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Protein S in circulation is in a dynamic equilibrium with C4b binding protein (C4bBP), thus affecting the measurement of free protein S antigen. We addressed the issue of overestimation of the free protein S concentration with current immunoassays due to the dynamic equilibrium and propose a new method for its accurate determination. Our assay system was tested at different reaction temperatures using purified free protein S, protein S-C4bBP complexes, plasma samples, and a commercially available free protein S assay kit. At a reaction temperature of 37°C, the free protein S fraction increased from 0.5 ng/ml (at 4°C) to 7.8 ng/ml, and from 4.5 ng/ml (at 4°C) to 56 ng/ml when the concentration of the assayed protein S-C4bBP complexes was 20 ng/ml and 200 ng/ml, respectively. In plasma samples, free protein S levels were approximately 0.8 μg/ml and 6 μg/ml higher at 25°C and 37°C, respectively compared to measurements at 4°C. Measurements of free protein S in plasma using a commercially available assay kit were approximately 0.6 μg/ml higher at 25°C than measurements performed at 4°C. Dynamic equilibrium between protein S and C4bBP affects the measurement of free protein S antigen. Measurement of free protein S antigen should be performed under conditions where protein S is not dissociated from protein S-C4bBP complexes, as exemplified by assay at low temperature (4°C).

Dynamic Equilibrium between Protein S and C4b Binding Protein Is Important for Accurate Determination of Free Protein S Antigen