A monoclonal antibody, specific for human fibrinogen and fibrinopeptide A-containing fragments. Its application in an enzyme-immunoassay for fibrinogen degradation products, suitable for use in plasma

Fibrinogen Degradation Products (FDP) are increased in the sera of patients with advanced cancer and other immunosuppressive states. We studied the effect of micromolecular FDP on the function of normal human lymphocytes.FDP were obtained by prolonged digestion of human fibrinogen with plasmin. Plasmin was prepared from plasminogen by activation with Streptokinase in 0.1M tris buffer, pH 8.0 at 37°C. After dialysis and lyophilization, terminal FDP were reconstituted in phosphate buffered saline, sterilized and added in varying concentration to a lymphocyte microculture system, sRBC and EAC rosette assays and a Lexy plate Migration Inhibition Factor assay (MIF).FDP exhibited dose dependent inhibition of lymphocyte blastogenesis induced by the mitogens Phytohemagglutinin (PHA), Concanavillin A (Con A) and Pokeweed (PWM). FDP incubated with lymphocytes had no effect on MIF production or on T and B lymphocyte Rosette formation. FDP were further separated by gel electrophoresis on G–25 Sephadex column. Three peaks were obtained. Peaks 1 and 2 inhibited mitogenic response and Peak 3 enhanced PHA induced lymphocyte blastogenesis. Peaks obtained from the column were further characterized by isoelectric focusing.Micromolecular FDP appear to play a role in the non-specific modulation of lymphocyte functions that depend on cell replication. Their role should be further investigated in clinical immunosuppressive states.

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Fibrinogen barcelona I. Congenital dysfibrinogenemia characterized by defective release of fibrinopeptide a and fibrinogen degradation products

Thrombosis Research ◽

10.1016/0049-3848(87)90307-0 ◽

1987 ◽

Vol 45 (5) ◽

pp. 437-449 ◽

Cited By ~ 3

Author(s):

Virtudes Vila ◽

Edelmiro Regañón ◽

Justo Aznar ◽

Gemma Navarro ◽

Manuel Salas

Keyword(s):

Degradation Products ◽

Fibrinopeptide A ◽

Fibrinogen Degradation Products

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A monoclonal antibody, specific for human fibrinogen, fibrinopeptide A- containing fragments and not reacting with free fibrinopeptide A

Blood ◽

10.1182/blood.v66.3.503.503 ◽

1985 ◽

Vol 66 (3) ◽

pp. 503-507 ◽

Cited By ~ 49

Author(s):

PW Koppert ◽

CM Huijsmans ◽

W Nieuwenhuizen

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Amino Acid ◽

Genetic Variants ◽

Mammalian Species ◽

Fibrinopeptide A ◽

Spleen Cells ◽

Human Fibrinogen ◽

Myeloma Cells ◽

Normal Human

Abstract Spleen cells of BALB/c mice, immunized with fragments Y of normal human fibrinogen, were fused with P3 X 63 Ag 8653 myeloma cells. A clone was found which produces monoclonal antibodies (Mab-Y18) of the IgM kappa type. Mab-Y18 is immunoreactive with normal human fibrinogen, and its fragments X, Y, N-terminal disulphide knot, A alpha-chain, and A alpha stretch 1–51. The immunoreactivity with these same fragments disappears upon treatment with thrombin or arvin. This strongly suggests that fibrinopeptide A is an essential component of the Mab-Y18 epitope. This is supported by the finding that Mab-Y18 prolongs the thrombin and arvin clotting times of human fibrinogen by inhibition of the fibrinopeptide A release. More detailed information about the nature of the Mab-Y18 epitope was obtained from studies with genetic variants of human fibrinogen (especially fibrinogen Metz) and with fibrinogens from other mammalian species. These studies show that amino acid residue A alpha 16 (arginine) of fibrinopeptide A is essential for the Mab-Y18 epitope. Mab-Y18 does not react with free fibrinopeptide A.

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A Quantitative Enzyme Immunoassay for Primary Fibrinogenolysis Products in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651055 ◽

1987 ◽

Vol 57 (01) ◽

pp. 025-028 ◽

Cited By ~ 40

Author(s):

P W Koppert ◽

W Kuipers ◽

B Hoegee-de Nobel ◽

E J P Brommer ◽

J Koopman ◽

...

