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. This assay was validated, compared with classical immunological methods and shown to be suitable for the routine clinical diagnosis of protein S deficiency. The method relies on the specific capture of free protein S directly from plasma by a monoclonal antibody (mAb), 34G2, immobilized on a sensor chip surface. A calibration curve was established with serial dilutions of standard plasma (working range 5-50%) and a linear 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 assay was confirmed using purified human protein S and polyethylene glycol treated plasma. In addition, it could be demonstrated that no dissociation of C4b-BP-protein S complexes occurred under the chosen experimental conditions. The technique was reproducible with inter-assay, intra-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 samples. Since all tests are automatic and long series of analyses can be performed with the same sensor chip, the method was applied to the determination of free protein S antigen in plasma from 20 normal blood donors and 38 thrombophilic patients. Results displayed excellent correlation with those of free protein S enzyme-linked immunosorbent assay (r = 0.99) and rocket immunoelectrophoresis of polyethylene glycol-treated plasma (r = 0.93).
INSERM U.311, Etablissement de Transfusion Sanguine de Strasbourg, France. catherine.ravanat@etss.u-strasbg.fr
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C
Ravanat
M L
ML
Wiesel
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S
Schuhler
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J
Dambach
J
J
Amiral
J P
JP
Cazenave
D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000097:blood; D015374:Biosensing Techniques; D002138:Calibration; D006801:Humans; D007159:Immunologic Tests / Q000379:methods*; D051379:Mice; D008875:Middle Aged; D017293:Protein S / Q000276:immunology*; D011786:Quality Control; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D000911:Antibodies, Monoclonal; D000941:Antigens; D017293:Protein S