WO1993000364A1 - ASSAY FOR DETECTING FIBRINOLYSIS AND FIBRINOGENOLYSIS BASED ON COOH-TERMINAL Aα CHAIN MARKERS - Google Patents
ASSAY FOR DETECTING FIBRINOLYSIS AND FIBRINOGENOLYSIS BASED ON COOH-TERMINAL Aα CHAIN MARKERS Download PDFInfo
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- WO1993000364A1 WO1993000364A1 PCT/US1992/005535 US9205535W WO9300364A1 WO 1993000364 A1 WO1993000364 A1 WO 1993000364A1 US 9205535 W US9205535 W US 9205535W WO 9300364 A1 WO9300364 A1 WO 9300364A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/745—Assays involving non-enzymic blood coagulation factors
- G01N2333/75—Fibrin; Fibrinogen
Definitions
- Human fibrinogen consists of 3 sets of peptide chains: 2 ⁇ chains; 2 ß chains; and 2 ⁇ chains.
- the ⁇ chain is known by different names depending on the state in which the chain exists. In free fibrinogen, the ⁇ chain is called the A ⁇ chain. In a mixture of fibrinogen and fibrin, the ⁇ chain is called the (A) ⁇ chain. In fibrin, the ⁇ chain is simply called the ⁇ chain.
- a ⁇ chain represents A ⁇ , (A) ⁇ and ⁇ chains.
- the carboxy (COOH) -terminal two-thirds of the fibrinogen A ⁇ chain is a substrate for both Factor XIII a and plasmin, and is therefore a source of biochemical markers that could form the basis of immunologic assays for the clinical detection of fibrinolysis and fibrinogenolysis.
- a monoclonal antibody that binds to the vicinity around A ⁇ residue 476 as the capture immunoglobulin
- an HRP-conjugated Mab that binds to the NH 2 -terminal, FPA region of the A ⁇ chain as the tagging antibody. While the tagging reagent renders that assay specific for fibrinogen derivatives, the capture antibody's specificity for an epitope somewhat distant from one of the earliest plasmin cleavage sites (A ⁇ #583-584) makes it a poor reagent for the exclusive capture of intact A ⁇ chain representatives.
- a number of radioimmunoassays that utilize monospecific antisera raised against COOH-terminal A ⁇ fragments have been described for the specific measurement of A ⁇ chain degradation products (10-12). All of these assays are serum-based and employ a preliminary clotting step, and in some cases heat precipitation, to remove cross-reactive fibrinogen and high molecular weight derivatives. Based on environmental considerations and the general undesirability of performing radioimmunoassays, the various sample manipulations and inevitable loss of sample material required in these A ⁇ chain-based assays render them impractical for routine clinical use.
- This invention provides a method of detecting the presence of an A ⁇ fragment in a fibrinogen or fibrinogen derivative-containing sample from a human subject which comprises: contacting the sample with an agent capable of forming a complex with intact fibrinogen, or or with a fibrinogen derivative which contains an A ⁇ chain under conditions permitting the formation of a complex; removing a complex so formed from the sample; contacting the resulting sample with a monoclonal antibody capable of forming a complex with an epitope containing amino acid residues present exclusively between amino acid residue 477 and the carboxy terminus of the A ⁇ fragment, under conditions permitting the formation of a monoclonal antibody-A ⁇ fragment complex; and detecting any such complex formed in the sample, thereby detecting the presence of A ⁇ fragment in the sample.
- This invention also provides a method of quantitatively determining the amount of A ⁇ fragment in a sample from a human subject which comprises: contacting the sample with an agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains an A ⁇ chain under conditions permitting the formation of a complex; removing any complex so formed from the sample; contacting the resulting sample with a monoclonal antibody capable of forming a complex with an epitope containing amino acid residues present exclusively between amino acid 477 and the carboxy terminal residue of the A ⁇ fragment, under conditions permitting the formation of a monoclonal antibody-A ⁇ fragment complex; and quantitatively determining the amount of complex formed and thereby quantitatively determining the amount of A ⁇ fragment in the sample.
- Plasma aliquots 1000 ⁇ l, including 1592 pmol fibrin-associated A ⁇ chain equivalents
- a ⁇ FDPs purified A ⁇ FDPs
- Serum FDP levels as measured in a tanned red cell hemagglutination assay based on monospecific antisera to fragments D and E, and BB 1-42 levels, as measured by radioimmunoassay, were determined at the time of the clinical trial (bottom panels).
- An hybridoma cell line designated F-102 producing a monoclonal antibody (also designated F-102) of the present invention has been deposited pursuant to the provisions of the Budapest Treaty On The International Recognition of The Deposit of Microorganisms For The Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, under ATCC Accession No. HB 10815.
- This invention provides a method of detecting the presence of an A ⁇ fragment in a fibrinogen- or fibrinogen derivative-containing sample from a human subject which comprises: contacting the sample with an agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains an A ⁇ chain under conditions permitting formation of a complex; removing a complex so formed from the sample; contacting the resulting sample a monoclonal antibody capable of forming a complex with an epitope containing amino acid residues present exclusively between amino acid residue 477 to the carboxy terminus of the A ⁇ fragment, under conditions permitting formation of a monoclonal antibody-A ⁇ fragment complex; and detecting any such complex formed in the sample, thereby detecting the presence of A ⁇ fragment in the sample.
- a sample is fluid taken from a human which may contain an A ⁇ fragment under normal or abnormal conditions.
- the sample is a plasma sample.
- the sample is a urine sample.
- Intact fibrinogen consists of two sets of 3 peptide chains: ⁇ , ß and ⁇ . Both ß and ⁇ chains possess carbohydrate moities (13, 14).
- the agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative containing an A ⁇ chain includes, but is not limited to, a carbohydrate-binding material.
- the carbohydrate-binding material may be a lectin.
- the lectin is Concanavalin A.
- Concanavalin A chromatography was employed for the large-scale purification of reduced, carboxymethylated A ⁇ chains free of their Bß and ⁇ partners (15).
- suitable conditions required for the formation of a carbohydrate-binding agent complex comprise, but are not limited to, proper pH, termperature, and salt concentration for such formation. Such conditions are well known to those of skill in the art.
- Removing the complexes formed between the carbohydrate-binding agent and intact fibrinogen, or fibrinogen derivatives may comprise passing a sample through a column of carbohydrate-binding agent, and collecting the elluted A ⁇ fragments.
- the A ⁇ carboxy terminus consists of amino acid residues 477 through the carboxy terminus of the A ⁇ fragment.
- Antibodies, such as F-102, directed to epitopes containing amino acid residues present exclusively in this region detect more A ⁇ fragments than do antibodies directed to epitopes centered around residues in the A ⁇ region consisting of residues 1 through 476.
- the F-102 antibody (16) recognizes an epitope that is localized in the COOH-terminal end of the A ⁇ chain, in the vicinity of residue A ⁇ #550.
- the determinant appears to be nearly equally expressed in fibrinogen and fibrin, as well as in a variety of its proteolytic derivatives.
- This offers the distinct advantage of being able to employ purified fibrinogen as both the plating antigen and the assay standard for quantitation, even though it is the smaller, A ⁇ fragment, that is being detected. (On a molar basis, homogeneous preparations of COOH-terminal A ⁇ chain derivatives, for use as an assay standard, are recovered in much lower yield than fibrinogen.)
- suitable conditions for antibody-antigen complex formation comprise, but are not limited to, conditions of proper pH, termperature and salt concentration for complex formation. Such conditions are well known to those of skill in the art.
- the monoclonal antibody used to contact the A ⁇ fragment is directed to an epitope centered around about amino acid residue 550 of the A ⁇ fragment.
- the monoclonal antobody used to contact the A ⁇ fragment is the monoclonal antibody F-102 (ATCC No. HB 10815).
