A novel specific immunoassay for plasma two-chain factor VIIa: Investigation of FVIIa levels in normal individuals and in patients with acute coronary syndromes

Abstract

We report the development of an enzyme-linked immunosorbent assay (ELISA) that is specific for factor VIIa (FVIIa). This assay uses a neoantigen specific capture antibody directed to the amino acid peptide sequence N terminal to the FVII cleavage activation site. The antibody exhibits ~3,000-fold greater reactivity to FVIIa than FVII on a molar basis. Experiments using plasma with added (exogenous) human FVIIa gave quantitative recovery in the ELISA over a range of 0.20 to 3.2 ng/mL of FVIIa. The intra- and inter-assay coefficient of variation (CVs) of the ELISA are 4.5% and 9.8%, respectively. The ELISA shows excellent correlation (r = .99) with a functional assay (using recombinant soluble tissue factor) in detecting FVIIa added to plasma over the range 0.05 to 18.0 ng/mL. However, a major discrepancy exists between the two assays when normal endogenous plasma concentrations of FVIIa are measured. Using normal plasma in = 14) the functional assay reported 3.10 ± 0.30 ng/mL (mean ± SE) whereas only 0.025 ± 0.010 ng/mL was detected in the same samples by the immunoassay. Patients (n = 43) presenting with acute coronary syndromes (myocardial infarction and unstable angina) exhibited elevations (P < .05) and correlating prothrombin fragment F1 + 2 levels (Spearman correlation coefficient r(s) = .4, P < .01), demonstrating that thrombin generation is certainly associated with, and may even be caused by, extrinsic pathway activation.