title: 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 approximately 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 (n = 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) in immunologically detected FVIIa, 0.093 +/- 0.013 ng/mL (mean +/- SE) compared to patient controls (n = 20) contemporaneously admitted with noncardiac chest pain, 0.048 +/- 0.007 ng/mL (mean +/- SE). These elevations in the acute coronary syndromes were accompanied by increased (P < .05) and correlating prothrombin fragment F1 + 2 levels (Spearman correlation coefficient rs = .4, P < .01), demonstrating that thrombin generation is certainly associated with, and may even be caused by, extrinsic pathway activation.
authors.affiliation: Department of Haematology, Charing Cross and Westminster Medical School, London, UK.
authors.forename: H
authors.initials: H
authors.lastname: Philippou
authors.forename: A
authors.initials: A
authors.lastname: Adami
authors.forename: R A
authors.initials: RA
authors.lastname: Amersey
authors.forename: P J
authors.initials: PJ
authors.lastname: Stubbs
authors.forename: D A
authors.initials: DA
authors.lastname: Lane
mesh_terms: D000208:Acute Disease; D000789:Angina, Unstable / Q000097:blood; D002637:Chest Pain / Q000097:blood; D003327:Coronary Disease / Q000097:blood* / Q000201:enzymology; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015942:Factor VIIa / Q000032:analysis* / Q000096:biosynthesis / Q000378:metabolism; D005260:Female; D005615:Freezing; D006801:Humans; D007118:Immunoassay; D008297:Male; D008875:Middle Aged; D009203:Myocardial Infarction / Q000097:blood; D018962:Phlebotomy / Q000009:adverse effects; D010949:Plasma / Q000378:metabolism; D011446:Prospective Studies; D011517:Prothrombin Time; D012189:Retrospective Studies; D013577:Syndrome
chemical_list: D015942:Factor VIIa