-related conformational change induced by metal ions, evidenced by the fact that half-maximal binding was observed at calcium concentration of 0.5, 0.6, and 0.7 mM, respectively, by the fact that these antibodies, even in the presence of Ca2+, do not react with gamma-carboxyglutamic acid domainless protein C, and by the fact that Zn2+ and Tb3+ support binding in essentially the same way. Each cell line was stabilized by recloning five times. In addition each antibody had a single isoelectric point and was of the IgG1 kappa class. The interaction of antibodies JTC-1, -2; and -3 with protein C-Ca2+ was characterized by a single class of binding sites with Kd of 3.98 X 10(-9) M, 4.01 X 10(-9) M, and 6.76 X 10(-9) M, respectively. However, antibodies JTC-1, -2, and -3 bound to prothrombin-Ca2+ with Kd of 7.81 X 10(-9) M, 2.0 X 10(-7) M, and higher than 1.0 X 10(-5) M, respectively. In addition they had weak affinity for factor X in the presence of Ca2+. The results indicate that the antibodies JTC-1, -2, and -3 are conformation-specific monoclonal antibodies directed against an at least partially common metal ion-induced three-dimensional structure in protein C, prothrombin, and factor X.
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D011486:Protein C; D011487:Protein Conformation; D011516:Prothrombin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D011486:Protein C; D011516:Prothrombin; D002918:Chymotrypsin; D002118:Calcium