Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assays (ELISA) for the Measurement of Vitamin K-Dependent Protein S: The Effect of Antibody Immunoreactivity on Plasma Protein S Antigen Determinations

Summary

Two monoclonal antibodies (Mabs) specifically directed to human protein S (PS) - named 5E9E9 and 3B10.25 - were produced and their properties compared to those of 2 previously characterized anti-PS-Mabs (HPS-2 and S10). 3B10.25, similar to S10, was directed to the calcium-free conformation of PS and had virtually identical affinity for free and C4b-binding protein (C4b-BP)-bound PS; 5E9E9 similar to HPS-2, had no calcium-dependency and was selectively directed to free PS. All Mabs were equally reactive to freshly purified and thrombin-cleaved PS. To evaluate the influence of C4b-BP bound PS on PS antigen determinations, ELISA systems employing the four Mabs individually as capture antibody (Ab) and peroxidase-conjugated polyclonal anti-PS IgG as detecting Ab were developed and compared to immunoelectrophoresis (EIA) and to an ELISA employing polyclonal anti-PS IgG as capture and detecting Ab, in the determination of PS in purified systems and in plasma. With all the ELISAs there was parallelism of dilution cuiyes obtained with normal plasma and purified PS; however, supplementation of plasma with purified C4b-BP resulted in loss of parallelism when employing the Mabs directed to free PS as capture Ab. Influence of high C4b-BP on PS antigen determinations was confirmed in a series of plasma samples from patients with C4b-BP levels ranging from 70% to over 200%. Compared to the values obtained with the S10- or 3B10.25 - based ELISAs - which were similar despite a 10-fold difference in sample dilution -plasma PS was underestimated by the ELISAs employing 5E9E9 or HPS-2 while it was overestimated by EIA. In addition, plasma PS and C4b-BP levels were significantly correlated only when it was measured by EIA or the ELISAs employing S10, 3B10.25 or polyclonal IgG. These results highlight the potential influence of high C4b-BP on plasma PS antigen determination. Accurate measurement of PS by ELISA requires selection of antibodies with identical affinity for all plasma PS forms.

• References

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