Degradation Products In Plasma Research Articles

A Monoclonal Antibody-Based Enzyme Immunoassay for Fibrin Degradation Products in Plasma

We 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 B beta-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.

New enzyme immunoassay of fibrin-fibrinogen degradation products in plasma using a monoclonal antibody.

In several diseases, such as disseminated intravascular coagulation, fibrin-fibrinogen degradation products (FDP) can be detected when serum is used. To avoid the use of serum with the risk of falsely positive and negative results, and to quantitatively measure routinely low levels of FDP in plasma, we developed a solid-phase enzyme immunoassay (EIA), using a monoclonal antibody (No. FDP-14). It reacts specifically with FDP, but not with the parent fibrinogen-fibrin molecule. To raise the monoclonal antibodies we injected into mice a whole mixture of fibrin degradation products isolated after complete lysis of a human blood clot by tissue-type plasminogen activator, because there is a chance that such products resemble FDP in patients' blood more closely than antigens derived from purified fibrinogen. The EIA developed is a two-step assay ("cap-tag procedure"), in which the monoclonal antibody is attached to the wells of microtiter plates. The monoclonal antibody is specific for a neoantigenic determinant in the fragment E moiety, exposed after degradation of fibrinogen-fibrin by plasmin. The assay discriminates neither between degradation products of fibrinogen and fibrin nor between fibrin degradation products that are or are not cross-linked. It is not disturbed by the presence of fibrinogen or fibrin monomer in plasma. The assay is accurate above 60 ng FDP per milliliter and has a detection limit down to approximately 10 ng FDP per milliliter when the supernatant of a lysed blood clot is used as calibration material. In frozen, then thawed plasma specimens from normal individuals, FDP values varied between 30 and 110 ng/ml. Plasma of patients with disseminated intravascular coagulation and of patients receiving thrombolytic therapy had increased FDP values. In patients with ovarian carcinoma, FDP concentrations in plasma varied with the clinical course of the disease. Results indicate that the EIA for FDP in plasma is promising as a diagnostic tool in different clinical situations.

A latex immunoassay of fibrin/fibrinogen degradation products in plasma using a monoclonal antibody.

We have developed a latex immunoassay using an anti D neo monoclonal antibody (F2C5) which recognizes an epitope present in fragment D but which is hidden in intact fibrinogen and in early fibrinogen degradation products. This technique was applied directly to plasma of both healthy donors and patients, and was shown to be very convenient for clinical investigation, especially in emergency cases for diagnosis of intravascular coagulation. The use of plasma samples instead of serum offers several advantages: it is not time consuming since blood clotting is not required; it avoids overestimation when fibrinogen cannot be totally clotted, and underestimation due to the binding of nonclottable fibrin degradation products to the clot during clotting in vitro. This monoclonal antibody, which reacts more with FbDP (expressed in fragment D) than with fragment D, does not allow fibrin and fibrinogen degradation products to be differentiated. However, this discrimination does not seem critical for its clinical use since the level of fragment D neo antigen remained within the normal range in 12 cases of spontaneous or venous occlusion-induced hyperfibrinolysis.

Rapid detection of cross-linked fibrin degradation products in plasma using monoclonal antibody-coated latex particles.

Conformational and structural changes on conversion of fibrinogen to fibrin and its cross-linking by Factor XIIIa lead to the development of new antigenic determinants that permit differentiation between their plasminolytic cleavage products. A monoclonal antibody (DD-3B6/22) that is specific for cross-linked fibrin derivatives containing the D dimer configuration has been used in developing a latex agglutination procedure that can detect fibrin degradation products in either plasma or serum. Fibrinogen or its degradation products do not cross-react with this antibody. Results were calibrated with an enzyme immunoassay, which used a purified D dimer standard. Plasmas from 40 normal subjects, all having D dimer levels below 250 ng/mL measured by enzyme immunoassay, were all negative by latex assay. In contrast, positive latex agglutination titers were obtained with 87 of 88 patients with demonstrated deep venous thrombosis, pulmonary embolism, or disseminated intravascular coagulation. Compared to enzyme immunoassay, latex agglutination assay is less sensitive, but this latex procedure provides a rapid and less elaborate test for elevated levels of cross-linked fibrin degradation products in patients with thrombosis. Plasma assays for fibrin degradation products are preferable to those using serum.