- detecting comprises direct detection of the complex using a reagent specific for the complex. In another embodiment of this invention, the detecting comprises indirect detection of the complex by detecting uncomplexed monoclonal antibody.
- the detecting comprises contacting the uncomplexed monoclonal antibody with an immobilized A ⁇ chain-containing protein under conditions permitting formation of an immobilized monoclonal antibody-A ⁇ chain-containing protein complex, contacting the complex with a detectable antibody directed to the monoclonal antibody under conditions permitting the formation of a detectable complex, and detecting the resulting detectable complex.
- the immobilized A ⁇ chain-containing protein is intact fibrinogen.
- the intact fibrinogen, or other A ⁇ fragment-containing protein may be immobilized by contacting it with standard ELISA wells under suitable conditions for it to adhere to the wells.
- the detectable antibody is a monoclonal antibody.
- a detectable antibody comprises a detectable marker.
- the detectable marker may be an enzyme, radiolabel, or flourescent marker.
- the detectable marker is an enzyme.
- the enzyme is coupled to the antibody and is an horse radish peroxidase-linked rabbit anti-mouse immunoglobulin (RAM-Ig-HRP).
- detecting the detectable antibody-A ⁇ fragment-containing protein complex may be performed by spectrophotometrically measuring an enzyme assay product released by an enzyme linked to an antibody, or counting the radioactivity emitted from a radiolabelled antibody.
- the reaction product can be detected using a colorimetic assay wherein the HRP-linked antibody is contacted with a substrate, orthophenylenediamine, and the reaction product is detected or measured spectrophotometrically.
- This invention also provides a method of quantitatively determining the amount of A ⁇ fragment in a sample from a human subject which comprises: contacting the sample with an agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains an A ⁇ chain under conditions permitting the formation of a complex; removing any complex so formed from the sample; contacting the resulting sample with a monoclonal antibody capable of forming a complex with an epitope containing amino acid residues present exclusively between amino acid residue 477 and the carboxy terminal residue of the A ⁇ fragment, under conditions permitting the formation of a monoclonal antibody-A ⁇ fragment complex; and quantitatively determining the amount of complex formed and thereby quantitatively determining the amount of A ⁇ fragment in the sample.
- the sample is a plasma sample.
- the sample is a urine sample.
- the agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains the A ⁇ chain is a carbohydrate-binding material.
- the carbohydrate binding material is a lectin.
- the lectin is Concanavalin A.
- the monoclonal antibody is the monoclonal antibody F-102 (ATCC No. HB 10815).
- quantitatively determining comprises directly determining the amount of the complex using a reagent specific for the complex.
- the quantitative determining comprises indirectly determining the amount of complex by detecting the amount of uncomplexed antibody.
- quantitatively determining the amount of uncomplexed monoclonal antibody comprises contacting the uncomplexed monoclonal antibody with an immobilized A ⁇ chain-containing protein under conditions permitting the formation of an immobilized antibody-A ⁇ chain-containing protein complex, contacting the complex with a detectable antibody directed to the monoclonal antibody under conditions permitting the formation of a detectable complex, determining the amount of the complex, and comparing the result with that of a known standard, thereby quantitatively determining the amount of A ⁇ fragment.
- the immobilized A ⁇ chain-containing protein is intact fibrinogen.
- the detectable antibody is a monoclonal antibody.
- detectable antibody comprises a detectable marker.
- the detectable marker can be an enzyme, radiolabel or fluorescent marker.
- the detectable marker is an enzyme.
- the enzyme is coupled to the antibody and is an HRP- linked rabbit anti mouse immunoglobulin (RAM-Ig-HRP).
- a known standard for the purposes of quantitative detection may comprise a colorimetric or radioemission measurement obtained using the method of detection on a known quantity of A ⁇ fragment.
- This invention also provides a method of monitoring fibrinogenolysis in a patient which comprises obtaining a sample from a patient, quantitatively determining the amount of A ⁇ fragment in the sample using the aforementioned method, and comparing the amount detected with a known standard, thereby monitoring fibrinogenolysis in a patient.
- the sample is a plasma sample.
- Obtaining a plasma sample is done according to the methods of obtaining plasma for protein analysis. Such methods are well known to those of skill in the art. An example of such a method is given in the Materials and Methods section of this application.
- This invention further provides a method of monitoring fibrinolysis in a patient which comprises obtaining a sample from a patient, quantitatively determining the amount of A ⁇ fragment in the sample using the aforementioned method, and comparing the amount detected with a known standard, thereby monitoring fibrinolysis in a patient.
- the sample is a plasma sample.
- Obtaining a plasma sample is done according to methods of obtaining plasma for protein analysis. Such methods are well known to those of skill in the art. An example of a method of plasma preparation is given in the Materials and Methods section of this application.
- This invention also provides a method of monitoring the effectiveness of thrombolytic therapy in a patient which comprises obtaining at different times during the course of therapy samples from the patient, quantitatively determining the amount of A ⁇ fragment present in the samples using the aforementioned method, and comparing the amounts so determined, thereby monitoring the effectiveness of thrombolytic therapy in a patient.
- the sample is a plasma sample.
- Obtaining a plasma sample is done according to methods of obtaining plasma samples for protein analysis. Such methods are well known to those of skill in the art. An example of a method of plasma preparation is given in the Materials and Methods section of this application.
- Thrombolytic therapy may comprise, but is not limited to, the administration of tissue plasminogen activator (PTA) or streptokinase to a patient.
- PTA tissue plasminogen activator
- streptokinase streptokinase
- This invention further provides a method of diagnosing a fibrinolytic disorder in a patient which comprises obtaining a sample from the patient, quantitatively determining the amount of A ⁇ fragment in the sample using the aforementioned method, and comparing the result with a known standard, thereby diagnosing a fibrinolytic disorder.
- the sample is a plasma sample.
- Obtaining a plasma sample is done according to methods of obtaining plasma for protein analysis. Such methods are well known to those of skill in the art. An example of a method of plasma preparation is given in the Materials and Methodssection of this application.
- Fibrinolytic disorders may include, but are not limited to, serosis, acute hepatitis, chronic hepatitis, and disseminated intravascular coagulation (D.I.C.).
- This invention also provides a method for diagnosing a fibrinogenolytic disorder in a patient which comprises obtaining a sample from the patient, quantitatively determining the amount of A ⁇ fragment in the sample using the aforementioned method, and comparing the result with a known standard, thereby diagnosing a fibrinogenolytic disorder in a patient.
- the sample is a plasma sample.
- Obtaining a plasma sample is done according to methods of obtaining plasma for protein analysis. Such methods are well known to those of skill in the art. An example of a method of plasma preparation is given in the Materials and Methods section of this application.
- Fibrinogenolytic disorders may include, but are not limited to, disseminated intravascular coagulation (D.I.C.).
- concanavalin A-Sepharo ⁇ e and DEAE-Sephacel were obtained from Fharmacia-LKB (Piscataway, NJ).
- Protein A agarose and DEAE-Affi-Gel Blue were from Bio- Rad (Richmond, CA).
- Human fibrinogen and plasmin were obtained from Kabi (Franklin, OH) and Trasylol was supplied by Mobay Pharmaceuticals (New York, NY).
- the monoclonal antibody, F-102 originally described by Ehrlich et al.
- Plasma adsorption on Con A A 100 ⁇ l volume of plasma, diluted to one ml in Con A binding buffer (0.02 M Tris - 0.2 M NaCl, pH 7.4, containing 0.05% SDS, 1mH CaCl 2 and 1mM MnCl 2 ), was incubated for 45 minutes at room temperature with 0.5 ml Con A, freshly equilibrated in this same buffer. The adsorption was conducted in a stoppered, mini column which was rotated constantly during the incubation period. Plasma components which do not bind to the lectin (including A ⁇ fragments) were then collected in a total 2 ml volume, which included the flow-through filtrate and two 0.5 ml buffer washes.
