title: Validity of enzyme-linked immunosorbent assays of cross-linked fibrin degradation products as a measure of clot lysis.
abstract: Concentrations of cross-linked fibrin degradation products (XL-FDPs) in plasma, measured by enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies (MAbs) raised against fragment D-dimer of cross-linked fibrin, increase when patients are given fibrinolytic agents. Whether XL-FDPs derive from circulating cross-linked fibrin polymers in plasma, compared with clot-associated fibrin, has been questioned because increases in XL-FDP are measured by some assays after fibrinolysis in vitro in the absence of clot. We characterized the source of XL-FDP immunoreactivity in plasma of patients with acute myocardial infarction and ischemic heart disease and the response to plasminogen activation in vitro induced by pharmacological concentrations of tissue-type plasminogen activator (t-PA) and streptokinase. XL-FDPs were measured with two different ELISA. One, "pan-specific tag ELISA," was based on a capture MAb specific for XL-FDP and a tag MAb that recognizes an epitope exposed in the fragment D region of both fibrin and fibrinogen, whereas the other, "fibrin-specific tag ELISA," was based on a capture and tag MAbs both specific for fibrin. After plasminogen activation was induced in vitro in plasma from patients with myocardial infarction, increased concentrations of XL-FDP were measured by the pan-specific tag ELISA; however, concentrations measured with the fibrin-specific tag ELISA were not increased. To determine the mechanism for this discrepancy, plasma was subjected to immunoadsorption with a MAb specific for fragment D-dimer before and after in vitro activation of the fibrinolytic system and immunoblotting with a fragment D-dimer-specific MAb and with the pan-specific MAb. Increased concentrations of fragment D-dimer, as well as fibrinogen fragment D at high concentrations, were recognized by the specific MAb. Non-cross-linked fragments were also shown by immunoblotting with the pan-specific MAb to coprecipitate with cross-linked fibrin fragments. This suggested the increased concentrations of XL-FDP measured by the pan-specific tag ELISA after in vitro activation of the fibrinolytic system were due to detection of non-cross-linked fibrinogen fragments. However, fibrin fragment D-dimer concentrations were found to increase in plasma of 15 patients given t-PA for acute myocardial infarction. We conclude fragment D-dimer in plasma of patients during thrombolysis does not originate from circulating soluble cross-linked fibrin but rather is a marker of solid-phase fibrin dissolution, which may be quantitated with assays based on capture and tag antibodies that do not detect fibrinogen or its degradation products.
authors.affiliation: Cardiovascular Division, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: P R
authors.initials: PR
authors.lastname: Eisenberg
authors.forename: A S
authors.initials: AS
authors.lastname: Jaffe
authors.forename: D C
authors.initials: DC
authors.lastname: Stump
authors.forename: D
authors.initials: D
authors.lastname: Collen
authors.forename: E G
authors.initials: EG
authors.lastname: Bovill
mesh_terms: D000911:Antibodies, Monoclonal; D000933:Antifibrinolytic Agents / Q000032:analysis*; D003327:Coronary Disease / Q000097:blood / Q000188:drug therapy; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D005069:Evaluation Studies as Topic; D005338:Fibrin Fibrinogen Degradation Products / Q000032:analysis*; D005342:Fibrinolysis; D006801:Humans; D015151:Immunoblotting; D009203:Myocardial Infarction / Q000097:blood / Q000188:drug therapy; D013300:Streptokinase / Q000627:therapeutic use*; D010959:Tissue Plasminogen Activator / Q000627:therapeutic use*
chemical_list: D000911:Antibodies, Monoclonal; D000933:Antifibrinolytic Agents; D005338:Fibrin Fibrinogen Degradation Products; C036309:fibrin fragment D; D013300:Streptokinase; D010959:Tissue Plasminogen Activator