Novel ELISA for the specific detection of protease NEXIN‐1 in human biological samples

Abstract

Abstract Introduction Serpin E2 or protease nexin‐1 (PN‐1) is a glycoprotein belonging to the serpin superfamily, whose function is closely linked to its ability to inhibit thrombin and proteases of the plasminergic system. Objectives In the absence of specific quantitative methods, an ELISA for the quantification of human PN‐1 was characterized and used in biological fluids. Methods The ELISA for human PN‐1 was developed using two monoclonal antibodies raised against human recombinant PN‐1. PN‐1 was quantified in plasma, serum, platelet secretion from controls and patients with hemophilia A and in conditioned medium of aortic tissue. Results A linear dose–response curve was observed between 2 and 35 ng/mL human PN‐1. Intra‐ and interassay coefficients of variation were 6.2% and 11.1%, respectively. Assay recoveries of PN‐1 added to biological samples were ≈95% in plasma, ≈97% in platelet reaction buffer, and ≈93% in RPMI cell culture medium. Levels of PN‐1 secreted from activated human platelets from controls was similar to that of patients with hemophilia A. PN‐1 could be detected in conditioned media of aneurysmal aorta but not in that of control aorta. Conclusion This is the first fully characterized ELISA for human serpin E2 level in biological fluids. It may constitute a relevant novel tool for further investigations on the pathophysiological role of serpin E2 in a variety of clinical studies.

Keywords

• ELISA
• hemophilia A
• human
• platelets
• serpin