Dynamic equilibrium between protein S and C4b binding protein is important for accurate determination of free protein...

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 degrees C, the free protein S fraction increased from 0.5 ng/ml (at 4 degrees C) to 7.8 ng/ml, and from 4.5 ng/ml (at 4 degrees 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 microg/ml and 6 pg/ml higher at 25 degrees C and 37 degrees C, respectively compared to measurements at 4 degrees C. Measurements of free protein S in plasma using a commercially available assay kit were approximately 0.6 microg/ml higher at 25 degrees C than measurements performed at 4 degrees 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 degrees C).