Keyword(s):

Myocardial Infarction ◽

Acute Myocardial Infarction ◽

Monoclonal Antibodies ◽

Detection Limit ◽

Enzyme Immunoassay ◽

Degradation Products ◽

Fibrinopeptide A ◽

Lower Detection Limit ◽

Lower Detection ◽

Low Levels

SummaryWe have developed a two-step enzyme immunoassay (EIA) that allows the quantitation of degradation products derived from fibrinogen (FbgDP) and that does not detect degradation products derived from cross-finked (XDP) or noncrosslinked fibrin (fdp).The EIA is based on two monoclonal antibodies (FDP-14 and Y-18), developed in our institute. FDP-14 is used as catching antibody. It complexes exclusively with degradation products, irrespective whether these are derived from fibrinogen or from fibrin. It does not complex with intact fibrinogen or fibrin. Y-18 is reactive with fibrinogen and fibrinopeptide A-comprising fibrinogen fragments. It is used, conjugated with horse-radish peroxy-dase, as tagging antibody.The FbgDP-EIA is highly specific, accurate and sensitive. The coefficient of variation is between 3 and 8%; the lower detection limit is less than 0.025 μg/ml.The assay has been applied to plasma from patients with suspected disseminated intravascular coagulation (DIC), to plasma from patients undergoing streptokinase (SK) therapy for acute myocardial infarction and to plasma from newborn babies.DIC patients had no or very low levels of FbgDP, but high levels of other degradation products, SK-treated patients showed high levels of degradation products two hours after termination of the SK infusion. A considerable fraction of these degradation products was shown to be FbgDP. Plasma from newborn babies contained elevated levels of FbgDP associated with prolonged prothrombin times.

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Measurement of fibrinogen degradation products by a sandwich enzyme immunoassay technique

Clinica Chimica Acta ◽

10.1016/0009-8981(80)90147-3 ◽

1980 ◽

Vol 103 (3) ◽

pp. 287-295 ◽

Cited By ~ 9

Author(s):

Tanaka Masao ◽

Minesaki Mitsue ◽

Kato Kanefusa

Keyword(s):

Enzyme Immunoassay ◽

Degradation Products ◽

Fibrinogen Degradation Products ◽

Immunoassay Technique ◽

Sandwich Enzyme Immunoassay ◽

Enzyme Immunoassay Technique

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

Thrombosis Research ◽

10.1016/0049-3848(86)90018-6 ◽

1986 ◽

Vol 44 (6) ◽

pp. 715-728 ◽

Cited By ~ 21

Author(s):

Massoud Mirshahi ◽

Jeannette Soria ◽

Claudine Soria ◽

Jean-Yves Perrot ◽

Claude Boucheix

Keyword(s):

Monoclonal Antibody ◽

Degradation Products ◽

Fibrinogen Degradation Products

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Solid-phase radioimmunoassay of fragment D of human fibrinogen by use of a low-affinity monoclonal antibody.

Clinical Chemistry ◽

10.1093/clinchem/29.5.778 ◽

1983 ◽

Vol 29 (5) ◽

pp. 778-781 ◽

Cited By ~ 3

Author(s):

S J Kennel ◽

P K Lankford

Keyword(s):

Monoclonal Antibody ◽

Solid Phase ◽

Degradation Products ◽

Test Sample ◽

Human Fibrinogen ◽

Phase Competition ◽

Disease States ◽

Affinity Constants ◽

Solid Phase Radioimmunoassay ◽

Solid Phase Assay

Abstract Increased concentrations of plasmin-catalyzed degradation products of fibrin and fibrinogen in body fluids have been correlated with many disease states. We have developed monoclonal antibodies that react with one of these products, fragment D. Antibodies binding fragment D but not fibrinogen were found to have low affinity constants (Ka = 10(7) L/mol). A double monoclonal-antibody assay was developed in which a specific antibody attached to Sepharose beads is used to concentrate fragment D selectively from a test sample. Fragment D bound to the washed beads was then quantified with a high affinity (Ka = 10(10) L/mol) monoclonal antibody that reacts equally well with fragment D and fibrinogen. This solid-phase assay is shown to be specific and about 10-fold more sensitive than a standard liquid-phase competition radioimmunoassay in which the same antibody was used.

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A Monoclonal Antibody-Based Qunatitative Enzyme Immunoassay for the Determination of Plasma Fibrinogen Concentartions

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646982 ◽

1988 ◽

Vol 60 (03) ◽

pp. 415-418 ◽

Cited By ~ 23

Author(s):

E Hoegee-de Nobel ◽

M Voskuilen ◽

E Briët ◽

E J P Brommer ◽

W Nieuwenhuizen

Keyword(s):

Monoclonal Antibody ◽

Enzyme Immunoassay ◽

Degradation Products ◽

Immunological Methods ◽

Response Curves ◽

Amino Terminal ◽

Fibrin Degradation Products ◽

Capture Antibody ◽

Immunological Assays ◽

Set Up

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.

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