Specific identification of fibrin polymers, fibrinogen degradation products, and crosslinked fibrin degradation products in plasma and serum with a new sensitive technique

A new method is described for identifying low concentrations of circulating derivatives of fibrinogen and fibrin, even when present in heterogeneous mixtures. This technique is applicable to plasma and serum and uses electrophoresis in 2% agarose in the presence of sodium dodecyl sulfate (SDS) followed by immunological identification of separated derivatives, using radiolabeled antifibrinogen antiserum and autoradiography. Unique electrophoretic patterns distinguish plasmic derivatives of crosslinked fibrin from those of fibrinogen and also identify crosslinked fibrin polymers produced by the combined action of thrombin and factor XIII on fibrinogen. The assay is sensitive to a concentration of 0.1 micrograms/mL of fibrinogen in serum or plasma. Fibrin polymers, plasmic degradation products of fibrinogen, and plasmic degradation products of crosslinked fibrin were detected in the plasma or serum of a patient with disseminated intravascular coagulation. Plasmic derivatives of both fibrinogen and crosslinked fibrin appeared in serum in the course of fibrinolytic therapy for pulmonary embolism, whereas during acute myocardial infarction a marked increase in the proportion of fibrin polymers in plasma was found in comparison with normal controls. Thus, the procedure can distinguish between the simultaneous processes of fibrin polymer formation, fibrinogenolysis, and fibrinolysis, and is sufficiently sensitive to detect relevant quantities of derivatives in pathologic conditions.

Specific Identification of Fibrin Polymers, Fibrinogen Degradation Products, and Crosslinked Fibrin Degradation Products in Plasma and Serum With a New Sensitive Technique

A new method is described for identifying low concentrations of circulating derivatives of fibrinogen and fibrin, even when present in heterogeneous mixtures. This technique is applicable to plasma and serum and uses electrophoresis in 2% agarose in the presence of sodium dodecyl sulfate (SDS) followed by immunological identification of separated derivatives, using radiolabeled antifibrinogen antiserum and autoradiography. Unique electrophoretic patterns distinguish plasmic derivatives of crosslinked fibrin from those of fibrinogen and also identify crosslinked fibrin polymers produced by the combined action of thrombin and factor XIII on fibrinogen. The assay is sensitive to a concentration of 0.1 micrograms/mL of fibrinogen in serum or plasma. Fibrin polymers, plasmic degradation products of fibrinogen, and plasmic degradation products of crosslinked fibrin were detected in the plasma or serum of a patient with disseminated intravascular coagulation. Plasmic derivatives of both fibrinogen and crosslinked fibrin appeared in serum in the course of fibrinolytic therapy for pulmonary embolism, whereas during acute myocardial infarction a marked increase in the proportion of fibrin polymers in plasma was found in comparison with normal controls. Thus, the procedure can distinguish between the simultaneous processes of fibrin polymer formation, fibrinogenolysis, and fibrinolysis, and is sufficiently sensitive to detect relevant quantities of derivatives in pathologic conditions.

Degradation of 125I-labelled prolactin in the rabbit: effect of nephrectomy and prolactin infusion.

Hyperprolactinaemia in patients with chronic renal disease undergoing dialysis has prompted the investigation of the relative roles of liver and kidney in the degradation of prolactin. Male rabbits were acutely nephrectomized, and compared with intact animals with or without prolactin infusion. Prolactin degradation was followed after intravenous injection of 125I-labelled ovine prolactin. Measurements were made of peptide-bound 125I and 125I-labelled degradation products in plasma, liver, kidney, bile, urine and muscle and total thyroid radioactivity. A significant (P less than 0.01) reduction in the metabolic clearance rate of 125I-labelled prolactin was observed due to nephrectomy, with double the accumulation of 125I-labelled peptides in the livers in this group. Prolactin infusion of nephrectomized animals had a further and larger effect than nephrectomy alone on prolactin degradation. Metabolic clearance rate significantly (P less than 0.01) decreased from 5.5 ml/min per kg in nephrectomized rabbits to 0.8 ml/min per kg with prolactin infusion. The accumulation of 125I-labelled prolactin degradation products in the blood was significantly (P less than 0.01) lower in this group of animals and the amount of peptide-bound 125I in plasma at 60 min after 125I-labelled prolactin administration was significantly (P less than 0.01) higher. Liver degradation of prolactin in the absence of exogenous hormone appears to be sufficient to maintain an approximately normal half-life for prolactin in plasma (intact t 1/2 = 6.8 min; nephrectomized t 1/2 = 8.5 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of erythromycin in biological fluids by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for the quantitative determination of major erythromycin components and their potential metabolites or degradation products in plasma and urine. An ether extract of alkalized plasma sample was chromatographed on a reversed-phase column and the components in the column effluent were monitored by an electrochemical detector. The recovery of the drug from extraction was virtually 100%. The detection limits for erythromycin A in plasma were 5–10 ng/ml and 30 ng/ml using 1 and 0.2 ml of sample, respectively. For urine samples, a simple one-step deproteinization with two volumes of acetonitrile was satisfactory for analysis. The method has been evaluated in plasma and urine from dogs receiving oral or intravenous erythromycin A. The standard curves for potential metabolites or degradation products were not constructed due to the lack of sufficient samples.