- Con A binding buffer 0.02 M Tris - 0.2 M NaCl, pH 7.4, containing 0.05% SDS, 1mH CaCl 2 and 1mM MnCl 2
- F-102 ELISA Reagent Purification. Purified F-102 was isolated from ascites fluid (prepared by Charles River; Wilmington, Mass.) in a two-step procedure that includes ion exchange chromatography on DEAE-Affi-Gel Blue, followed by affinity chromatography on Protein A. The methodology is detailed in Sobel et al (17) and is based on procedures originally reported by Bruck (18) and Ey (19). The assay standard, purified fibrinogen with predominantly intact A ⁇ chain populations, was isolated from heterogeneous starting material by successive chromatography on lysine-Sepharose and DEAE-Sephacel. The methodology is detailed in Koehn & Canfield (20) and 13 based on procedures originally reported by Deutsch 6 Martz (21) and by Finlayson & Mosesson (22).
- 125 ⁇ l F-102 IgG 34 ng/ml in assay buffer, 1% BSA dissolved in 0.01M phosphate - 0.15 M NaCl, pH 7.2 [PBS]
- 125 ⁇ l test sample serum diluted, 1:2 - 1:32, in assay buffer
- test sample serum diluted, 1:2 - 1:32, in assay buffer
- One hundred microliter volumes from each tube were then delivered, in duplicate, into the wells of microtiter plates that have been precoated for 18h at 4oC with 100 ⁇ l of purified fibrinogen solution (10 ng/ml in 0.1M carbonate buffer, pH 9.0) and treated for 1h at room temperature with 5% BSA in PBS to minimize non-specific binding. Three PBS washes are included between each of these and all subsequent incubations in the ELISA protocol.
- One hundred microliters RAM-Ig-HRP, diluted 1:500 in assay buffer, were then added to the wells and incubated for 3h at room temperature.
- the ratio of RAM Ig-HRP complexed to fibrinogen-bound F- 102 in the presence and absence of test sample was calculated using the software program. Automate (Flow- ICN; Costa Mesa, CA). A ⁇ fragment levels were quantified from these assay data based on an eleven point, logit- transformed, dose-response curve that was generated from 0.67-fold serial dilutions of a purified fibrinogen standard. The concentration of the assay standard stock solution was determined by amino acid analysis. Dose is expressed as pmol A ⁇ chain equivalents per ml, based on a molecular weight of 340kD for fibrinogen and 2 moles A ⁇ chain/mole fibrinogen.
- FIG. 1 illustrates the results obtained when 100 ⁇ l volumes of normal plasma are "spiked” with increasing amounts of A ⁇ fragments (generated in vitro by plasmin digestion of purified fibrinogen at an enzyme/substrate ratio of 0.009 CU/mg for 20 min and then separated free of residual, partially degraded fibrinogen by Con A treatment) and these "test samples" are then processed in the A ⁇ fragment ELISA.
- the immunoreactivity recovered in the Con A filtrates is directly proportional to the A ⁇ fragment content of each "load” (see Fig. 1, closed circles) and approximately 70-80% of the applied A ⁇ FDP immunoreactivity is recovered for each test sample examined.
- F-102 ELISA for the detection of A ⁇ FDPs: It has previously been shown, using a liquid phase radioimmunoassay (16), that the F-102 epitope is nearly, equally expressed in fibrinogen and in its chemically-derived, COOH-terminal A ⁇ chain fragment, A ⁇ #518-584 (CNBr X). The parallel and nearly coincident dose-response curves obtained in the F-102 ELISA for purified fibrinogen, CNBr X, and serial dilutions of A ⁇ FDPs (see Fig.
- Either of these reagents can be employed as the assay standard for the detection of plasma A ⁇ fragments (i.e., following Con A treatment) but because the purified fibrinogen is easier to prepare in high yield, it has been selected as the reagent of choice.
- the A ⁇ FDP ELISA has been applied to measure the extent of A ⁇ chain proteolysis occurring in vivo in normal individuals and in a group of patients who participated in Phase 1 of the TIMI Trial comparing thrombolysis with rt-PA and streptokinase (SK), held at the Columbia Presbyterian Medical Center.
- a ⁇ fragment levels in normal plasmas ranged from 0-138 pmol/ml and represented less than 1% of the circulating fibrinogen A ⁇ chain innumoreactivity, as measured in the capture-tag ELISA for intact fibrinogen referred to earlier. This is consistent with a low-level of fibrinogen proteolysis occurring under physiologic conditions.
- Figure 3 illustrates the results obtained for two patients when the A ⁇ fragment ELISA was applied to characterize the fibrinogenolytic response created during thrombolytic therapy.
- the Figure also includes the serum fragment (dashed line) and Bß 1-42 (closed boxes) profiles determined at the time of the TIMI trial, for comparison.
- serum fragment dashed line
- Bß 1-42 closed boxes
- Serum A ⁇ fragment concentrations reach a maximum slightly later (30 min) and remain high until after thrombolytic therapy is terminated.
- Bß 1-42 levels peak at the same time as the A ⁇ fragments, but the magnitude of the response is significantly lower.
- the pattern of A ⁇ chain proteolysis observed during thrombolytic therapy with rt-PA is markedly different.
- a ⁇ fragment release is slower and more sustained, with peak levels representing 30-40% of the pre-treatment fibrinogen-associated A ⁇ chain immunoreactivity, evident at 30 min and maintained for at least 45-60 min thereafter. 6) Circulating fibrinogen levels also decrease more gradually than in the SK-treatment group.
- At least 10-40% of the pre-treatment-plasma fibrinogen concentration is maintained after 1h of thrombolytic therapy with rt-PA, in contrast to less than 1% detectable in the SK-treated patients at this same time point.
- Serum A ⁇ fragment and Bß 1-42 plasma concentrations reach peak levels after A ⁇ FDP levels have already begun to decrease (2h). Discussion
- Assays that are currently employed to evaluate changes in hemostatic parameters during thrombolytic therapy include the measurement of functionally clottable fibrinogen and the quantitation of FDPs based on the immunologic detection of fragment D- and E-containing derivatives.
- the A ⁇ FDP ELISA described here, as well as the new assay developed for intact fibrinogen A ⁇ chains provide the opportunity to measure an additional hemostatic parameter by focusing on biochemical markers contained within the COOH-terminal two-thirds of the fibrinogen A ⁇ chain. This region includes Factor XIII a -sensitive domains and, therefore, plays a critical role in fibrin stabilization.
- a ⁇ chain structural integrity could contribute to the risk factors associated with current thrombolytic therapy, i.e., bleeding and rethrombosis, and would appear to be a useful parameter to be able to evaluate.
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Abstract
This invention provides a method of detecting the presence of an Aα fragment in a fibrinogen- or fibrinogen derivative-containing sample from a human subject which comprises: contacting the sample with an agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains an Aα chain under conditions permitting formation of a complex; removing a complex so formed from the sample; contacting the resulting sample with a monoclonal antibody capable of forming a complex with an epitope containing amino acid residues present exclusively between amino acid residue 477 and the carboxy terminus of the Aα fragment, under conditions permitting formation of a monoclonal antibody Aα fragment complex; and detecting any such complex formed in the sample, thereby detecting the presence of Aα fragment in the sample.
Description
ASSAY FOR DETECTING FIBRINOLYSIS AND FIBRINOGENOLYSIS BASED ON COOH-TERMINAL Aα CHAIN MARKERS
This invention was made with government support under Grant Number HL 45936 from the National Institute of Health. The United States Government has certain rights in this invention.
Background of the Invention
Throughout this application, various publications are referenced by arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Human fibrinogen consists of 3 sets of peptide chains: 2 α chains; 2 ß chains; and 2 γ chains. The α chain is known by different names depending on the state in which the chain exists. In free fibrinogen, the α chain is called the Aα chain. In a mixture of fibrinogen and fibrin, the α chain is called the (A)α chain. In fibrin, the α chain is simply called the α chain. For the purposes of this invention, the term "Aα chain" represents Aα, (A)α and α chains.
