Monoclonal antibodies to human plasma protein X alias complement S-protein

Protein X alias complement S-protein was isolated by dissociation from purified XC5b-9 (fluid-phase terminal C5b-9) complexes with 250 mM deoxycholate and subsequent sucrose density gradient centrifugation and Sephacryl gel chromatography. Polyclonal rabbit and monoclonal mouse antibodies were used to preliminarily characterize the protein in human serum and plasma. In plasma, Protein X yielded a symmetrical immunoprecipitate of alpha 2-mobility in a crossed immunoelectrophoresis assay. However, a second immunoprecipitate of alpha 1-mobility was observed when serum was analysed; this precipitate represented Protein X in complex with antithrombin-III. The co-precipitation of Protein X with serum antithrombin-III was exploited for establishing a simple screening test for unequivocal identification of monoclonal anti-Protein X antibodies. SDS-PAGE immunoblotting with monoclonal antibodies showed that Protein X exhibits pronounced microheterogeneity, migrating as a diffuse moiety of approx. Mr 80-90 000. Additionally, a small amount of polymeric aggregates appear to be present in plasma. Reduction of disulfide bonds led to liberation of a polypeptide of approx. 15 K as discerned by two-dimensional SDS-PAGE immunoblotting. Protein X is not cleaved to lower molecular weight entities during the process of blood coagulation or during formation of fluid-phase terminal complement complexes. The plasma concentrations in healthy adults were in the range of 500-700 micrograms/ml. The availability of methods for isolating Protein X and raising monoclonal antibodies will facilitate further studies on the dual role of this protein in the terminal complement and coagulation cascades.