A novel factor IXa-specific enzyme-linked immunosorbent assay detects factor IXa in human plasma.

Background: Factor (F)IXa activity has been detected in human plasma and may impact thrombotic risk. Current FIXa activity assays are complex and cumbersome.

Objectives: To develop a reproducible enzyme-linked immunosorbent assay (ELISA) using a novel monoclonal antibody that detects total FIXa in human plasma.

Methods: A monoclonal antibody was raised against the new N-terminus exposed upon activation of FIX to FIXa by cleavage after R226. This antibody is specific for FIXa protease and does not recognize FIX zymogen or FIXα. The antibody was used to develop a FIXa-specific ELISA capable of quantifying total FIXa (free FIXa and FIXa-antithrombin complex) in human plasma. Total FIXa quantified using the ELISA was compared to that of FIXa-antithrombin quantified using modifications of a previously described ELISA.

Results: The FIXa-specific ELISA was reproducible and quantified total FIXa in human plasma. Total FIXa levels correlated with FIXa-antithrombin levels.

Conclusion: A monoclonal antibody was developed that specifically detects human FIXa protease. A FIXa-specific ELISA using the new antibody is capable of reproducibly measuring total FIXa in human plasma (both free FIXa and FIXa-antithrombin). This assay should facilitate the evaluation of total FIXa levels in a variety of clinical circumstances.