The carboxy (COOH) -terminal two-thirds of the fibrinogen Aα chain is a substrate for both Factor XIIIa and plasmin, and is therefore a source of biochemical markers
that could form the basis of immunologic assays for the clinical detection of fibrinolysis and fibrinogenolysis.
Current methods for quantifying fibrinogen proteolysis, either by measuring intact fibrinogen or its degradation products, rely on clottability end points (1) and/or on the immunological detection of fragments released from the E and D domains of the fibrin or fibrinogen molecule (2-5). Among these, serum-based assays suffer from quantitative artifacts introduced because some degradation products can interfere with fibrin polymerization while others are clottable. Assays that are based on the detection of epitopes within the D and E domains of fibrin and fibrinogen measure derivatives that are released late in the fibrinolytic and fibrinogenolytic processes. These "core" fragments circulate only after Fragments Y and X (or their crosslinked counterparts) have already formed (6-8).
The plasmin-mediated release of the COOH-terminal Aα chain fragments during Fragment X formation is the initial event in fibrinogen proteolysis. (A)α chain markers, as distinct from those within the γ chain, would be expected to be the earliest circulating hallmarks of fibrinolysis and fibrinogenolysis. To date, only one assay has been reported that takes advantage of an epitope within the COOH-terminal two thirds of the Aα chain for the measurement of intact plasma fibrinogen (9). That assay is a capture-tap, plasma enzyme-linked immunosorbent assay-based-assay (ELISA). It employs a monoclonal antibody (Mab) that binds to the vicinity around Aα residue 476 as the capture immunoglobulin, and an HRP-conjugated Mab that binds to the NH2-terminal, FPA region of the Aα chain as the tagging antibody. While
the tagging reagent renders that assay specific for fibrinogen derivatives, the capture antibody's specificity for an epitope somewhat distant from one of the earliest plasmin cleavage sites (Aα #583-584) makes it a poor reagent for the exclusive capture of intact Aα chain representatives.
A number of radioimmunoassays that utilize monospecific antisera raised against COOH-terminal Aα fragments have been described for the specific measurement of Aα chain degradation products (10-12). All of these assays are serum-based and employ a preliminary clotting step, and in some cases heat precipitation, to remove cross-reactive fibrinogen and high molecular weight derivatives. Based on environmental considerations and the general undesirability of performing radioimmunoassays, the various sample manipulations and inevitable loss of sample material required in these Aα chain-based assays render them impractical for routine clinical use.
Summary of the Invention
This invention provides a method of detecting the presence of an Aα fragment in a fibrinogen or fibrinogen derivative-containing sample from a human subject which comprises: contacting the sample with an agent capable of forming a complex with intact fibrinogen, or or with a fibrinogen derivative which contains an Aα chain under conditions permitting the formation of a complex; removing a complex so formed from the sample; contacting the resulting sample with a monoclonal antibody capable of forming a complex with an epitope containing amino acid residues present exclusively between amino acid residue 477 and the carboxy terminus of the Aα fragment, under conditions permitting the formation of a monoclonal antibody-Aα fragment complex; and detecting any such complex formed in the sample, thereby detecting the presence of Aα fragment in the sample.
This invention also provides a method of quantitatively determining the amount of Aα fragment in a sample from a human subject which comprises: contacting the sample with an agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains an Aα chain under conditions permitting the formation of a complex; removing any complex so formed from the sample; contacting the resulting sample with a monoclonal antibody capable of forming a complex with an epitope containing amino acid residues present exclusively between amino acid 477 and the carboxy terminal residue of the Aα fragment, under conditions permitting the formation of a monoclonal antibody-Aα fragment complex; and quantitatively determining the
amount of complex formed and thereby quantitatively determining the amount of Aα fragment in the sample.
Brief Description of the Figures
Figure 1:
Selective recovery of Aα chain fragments in Con A filtrates, demonstrated by ELISA. Plasma aliquots (1000 μl, including 1592 pmol fibrin-associated Aα chain equivalents) containing increasing amounts of purified Aα FDPs (0-4316 pmol Aα equivalents) were incubated with Con A as described in the text.
Samples of the filtrate, from each incubation, were then assayed in the F-102 ELISA (to determine Aα FDP concentration) and in the intact fibrinogen Aα chain assay (to determine plasma fibrinogen concentration). Plasma fibrinogen, present at a level of 1592 pmol in each Con A "load" Was not detected in any of the Con A filtrates (closed triangels) because it bound to the lectin via carbohydrate moieties in its Bß and γ components. Aα FDPs (closed circles) were recovered in each of the Con A filtrates at approximately 72% yield, based on the amount originally present in each load.
Figure 2:
Quantitation in the F-102 ELISA for the measurement of Aα FDP's. Purified fibrinogen (130.9-2.4 pmol/ml) and its CNBr Aα chain derivative CNBr X, Aα 518-584, (75.3-2.1 pmol/ml) were incubated with F-102 and excess antibody then measured in a second incubation on fibrinogen-coated plates as described in the text. The dose response curves obtained yield statistically similar slopes, indicating parallelism and therefore that a common epitope is recognized in each antigen by F-102. Serial dilutions of a preparation of purified Aα FDPs (open
triangles) yield a dose response curve that is parallel to the ones for fibrinogen (closed circles) and CNBr X (closed triangles).
Figure 3:
Detection of Aα chain fibrinogenolysis during thrombolytic therapy. Plasma from two patients who participated in the TIMI (Phase 1) trial held at the Columbia Presbyterian Medical Center (one of whom was treated with streptokinase (left panels)), the other with re-PA (right penels), were assayed for Aα FDPs (closed circles) and intact fibrinogen Aα chains (closed triangles) as described in the text (top panels). Serum FDP levels, as measured in a tanned red cell hemagglutination assay based on monospecific antisera to fragments D and E, and BB 1-42 levels, as measured by radioimmunoassay, were determined at the time of the clinical trial (bottom panels).
Detailed Description of the Invention
An hybridoma cell line designated F-102 producing a monoclonal antibody (also designated F-102) of the present invention has been deposited pursuant to the provisions of the Budapest Treaty On The International Recognition of The Deposit of Microorganisms For The Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, under ATCC Accession No. HB 10815.
This invention provides a method of detecting the presence of an Aα fragment in a fibrinogen- or fibrinogen derivative-containing sample from a human subject which comprises: contacting the sample with an agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains an Aα chain under conditions permitting formation of a complex; removing a complex so formed from the sample; contacting the resulting sample a monoclonal antibody capable of forming a complex with an epitope containing amino acid residues present exclusively between amino acid residue 477 to the carboxy terminus of the Aα fragment, under conditions permitting formation of a monoclonal antibody-Aα fragment complex; and detecting any such complex formed in the sample, thereby detecting the presence of Aα fragment in the sample.
For the purposes of this invention, a sample is fluid taken from a human which may contain an Aα fragment under normal or abnormal conditions. In one embodiment of the invention, the sample is a plasma sample. In another
embodiment of the invention, the sample is a urine sample.
Intact fibrinogen consists of two sets of 3 peptide chains: α , ß and γ. Both ß and γ chains possess carbohydrate moities (13, 14). Thus, intact fibrinogen and ß and γ chain-containing protein can be removed from Aα chains using an agent which binds to carbohydrate moities. For the purposes of this invention, the agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative containing an Aα chain includes, but is not limited to, a carbohydrate-binding material. In one embodiment of the invention, the carbohydrate-binding material may be a lectin. In the preferred embodiment of the invention, the lectin is Concanavalin A. In a single report illustrating the practical utility of this structural feature of the fibrinogen molecule, Concanavalin A chromatography was employed for the large-scale purification of reduced, carboxymethylated Aα chains free of their Bß and γ partners (15).
