ONE-STEP ASSAY FOR THE DETERMINATION OF FREE PROTEIN-S ANTIGEN IN PLASMA USING REAL-TIME BIOSPECIFIC INTERACTION ANALYSIS

Real-time biospecific interaction analysis based on optical detection by surface plasmon resonance was used to develop an accurate one-step method for the direct measurement of free protein S in human plasma. T his assay was validated, compared with classical immunological methods and shown to be suitable for the routine clinical diagnosis of protei n S deficiency. The method relies on the specific capture of free prot ein S directly from plasma by a monoclonal antibody (mAb), 34G2, immob ilized on a sensor chip surface. A calibration curve was established w ith serial dilutions of standard plasma (working range 5-50%) and a li near relationship was found to exist between the relative response in resonance units (RU) and the concentration of free protein S expressed as percentage plasma dilution (r = 0.99). The specificity of the assa y was confirmed using purified human protein S and polyethylene glycol treated plasma. In addition, it could be demonstrated that no dissoci ation of C4b-BP-protein S complexes occurred under the chosen experime ntal conditions. The technique was reproducible with inter-assay, intr a-assay and inter-sensor chip variation coefficients of 1.5-5.4%, 2-3. 1% and 4.4-4.9%, respectively, as evaluated in two different plasma sa mples. Since all tests are automatic and long series of analyses can b e performed with the same sensor chip, the method was applied to the d etermination of free protein S antigen in plasma from 20 normal blood donors and 38 thrombophilic patients. Results displayed excellent corr elation with those of free protein S enzyme-linked immunosorbent assay (r = 0.99) and rocket immunoelectrophoresis of polyethylene glycol-tr eated plasma (r = 0.93). Blood Coag Fibrinol 9:333-341 (C) 1998 Lippin cott-Raven Publishers.