SummaryThe most commonly used fibrinogen assays in the clinic are clotting rate assays, e.g. the Clauss method. Such functional assays may be disturbed by e.g. heparin, anticoagulant fibrinogen degradation products (FgDP) and in the case of a dysfi-brinogenemia. Immunological methods would not suffer from these interferences. However, immunological assays for fibrinogen, which do not measure FgDPs, do not exist.To set up such an enzyme immunoassay (EIA) we developed two monoclonal antibodies. The first monoclonal antibody (G8) has its epitope in the carboxyl-terminal 150 amino acid stretches of the fibrinogen Aa-chains. G8 is used to coat the wells of microtitration plates, and is the capture antibody in this EIA. The second antibody (Y18) has been described by us previously (Blood 1985; 66: 503). It is directed against fibrinopeptide A, covalently bound to the ±-chains i.e. against the amino-terminal stretches of the A±-chains. Y18 is conjugated with horse-radish peroxidase, and used as tagging antibody.The EIA does not react with, and is not interfered by FgDP such as purified fragments X and Y, up to a concentration of 800 μg/ml. An FgDP mixture such as generated by Streptokinase treatment of plasma does not respond. Fibrin degradation products (whole blood lysate) up to 800 μg/ml do not interfere nor do heparin, EDTA or oxalate. The time-to-result of the EIA is only 45 minutes. Some patient plasmas yielded dose-response curves which are not parallel with the calibration curve of the EIA. An explanation for this phenomenon could not be given. Our fibrinogen EIA may be especially suitable to monitor patients with conditions which favour proteolytic damage to fibrinogen such as thrombolytic therapies.

SummaryWe have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes.* The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the capture antibody, binds both fibrinogen degradation products (FbgDP) and FbDP, but does not react with the parent fibrin(ogen) molecules. It has its epitope in the E-domain of the fibrinogen molecule on the Bβ-chain between amino acids 54-118. Antibody DD-13 was raised using D-dimer as antigen and is used as a tagging antibody, conjugated with horse-radish peroxidase. A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard. The EIA does virtually not detect FbgDP i. e. purified fragments X, Y, or FbgDP generated in vitro in plasma by streptokinase treatment. This indicates that the assay is specific for fibrin degradation products.We have successfully applied this assay to the plasma of patients with a variety of diseased states. In combination with the assay previously developed by us for FbgDP and for the total amount of FbgDP + FbDP (TDP) in plasma, we are now able to study the composition of TDP in patients plasma in terms of FbgDP and FbDP.

Summary. Background: The development of global tests for the fibrinolytic capacity in blood is hampered by the low base-line fibrinolytic activity in blood, by the involvement of both plasmatic components and blood cells in the fibrinolytic system and by the loss of fibrinolytic activity as a result of the action of plasminogen activator inhibitor-1 (PAI-1). Objective: To develop a new test for the global fibrinolytic capacity (GFC) of whole blood samples. Methods and results: Collection of blood in thrombin increased the subsequent generation of fibrin degradation products. This was ascribed to rapid clot formation and concomitant reduction of in vitro neutralization of tissue-type plasminogen activator (tPA) by PAI-1. On the basis of this observation, the following test was designed: blood samples were collected in thrombin with and without aprotinin and clots were incubated for 3 h at 37°C. The GFC was assessed from the difference between the fibrin degradation products in the two sera. The assay was applied to blood samples from patients and healthy subjects. Other hemostasis parameters were determined in plasma samples taken simultaneously. The GFC varied considerably (normal range 0.13-13.6 lg mL )1 ); physical exercise strongly increased the GFC. Statistically significant correlations were found with tPA activity, PAI-1 activity and fibrinogen level. A mixture of antibodies against tPA and urokinase-type plasminogen activator (uPA) completely inhibited the GFC. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFI) accelerated fibrinolysis 8-fold. Conclusion: The new test represents a global assessment of the main fibrinolytic factors in plasma and potentially those associated with blood cells.

SummarySoluble fibrin is considered as a molecular marker for intravascular fibrin formation, and impending thrombotic events. Most of the existing assays are less suitable for routine clinical applications and their specificity may be limited.We have developed a sandwich-type EIA with a fibrin-specific MoAb described by us before (Proc Natl Acad Sci 1989; 86: 8951) as the capture antibody. An other MoAb (G8; Thromb Haemostas 1988; 60: 415) with an epitope in the carboxyl-terminal sections of the fibrin α-chains, was labeled with peroxidase and used as the tagging antibody. The EIA is calibrated against plasma spiked with known concentrations of soluble fibrin. The time-to-result of the EIA is only 1.5 h. Concentrations as low as 0.5 µg soluble fibrin/ml plasma are readily measurable. Heparin has no effect on the results. Fibrinogen and fibrin(ogen) degradation products are not detected. The values of fibrinopeptide A and soluble fibrin values found with the “COA-SET soluble fibrin” assay correlated well with the soluble fibrin values found with our EIA i.e. r = 0.998 and 0.984, respectively. The run-to-run variabilities were 7.9% and 6.6% for samples with low and high soluble fibrin concentrations, respectively. The within-run variabilities were 2.5, 1.8, 4.0 and 4.6% for samples with 1, 0.5, 0.25 and 0.125 µg soluble fibrin/ml, respectively.The sensitivity, specificity, accuracy and short time-to-result make our EIA suitable for routine clinical applications and the monitoring of the effectivity of heparinization.

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