Also for the purposes of this invention, suitable conditions required for the formation of a carbohydrate-binding agent complex comprise, but are not limited to, proper pH, termperature, and salt concentration for such formation. Such conditions are well known to those of skill in the art.
Removing the complexes formed between the carbohydrate-binding agent and intact fibrinogen, or fibrinogen derivatives may comprise passing a sample through a column of carbohydrate-binding agent, and collecting the elluted Aα fragments.
For the purposes of this invention, the Aα carboxy terminus consists of amino acid residues 477 through the carboxy terminus of the Aα fragment. Antibodies, such as F-102, directed to epitopes containing amino acid residues present exclusively in this region detect more Aα fragments than do antibodies directed to epitopes centered around residues in the Aα region consisting of residues 1 through 476.
The F-102 antibody (16) recognizes an epitope that is localized in the COOH-terminal end of the Aα chain, in the vicinity of residue Aα #550. The determinant appears to be nearly equally expressed in fibrinogen and fibrin, as well as in a variety of its proteolytic derivatives. This offers the distinct advantage of being able to employ purified fibrinogen as both the plating antigen and the assay standard for quantitation, even though it is the smaller, Aα fragment, that is being detected. (On a molar basis, homogeneous preparations of COOH-terminal Aα chain derivatives, for use as an assay standard, are recovered in much lower yield than fibrinogen.)
Also for the purposes of this invention, suitable conditions for antibody-antigen complex formation comprise, but are not limited to, conditions of proper pH, termperature and salt concentration for complex formation. Such conditions are well known to those of skill in the art.
IN one embodiment of this invention, the monoclonal antibody used to contact the Aα fragment is directed to an epitope centered around about amino acid residue 550 of the Aα fragment. In the preferred embodiment of this invention, the monoclonal antobody used to contact the Aα
fragment is the monoclonal antibody F-102 (ATCC No. HB 10815).
In one embodiment of this invention, detecting comprises direct detection of the complex using a reagent specific for the complex. In another embodiment of this invention, the detecting comprises indirect detection of the complex by detecting uncomplexed monoclonal antibody.
In the prefered embodiment of this invention, the detecting comprises contacting the uncomplexed monoclonal antibody with an immobilized Aα chain-containing protein under conditions permitting formation of an immobilized monoclonal antibody-Aα chain-containing protein complex, contacting the complex with a detectable antibody directed to the monoclonal antibody under conditions permitting the formation of a detectable complex, and detecting the resulting detectable complex.
In the preferred embodiment of this invention, the immobilized Aα chain-containing protein is intact fibrinogen. The intact fibrinogen, or other Aα fragment-containing protein, may be immobilized by contacting it with standard ELISA wells under suitable conditions for it to adhere to the wells.
In one embodiment of this invention, the detectable antibody is a monoclonal antibody.
In this invention, a detectable antibody comprises a detectable marker. The detectable marker may be an enzyme, radiolabel, or flourescent marker. In one embodiment of the invention, the detectable marker is an enzyme. In the preferred embodiment of this invention,
the enzyme is coupled to the antibody and is an horse radish peroxidase-linked rabbit anti-mouse immunoglobulin (RAM-Ig-HRP).
In this invention, detecting the detectable antibody-Aα fragment-containing protein complex may be performed by spectrophotometrically measuring an enzyme assay product released by an enzyme linked to an antibody, or counting the radioactivity emitted from a radiolabelled antibody. In the case of RAM-Ig-HRP, the reaction product can be detected using a colorimetic assay wherein the HRP-linked antibody is contacted with a substrate, orthophenylenediamine, and the reaction product is detected or measured spectrophotometrically.
This invention also provides a method of quantitatively determining the amount of Aα fragment in a sample from a human subject which comprises: contacting the sample with an agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains an Aα chain under conditions permitting the formation of a complex; removing any complex so formed from the sample; contacting the resulting sample with a monoclonal antibody capable of forming a complex with an epitope containing amino acid residues present exclusively between amino acid residue 477 and the carboxy terminal residue of the Aα fragment, under conditions permitting the formation of a monoclonal antibody-Aα fragment complex; and quantitatively determining the amount of complex formed and thereby quantitatively determining the amount of Aα fragment in the sample.
As noted hereinabove, in one embodiment of this invention, the sample is a plasma sample. In another embodiment of this invention, the sample is a urine sample.
In one embodiment of this invention, the agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains the Aα chain is a carbohydrate-binding material. In one embodiment of the invention, the carbohydrate binding material is a lectin. In the preferred embodiment of this invention, the lectin is Concanavalin A.
In a prefered embodiment of this invention, the monoclonal antibody is the monoclonal antibody F-102 (ATCC No. HB 10815).
In one embodiment of this invention, quantitatively determining comprises directly determining the amount of the complex using a reagent specific for the complex. In another embodiment of the invention, the quantitative determining comprises indirectly determining the amount of complex by detecting the amount of uncomplexed antibody. In the preferred embodiment of this invention, quantitatively determining the amount of uncomplexed monoclonal antibody comprises contacting the uncomplexed monoclonal antibody with an immobilized Aα chain-containing protein under conditions permitting the formation of an immobilized antibody-Aα chain-containing protein complex, contacting the complex with a detectable antibody directed to the monoclonal antibody under
conditions permitting the formation of a detectable complex, determining the amount of the complex, and comparing the result with that of a known standard, thereby quantitatively determining the amount of Aα fragment.
In the preferred embodiment of this invention, the immobilized Aα chain-containing protein is intact fibrinogen.
In one embodiment of this invention, the detectable antibody is a monoclonal antibody.
In one embodiment of this invention, detectable antibody comprises a detectable marker. The detectable marker can be an enzyme, radiolabel or fluorescent marker. In one embodiment of the invention, the detectable marker is an enzyme. In the preferred embodiment of this invention, the enzyme is coupled to the antibody and is an HRP- linked rabbit anti mouse immunoglobulin (RAM-Ig-HRP).
A known standard for the purposes of quantitative detection may comprise a colorimetric or radioemission measurement obtained using the method of detection on a known quantity of Aα fragment.
This invention also provides a method of monitoring fibrinogenolysis in a patient which comprises obtaining a sample from a patient, quantitatively determining the amount of Aα fragment in the sample using the aforementioned method, and comparing the amount detected with a known standard, thereby monitoring fibrinogenolysis in a patient. In a prefered embodiment of the invention, the sample is a plasma sample.
Obtaining a plasma sample is done according to the methods of obtaining plasma for protein analysis. Such methods are well known to those of skill in the art. An example of such a method is given in the Materials and Methods section of this application.
This invention further provides a method of monitoring fibrinolysis in a patient which comprises obtaining a sample from a patient, quantitatively determining the amount of Aα fragment in the sample using the aforementioned method, and comparing the amount detected with a known standard, thereby monitoring fibrinolysis in a patient. In a preferred embodiment of this invention, the sample is a plasma sample. Obtaining a plasma sample is done according to methods of obtaining plasma for protein analysis. Such methods are well known to those of skill in the art. An example of a method of plasma preparation is given in the Materials and Methods section of this application.
This invention also provides a method of monitoring the effectiveness of thrombolytic therapy in a patient which comprises obtaining at different times during the course of therapy samples from the patient, quantitatively determining the amount of Aα fragment present in the samples using the aforementioned method, and comparing the amounts so determined, thereby monitoring the effectiveness of thrombolytic therapy in a patient. In the preferred embodiment of this invention, the sample is a plasma sample. Obtaining a plasma sample is done according to methods of obtaining plasma samples for protein analysis. Such methods are well known to those of skill in the art. An example of a method of plasma
preparation is given in the Materials and Methods section of this application.