Effect of fibrin degradation products and thrombin on fibrinogen synthesis.

The effect of intravenous administration of homologous fibrin degradation products and thrombin on fibrinogen synthesis was assessed in rabbits. The relative fibrinogen synthesis rate was calculated as a ratio of the amount of radiolabelled lysine incorporated into fibrinogen to the amount incorporated into albumin during the same measurement period. An increase in this ratio above control would indicate a relatively specific stimulation of fibrinogen synthesis as compared with albumin, which is not an acute-phase reactant. Injection of 45 mg of 'early' or 'late' fibrin degradation products failed to produce a significant increase in the relative fibrinogen synthesis rate, suggesting that fibrin degradation products play no feedback role in controlling fibrinogen synthesis. Infusion of small amounts of homologous thrombin (15--25 NIH u) was followed by a small but statistically significant elevation of the relative fibrinogen synthesis rate. This was not accompanied by any increase in the levels of fibrinogen degradation products in plasma, or by any decrease in plasma fibrinogen concentration, possibly suggesting that thrombin can stimulate fibrinogen synthesis by a mechanism independent of significant fibrinogenolysis or intravascular coagulation.

Precipitation of fibrin monomers and fibrin degradation products by ristocetin.

Ristocetin, at relatively low concentrations (1.0 mg/ml-1.5 mg/ml), can selectively precipitate fibrin monomers and fibrin degradation products (fdp) from plasma without effect on fibrinogen or fibrinogen degradation products (FDP). 125I-labeled fibrin monomers and fibrin degradation products were precipitated by ristocetin when their plasma concentrations were greater than 0.25 microgram/ml and 50 microgram/ml, respectively. In order to obtain a visible precipitated, 2 microgram/ml of fibrin monomers of 50 to 100 microgram/ml of fibrin degradation products were necessary. These effects were optimally observed under the following conditions: (1) temperature, 20 C to 37 C; (2) pH, 7.0 to 7.5; and (3) incubation time, 15 to 60 minutes. Late-fibrin degradation products are approximately eight times less sensitive to ristocetin-induced precipitation than early-fibrin degradation products. Plasma medium is essential for the differentiation of fibrin monomers and fibrin degradation products from fibrinogen and fibrinogen degradation products by ristocetin. These results suggest that the specific detection of fibrin monomers and fibrin degradation products in plasma may be easily performed by ristocetin.

Distinction between fibrinogen and fibrin degradation products in plasma

Polyacrylamide (PA) gel electrophoresis in sodium dodecyl sulphate (SDS) has been shown to distinguish between the major components in the plasmin induced lysates of purified crosslinked fibrin and fibrinogen. The D dimer fragment from crosslinked fibrin separated on SDS-PA gels from the fibrinogen fragments X, Y, D g and E. Following immuno-absorption of plasmin induced lysates of plasma clots, plasma fibrinogen, and mixtures of both, the major fragments of fibrinogen and fibrin were capable of being discriminated by SDSPA gel electrophoresis. The presence of D dimer in patient plasma suggested the efficacy of heparin in the management of haemorrhage associated with amniotic fluid embolism.

Immunonephelometric determination of fibrinogen and its derivatives in plasma

An immunonephelometric method for determination of fibrinogen and fibrin degradation products in plasma is described. Test plasms are diluted 1/600 in a solution containing 17 g NaCl and 3.8 g trisodium citrate/100 ml. To 3 cm 3 of this mixture 0.2 cm 3 of a concentrated antifibrin gamma globulin preparation is added. The reading of this sample minus the γ-globulin blank in a Thorp Nephelometer is directly proportional to the fibrinogen content of the plasma or to its FDP concentration in terms of intact fibrinogen. This paper examines the mechanism, accuracy and reproducibility of this method.