Monoclonal antibodies to human vitamin K-dependent protein S.
Monoclonal antibodies to human protein S have been prepared using established hybridoma technology. One antibody was isolated that binds protein S only when Ca2+ is present; others bind antigen equally well in the presence or absence of EDTA. Other antibodies display a diminished affinity for protein S in the presence of EDTA. Purified immunoglobulins from cell lines displaying Ca2+ dependence were immobilized and used to purify protein S from fractions obtained by barium precipitation of citrated plasma, ammonium sulfate fractionation, and chromatography on diethylaminoethanol (DEAE)-Sephadex and dextran sulfate agarose. Essentially homogeneous protein S was isolated from the barium-citrate-adsorbed, 35% ammonium-sulfate-soluble proteins using a totally Ca2+-dependent antibody and EDTA elution. Protein S and several substances of higher mol wt were bound directly from plasma by a partially Ca2+-dependent antibody and were eluted partially with EDTA and NaCl and finally with NaSCN. The largest and most abundant of the high mol wt materials is likely protein S-complement C4b-binding protein complex. The immunoaffinity-isolated protein S was found to be indistinguishable from conventionally isolated protein S in terms of activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility, and by high-performance liquid chromatography (HPLC). These studies establish reagents that can probe the structure of protein S and isolate protein S in its free and complexed forms.
D000911:Antibodies, Monoclonal; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005164:Factor IX / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D006023:Glycoproteins / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008433:Mathematics; D011486:Protein C; D017293:Protein S; D011516:Prothrombin / Q000378:metabolism; D014812:Vitamin K / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D011486:Protein C; D017293:Protein S; D014812:Vitamin K; D011516:Prothrombin; D005164:Factor IX; D005170:Factor X