Thrombolytic therapy may comprise, but is not limited to, the administration of tissue plasminogen activator (PTA) or streptokinase to a patient.
This invention further provides a method of diagnosing a fibrinolytic disorder in a patient which comprises obtaining a sample from the patient, quantitatively determining the amount of Aα fragment in the sample using the aforementioned method, and comparing the result with a known standard, thereby diagnosing a fibrinolytic disorder. In as preferred embodiment of the invention, the sample is a plasma sample. Obtaining a plasma sample is done according to methods of obtaining plasma for protein analysis. Such methods are well known to those of skill in the art. An example of a method of plasma preparation is given in the Materials and Methodssection of this application.
Fibrinolytic disorders may include, but are not limited to, serosis, acute hepatitis, chronic hepatitis, and disseminated intravascular coagulation (D.I.C.).
This invention also provides a method for diagnosing a fibrinogenolytic disorder in a patient which comprises obtaining a sample from the patient, quantitatively determining the amount of Aα fragment in the sample using the aforementioned method, and comparing the result with a known standard, thereby diagnosing a fibrinogenolytic disorder in a patient. In a preferred embodiment of the invention, the sample is a plasma sample. Obtaining a plasma sample is done according to methods of obtaining
plasma for protein analysis. Such methods are well known to those of skill in the art. An example of a method of plasma preparation is given in the Materials and Methods section of this application.
Fibrinogenolytic disorders may include, but are not limited to, disseminated intravascular coagulation (D.I.C.).
Experimental Details
Materials and Methods
Reagents: concanavalin A-Sepharoεe and DEAE-Sephacel were obtained from Fharmacia-LKB (Piscataway, NJ). Protein A agarose and DEAE-Affi-Gel Blue were from Bio- Rad (Richmond, CA). Human fibrinogen and plasmin were obtained from Kabi (Franklin, OH) and Trasylol was supplied by Mobay Pharmaceuticals (New York, NY). The human Aα chain cyanogen bromide fragment, Aα #518-584 (CNBr X) vas isolated and purified as described by sobel et al (17). The monoclonal antibody, F-102, originally described by Ehrlich et al. (16), vas purified from ascites fluid for use here. Rabbit anti-mouse immunoglobulins conjugated to HRP (RAM-Ig-HRP) were obtained from Dako (Santa Barbara, CA). Bovine serum albumin, ELISA grade, was from Sigma (St. Louis, MO) and the HRP substrate, ortho-phenylenediamine, was from Fisher (Fairlawn, NJ). Disposable, polypropylene minicolumns were obtained from Bio Rad (Richmond, CA) and polyvinylchloride microtiter plates were from Dynatech (Chantilly, VA). All other chemicals were reagent-grade.
Plasma adsorption on Con A: A 100 μl volume of plasma, diluted to one ml in Con A binding buffer (0.02 M Tris - 0.2 M NaCl, pH 7.4, containing 0.05% SDS, 1mH CaCl2 and 1mM MnCl2), was incubated for 45 minutes at room temperature with 0.5 ml Con A, freshly equilibrated in this same buffer. The adsorption was conducted in a stoppered, mini column which was rotated constantly during the incubation period. Plasma components which do not bind to the lectin (including Aα fragments) were then collected in a total 2 ml volume, which included the flow-through filtrate and two 0.5 ml buffer washes. This material, now diluted 20 times relative to its original plasma volume, is the processed sample ready for assaying in the F-102 ELISA.
F-102 ELISA: Reagent Purification. Purified F-102 was isolated from ascites fluid (prepared by Charles River; Wilmington, Mass.) in a two-step procedure that includes ion exchange chromatography on DEAE-Affi-Gel Blue, followed by affinity chromatography on Protein A. The methodology is detailed in Sobel et al (17) and is based on procedures originally reported by Bruck (18) and Ey (19). The assay standard, purified fibrinogen with predominantly intact Aα chain populations, was isolated from heterogeneous starting material by successive chromatography on lysine-Sepharose and DEAE-Sephacel. The methodology is detailed in Koehn & Canfield (20) and 13 based on procedures originally reported by Deutsch 6 Martz (21) and by Finlayson & Mosesson (22).
ASSAY; In the first step, 125 μl F-102 IgG (34 ng/ml in assay buffer, 1% BSA dissolved in 0.01M phosphate - 0.15 M NaCl, pH 7.2 [PBS]) and 125 μl test sample (serially diluted, 1:2 - 1:32, in assay buffer) or standard were mixed together in polypropylene test tubes and then incubated for 2h at 37ºC. One hundred microliter volumes from each tube were then delivered, in duplicate, into the wells of microtiter plates that have been precoated for 18h at 4ºC with 100 μl of purified fibrinogen solution (10 ng/ml in 0.1M carbonate buffer, pH 9.0) and treated for 1h at room temperature with 5% BSA in PBS to minimize non-specific binding. Three PBS washes are included between each of these and all subsequent incubations in the ELISA protocol. One hundred microliters RAM-Ig-HRP, diluted 1:500 in assay buffer, were then added to the wells and incubated for 3h at room temperature. Following a final wash series, including a distilled water rinse to lower the pH of the solid phase environment prior to the substrate reaction, 100 μl
phenylenediamine (0.06% [w/v] in 0.05M sodium phosphate- 0.025M citric acid, pH 5.0, containing 0.001% [v/v] H2O2) was added. Color development is allowed to proceed for 1h at room temperature, and the absorbance at 414run was read in a Multiskan MC microtiter plate reader (Flow-ICN; Costa Mesa, CA).
The ratio of RAM Ig-HRP complexed to fibrinogen-bound F- 102 in the presence and absence of test sample (% B) was calculated using the software program. Automate (Flow- ICN; Costa Mesa, CA). Aα fragment levels were quantified from these assay data based on an eleven point, logit- transformed, dose-response curve that was generated from 0.67-fold serial dilutions of a purified fibrinogen standard. The concentration of the assay standard stock solution was determined by amino acid analysis. Dose is expressed as pmol Aα chain equivalents per ml, based on a molecular weight of 340kD for fibrinogen and 2 moles Aα chain/mole fibrinogen.
Results
Plasma adsorption on Con A: Figure 1 illustrates the results obtained when 100 μl volumes of normal plasma are "spiked" with increasing amounts of Aα fragments (generated in vitro by plasmin digestion of purified fibrinogen at an enzyme/substrate ratio of 0.009 CU/mg for 20 min and then separated free of residual, partially degraded fibrinogen by Con A treatment) and these "test samples" are then processed in the Aα fragment ELISA. The immunoreactivity recovered in the Con A filtrates is directly proportional to the Aα fragment content of each "load" (see Fig. 1, closed circles) and approximately 70-80% of the applied Aα FDP immunoreactivity is recovered
for each test sample examined. Although 1532 pmols of (plasma) fibrinogen-associated Aα chain immunoreactivity is also present in each of the "loads", as measured in a recently developed capture-tag ELISA for intact Aα chains (manuscript in preparation), none of this material emerges in the Con A filtrates (See Fig. 1, closed triangles). Rather, it is bound to the lectin via the carbohydrate moieties contained within the D and E domains of the fibrinogen molecules. Fibrinogen-associated Aα chain immunoreactivity appears in material eluted from Con A with 0.25 M methyl-mannoside and is recovered at a constant level for each of the test samples examined (30-40% of the applied plasma fibrinogen Aα chain content; data not shown).
Up to 200 μl plasma can be treated with 0.5 ml Con A before the capacity of the lectin for fibrinogen (in the presence of other carbohydrate-containing plasma proteins) is exceeded. This was determined from immunoblotting studies (not shown) in which the appearance of high molecular weight bands of F-102 immunoreactivity (i.e., fibrinogen) in Con A filtrates was indicative of ligand excess. Based on these findings and considerations of ease of handling and cost of reagents, the Con A adsorption step was developed for a 100 μl plasma sample and a 0.5 ml volume of lectin.
F-102 ELISA for the detection of Aα FDPs: It has previously been shown, using a liquid phase radioimmunoassay (16), that the F-102 epitope is nearly, equally expressed in fibrinogen and in its chemically-derived, COOH-terminal Aα chain fragment, Aα #518-584 (CNBr X). The parallel and nearly coincident dose-response curves obtained in the F-102 ELISA for purified
fibrinogen, CNBr X, and serial dilutions of Aα FDPs (see Fig. 2) suggest that plasmin derivatives that are likely to be circulating in vivo under a variety of pathophysiologic conditions may also harbor the F-102 epitope in an exposed, and therefore, detectable conformation. The F-102 ELISA is sensitive, as characterized by an ED50%B of 9.75±2.45 pmol/ml (n=14) and 6.40±0.98 pmol/ml (n=12) for the purified fibrinogen and CNDr X standards, respectively. Either of these reagents can be employed as the assay standard for the detection of plasma Aα fragments (i.e., following Con A treatment) but because the purified fibrinogen is easier to prepare in high yield, it has been selected as the reagent of choice.
Application of the Aα fragment ELISA: The Aα FDP ELISA has been applied to measure the extent of Aα chain proteolysis occurring in vivo in normal individuals and in a group of patients who participated in Phase 1 of the TIMI Trial comparing thrombolysis with rt-PA and streptokinase (SK), held at the Columbia Presbyterian Medical Center.
In a preliminary sampling, Aα fragment levels in normal plasmas ranged from 0-138 pmol/ml and represented less than 1% of the circulating fibrinogen Aα chain innumoreactivity, as measured in the capture-tag ELISA for intact fibrinogen referred to earlier. This is consistent with a low-level of fibrinogen proteolysis occurring under physiologic conditions. Figure 3 illustrates the results obtained for two patients when the Aα fragment ELISA was applied to characterize the fibrinogenolytic response created during
thrombolytic therapy. In addition to Aα fragment (closed circles) and intact fibrinogen Aα chain (closed triangles, upright) levels, the Figure also includes the serum fragment (dashed line) and Bß 1-42 (closed boxes) profiles determined at the time of the TIMI trial, for comparison. To date, the plasmas of 6 patients (three from each treatment group) have been analyzed. The data are consistent with the following distinctive features shown in Figure 3. 1) A rapid (by 15 min) release of Aα FDPs, corresponding to 40-50% of the pre-treatment fibrinogen-associated Aα immunoreactivity, occurs during thrombolytic therapy with SK. 2) This is paralleled by a rapid drop (by 15 min) in circulating fibrinogen, to 1-5% of the pre-treatment plasma concentration. 3) Serum Aα fragment concentrations reach a maximum slightly later (30 min) and remain high until after thrombolytic therapy is terminated. 4) In the case of the patient shown, Bß 1-42 levels peak at the same time as the Aα fragments, but the magnitude of the response is significantly lower. The pattern of Aα chain proteolysis observed during thrombolytic therapy with rt-PA is markedly different. 5) Aα fragment release is slower and more sustained, with peak levels representing 30-40% of the pre-treatment fibrinogen-associated Aα chain immunoreactivity, evident at 30 min and maintained for at least 45-60 min thereafter. 6) Circulating fibrinogen levels also decrease more gradually than in the SK-treatment group. At least 10-40% of the pre-treatment-plasma fibrinogen concentration is maintained after 1h of thrombolytic therapy with rt-PA, in contrast to less than 1% detectable in the SK-treated patients at this same time point. 7) Serum Aα fragment and Bß 1-42 plasma concentrations reach peak levels after Aα FDP levels have already begun to decrease (2h).
Discussion
Assays that are currently employed to evaluate changes in hemostatic parameters during thrombolytic therapy include the measurement of functionally clottable fibrinogen and the quantitation of FDPs based on the immunologic detection of fragment D- and E-containing derivatives. The Aα FDP ELISA described here, as well as the new assay developed for intact fibrinogen Aα chains (referred to in this application) provide the opportunity to measure an additional hemostatic parameter by focusing on biochemical markers contained within the COOH-terminal two-thirds of the fibrinogen Aα chain. This region includes Factor XIIIa-sensitive domains and, therefore, plays a critical role in fibrin stabilization. In view of this, Aα chain structural integrity (or lack of it) could contribute to the risk factors associated with current thrombolytic therapy, i.e., bleeding and rethrombosis, and would appear to be a useful parameter to be able to evaluate.
References
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4. Koppert, P.W., Koopman, J., Haverkate, F. & Nieuwenhuizen, W. (1986) Production and characterization of a monoclonal antibody reactive with a specific neoantigenic determinant (comprising Bß 54-118) in degradation products of fibrin and of fibrinogen. Blood. 68:437-441.
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9. Hoegee-de-Nobel, M., Voskuilen, E., Briet, E., Brommer, E.J.P. & Nieuwenhuizen, W. (1988) A monoclonal antibody-based quantitative enzyme immunoassay for the determination of plasma fibrinogen concentrations. Thromb. Haemost. 60:415- 418.
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(1978) Radioimmunoassay of a large cyanogen bromide
peptide of the human fibrinogen Aα chain. Recent Progress in Blood Coagulation and Thrombosis Research. Biblthea hemat. 44:114-116. Karger, Basel.
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13. Topfer-Petersen, E., Lottspeich, F. and Henschen, A.
(1976) Carbohydrate linkage site in the Bß-chain of human fibrin. Hoppe-Seyler's Z. Physiol. Chem. 357:1509-13.
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Canfield (1983) Monoclonal antibodies to α chain regions of human fibrinogen that participate in polymer formation. Biochemistry 22:4184-4192.
17. Sobel, J.H., Koehn, J.A., Friedman, R. and R.E.
Canfield (1982) Alpha chain crosslinking of human fibrin: Purification and radioimmunoassay
development for two Aα chain regions involved in crosslinking. Thromb. Res. 26:411-424.
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Claims
1. A method of detecting the presence of an Aα fragment in a fibrinogen- or fibrinogen derivative-containing sample from a human subject which comprises:
a) contacting the sample with an agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains an Aα chain under conditions permitting formation of a complex;
b) removing a complex so formed from the sample; c) contacting the resulting sample with a monoclonal antibody capable of forming a complex with an epitope containing amino acid residues present exclusively between amino acid residue 477 and the carboxy terminus of the Aα fragment, under conditions permitting formation of a monoclonal antibody-Aα fragment complex; and
d) detecting any such complex formed in the sample, thereby detecting the presence of Aα fragment in the sample.
2. A method of claim 1, wherein the sample is a plasma sample.
3. A method of claim 1, wherein the sample is a urine sample.
4. A method of claim 1, wherein the agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains an Aα chain is a carbohydrate-binding material.
5. A method of claim 4, wherein the carbohydrate- binding material is a lectin.
6. A method of claim 5, wherein the lectin is Concanavalin A.
7. A method of claim 1, wherein the monoclonal antibody is directed to an epitope centered around about amino acid residue 550.
8. A method of claim 7, wherein the monoclonal antibody is the monoclonal antibody F-102 (ATCC No. HB 10815).
9. A method of claim 1, wherein the detecting comprises direct detection of the complex using a reagent specific for the complex.
10. A method of claim 1, wherein the detecting comprises indirect detection of the complex by detecting uncomplexed monoclonal antibody.
11. A method of claim 10, wherein the detecting comprises contacting the uncomplexed monoclonal antibody with an immobilized Aα chain-containing protein under conditions permitting formation of an immobilized monoclonal antibody-Aα chain-containing protein complex, contacting the complex with a detectable antibody directed to the monoclonal antibody under conditions permitting formation of a detectable complex, and detecting the resulting detectable complex.
12. A method of claim 11, wherein the immobilized Aα chain-containing protein is intact fibrinogen.
13. A method of claim 11, wherein the detectable
antibody is a monoclonal antibody.
14. A method of claim 11, wherein the detectable antibody comprises a detectable marker.
15. A method of claim 14, wherein the detectable marker is an enzyme, radiolabel, or fluorescent marker.
16. A method of claim 15, wherein the detectable marker is an enzyme.
17. A method of claim 16, wherein the enzyme is coupled to the antibody and is an HRP-linked rabbit antimouse immunoglobulin (RAM-Ig-HRP).
18. A method of quantitatively determining the amount of Aα fragment in a sample from a human subject which comprises:
a) contacting the sample with an agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains an Aα chain under conditions permitting formation of a complex;
b) removing any complex so formed from the sample; c) contacting the resulting sample with a monoclonal antibody capable of forming a complex with an epitope containing amino acid residues present exclusively between amino acid residue 477 and the carboxy terminal residue of the Aα fragment, under conditions permitting the formation of a monoclonal antibody-Aα fragment complex; and
d) quantitatively determining the amount of complex formed and thereby quantitatively determining the amount of Aα fragment in the sample.
19. A method of claim 18, wherein the sample is a plasma sample.
20. A method of claim 18, wherein the sample is a urine sample.
21. A method of claim 18, wherein the agent capable of forming a complex with intact fibrinogen, or with a fibrinogen derivative which contains an Aα chain is a carbohydrate-binding material.
22. A method of claim 21, wherein the carbohydrate- binding material is a lectin.
23. A method of claim 22, wherein the lectin is Concanavalin A.
24. A method of claim 18, wherein the monoclonal antibody is the monoclonal antibody F-102
(ATCC No. HB 10815).
25. A method of claim 18, wherein quantitatively
determining comprises directly determining the amount of the complex using a reagent specific for the complex.
26. A method of claim 18, wherein quantitative
determining comprises indirectly determining the amount of complex by detecting the amount of uncomplexed monoclonal antibody.
27. A method of claim 26, wherein quantitatively
determing the amount of uncomplexed monoclonal antibody comprises contacting the uncomplexed monoclonal antibody with an immobilized Aα chain- containing protein under conditions permitting formation of an immobilized monoclonal antibody-Aα chain-containing protein complex, contacting the complex with a detectable antibody directed to the monoclonal antibody under conditions permitting formation of a detectable complex, determining the amount of the complex, and comparing the result with that of a known standard, thereby quantitatively determining the amount of Aα fragments.
28. A method of claim 27, wherein the immobilized Aα chain-containing protein is intact fibrinogen.
29. A method of claim 27, wherein the detectable
antibody is a monoclonal antibody.
30. A method of claim 27, wherein the detectable antibody comprises a detectable marker.
31. A method of claim 30, wherein the detectable marker is an enzyme, radiolabel, or fluorescent marker.
32. A method of claim 31, wherein the detectable marker is an enzyme.
33. A method of claim 32, wherein the enzyme is coupled to the antibody and is an HRP-linked rabbit anti- mouse immunoglobulin (RAM-Ig-HRP).
34. A method of monitoring fibrinogenolysis in a patient which comprises obtaining a sample from the patient, quantitatively determining the amount of Aα fragment in the sample using the method of claim 18, and comparing the amount detected with a known standard, thereby monitoring fibrinogenolysis in a patient.
35. A method of claim 34, wherein the sample is plasma.
36. A method of monitoring fibrinolysis in a patient which comprises obtaining a sample from the patient, quantitatively determining the amount of Aα fragment in the sample using the method of claim 18, and comparing the amount detected with a known standard, thereby monitoring fibrinolysis in a patient.
37. A method of claim 36, wherein the sample is plasma.
38. A method of monitoring the effectiveness of thrombolytic therapy in a patient which comprises obtaining at different times during the course of therapy samples from the patient, quantitatively determining the amount of Aα fragment present in the samples using the method of claim 18, and comparing the amounts so determined, thereby monitoring the effectiveness of thrombolytic therapy in a patient.
39. A method of claim 38, wherein the sample is plasma.
40. A method of diagnosing a fibrinolytic disorder in a patient which comprises obtaining a sample from a patient, quantitatively determining the amount of Aα fragment in the sample using the method of claim 18, and comparing the result with a known standard, thereby diagnosing a fibrinolytic disorder.
41. A method of claim 40, wherein the sample is plasma.
42. A method of diagnosing a fibrinogenolytic disorder in a patient which comprises obtaining a sample from a patient, quantitatively determining the amount of Aα fragment in the sample using the method of claim 18, and comparing the result with a known standard, thereby diagnosing a fibrinogenolytic disorder in a patient.
43. A method of claim 42, wherein the sample is plasma.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002112228A CA2112228A1 (en) | 1991-06-28 | 1992-06-29 | Assay for detecting fibrinolysis and figrinogenolysis based on cooh-terminal a chain markers |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US72268191A | 1991-06-28 | 1991-06-28 | |
US722,681 | 1991-06-28 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1993000364A1 true WO1993000364A1 (en) | 1993-01-07 |
Family
ID=24902908
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1992/005535 WO1993000364A1 (en) | 1991-06-28 | 1992-06-29 | ASSAY FOR DETECTING FIBRINOLYSIS AND FIBRINOGENOLYSIS BASED ON COOH-TERMINAL Aα CHAIN MARKERS |
Country Status (3)
Country | Link |
---|---|
AU (1) | AU2307692A (en) |
CA (1) | CA2112228A1 (en) |
WO (1) | WO1993000364A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2769093A1 (en) * | 1997-09-30 | 1999-04-02 | Pasteur Sanofi Diagnostics | Detecting platelet activation and predicting risk of thrombosis |
WO2009082340A1 (en) * | 2007-12-20 | 2009-07-02 | Astrazeneca Ab | Method for monitoring the progression of fibrinolysis'646 |
-
1992
- 1992-06-29 AU AU23076/92A patent/AU2307692A/en not_active Abandoned
- 1992-06-29 WO PCT/US1992/005535 patent/WO1993000364A1/en active Application Filing
- 1992-06-29 CA CA002112228A patent/CA2112228A1/en not_active Abandoned
Non-Patent Citations (5)
Title |
---|
BIOCHEMISTRY, Vol. 22, issued 30 August 1983, P. EHRLICH et al., "Monoclonal Antibodies to Alpha-Chain Regions of Human Fibrinogen that Participate in Polymer Formation", pages 4184-4192. * |
SIGMA DIAGNOSTICS 1991 CATALOG, page 120, "Fibrin Degradation Products (FDP) and (Fibrinogen)". * |
THROMBOSIS AND HAEMOSTASIS, (Stuttgart), Vol. 39, issued 1978, K. TAKAGI et al., "Radioimmunoassay of an Early Plasmin Degradation Product of Human Fibrinogen, 'Fragment A', and its Clinical Application", pages 1-11. * |
THROMBOSIS AND HAEMOSTASIS, (Stuttgart), Vol. 60, No. 2, issued 1988, C. BOGLI et al., "Isolation of Fibrinogen A Alpha-Chain by Affinity Chromatography on Concanavalin A-Sepharose", pages 308-310. * |
THROMBOSIS RESEARCH, Vol. 8, issued 1976, M. BLOMBACK et al., "Immunological Characterization of Early Fibrinogen Degradation Products", pages 567-577. * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2769093A1 (en) * | 1997-09-30 | 1999-04-02 | Pasteur Sanofi Diagnostics | Detecting platelet activation and predicting risk of thrombosis |
WO2009082340A1 (en) * | 2007-12-20 | 2009-07-02 | Astrazeneca Ab | Method for monitoring the progression of fibrinolysis'646 |
Also Published As
Publication number | Publication date |
---|---|
AU2307692A (en) | 1993-01-25 |
CA2112228A1 (en) | 1993-01-07 |
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