CA1247022A

CA1247022A - Monoclonal antibodies with specificity for crosslinked fibrin derivatives and assay for said derivatives

Info

Publication number: CA1247022A
Application number: CA000449733A
Authority: CA
Inventors: Dennis B. Rylatt; Peter G. Bundesen
Original assignee: MABCO Ltd
Current assignee: MABCO Ltd
Priority date: 1983-03-17
Filing date: 1984-03-16
Publication date: 1988-12-20
Also published as: PT78267A; EP0122478A3; DE3486262T3; DK158284D0; NZ207459A; AU572125B2; PT78267B; NO841023L; KR840008288A; EP0122478A2; DE3486262T2; EP0122478B2; IL71264A0; DE3486262D1; ES530691A0; IL71264A; NO167338B; ATE99738T1; US4758524A; IN158324B

Abstract

ABSTRACT

This invention relates to a method of preparation of monoclonal antibody derived from a crosslinked fibrin derivative including the steps of;
(i) obtaining a crosslinked fibrin derivative or extract containing same; and (ii) forming antibody to said derivative or extract by cloning antibody producing cells from an animal having been administered thereto said derivative or extract.
There is also provided a screening assay for use in the abovementioned method which includes the steps of:
(A) coating a surface with antigen selected from (i) crosslinked fibrin derivative;
(ii) extract containing said derivative, or (iii) fibrinogen degradation product.
(B) contacting said antigen with monoclonal antibody derived from crosslinked fibrin derivative; and (C) subjecting the complex formed in step (A) to a signal amplification step.
There is also provided an assay procedure for detection of the presence of a crosslinked fibrin derivative in an animal body fluid including the steps of:
(i) contacting monoclonal antibody prepared from a crosslinked fibrin derivative with a fluid sample suspected of containing an antigen derived from a crosslinked fibrin derivative or comprising a crosslinked fibrin derivative per se; and (ii) subjecting the complex formed in step (i) to a signal amplification step.

Description

761~

THIS INVENTION relates to monoclonal antibodies derived from crosslinked fibrin derivatives and assays for said cross linked derivatives which may be used as a diagnostic test for fibrin breakdown products in fibrinolysis generally and pre-thrombotic states and thrombotic states includiny Disseminated Intravascular Coagulation (DIC).
Fibrinogen is a large protein molecule that normally circulates in blood plasma in a dissolved state.
Under attack from an enzyme thrombin the fibrinogen molecules link up, spontaneously aligning themselves into the long thread like polymer or network called fibrin which is the primary ingredient of blood clots.
It h~s been discovered that upon digestion with an enzyme called plasmin twhich ~unctions in the blood to destroy the fibrin network and restore the fluidity of the plasma) that fibrinogen breaks down into fragments designated A-E. Fragments D and E made up the bulk of the recovered mass and there was about twice as much D as there was of E.
Fibrinogen also has been discovered to have a trinodular shape wherein E is a central component and D is a terminal component.
Plasmin digests of fibrin and fibrinogen can be differentiated from each other using polyacrylamide gel electrophoresis (PAGE). Crosslinking of fibrin with an enzyme called Factor XIIIa forms dimers of fragment D called D dimer. Factor XIIIa is an enzyme which introduces covalent bonds between adjacent monomers in fibrin and thus may stabil-ize the fibrin structure. For a more detailed explanation of the nature of the crosslinking between fibrin monomers we refer to Budzynski et al Blood Vol 54 No. 4 (October)1979.
Factor XIIIa is activated by the thrombin-catalyzed removal of a peptide from a precur~or in the pla~ma ~nd qn blood platelets. D dimer is a molecule of about 189,000 daltons which consists essentially of two fragment D moieties derived from diEferent fibrin molecules covalently bound by cross link bonds between the gamma chain remnants of fibrinogen.

Fibrinogen itself comprises 6 chains comprising two copies of an alpha, beta and gamma chain.
Another complex (DD)E is formed by plasmin degradation of cross linked human fibrin and comprises a combination of two D fragments and fragment E.
Other cross linked derivatives may be prepared as described in an article from Seminars in Thrombosis and Hemostasis Vol 8, No. 1 tl982) entitled "Detection and Relevance of Crosslinked Fibrin Derivatives in Blood" by Graeff and Halfer. These include high molecular weight cross linked derivatives and may be referred to in the above reference as derivatives DY, YY, XD~ XY, DXD and YXD.
Normal haemostasis or coagulation of blood involves maintaining intravascular constituents in a liquid phase or suspension while concomitantly permitting local deposition of solid phase blood components in areas of vessel damage.
In health it has been assumed, but never experimentally demonstrated, that a balance exists between a low-grade intravascular deposition of fibrin and its removal by fibrinolysis or cellular phagocytosis.
Early clinical observations revealed that some severely ill patients developed signs of haemorrhage and massive bruising and had prolonged clotting times and thrombocytopenia. At postmortem, in some cases, fibrin thrombi were demonstrated in the microv~sculature. The diffuse nature of these thrombi gave rise to the term ~isseminated intravascular coagulation" (DIC). Subsequently, coagulation factors were shown to be reduced. These findings give rise to the concept of "consu~ptive coagulopathy", a term sometimes used as a synonym for DIC.
The currently accepted sequence of events in DIC
involves activation of the coagulation system resulting in platelet consumption, thrombin yeneration, fibrin deposition, and secondary fibrinolysis. The net biologic effect of this process reflects a balance between fibrin deposition and Z~ , fibrin clearance. The resulting clinical manifestations may be haemorrhage~when depletion of coagulation factors pre-dominates, or ischemic tissue damage due to the effects of vascular occlusion.
DIC has been rep~rted as a secondary phenomenon in a wide variety of disorders, particularly those accompan-ied by a combination of shock, acidosis, and hypoxemia.
The well-recognized clinical associations are sepsis, major trauma, malignancy, and obstetric disorders. Xn these clinical settings activation of the coagulation sequence results in consumption of coagulation protein and platelets, leading to fibrin deposition in the micro-circulation.
The precise factors that initiate the DIC are unknown, but many potential mechanisms have been demonstrated in animal experiments.
Ideally a definitive diagnosis of DIC should requi~e the direct demonstration of diffuse fibrin deposition. The practical difficulty of obtaining multiple direct biopsy evidence to differentiate between localized and generalized fibrin formation has led to the development of indirect tests that are substituted as diagnostic end points. ~owever these tests are not specific for the syndrome of intravascular fibrin deposition. Their specificity is further reduced by the action of other enzymes that although not able to convert fibrinogen to ~ibrin can cause similar alterations to thrombin on the other coagulation factors involved in thrombosis. All of the indirect tests are based on the principle that thrombin is the only enzyme (snake venoms excluded) capable of convert-ing fibrinogen to fibrin in man.
Also apart from the p~racoagulation tests that detect the presence of circulating soluble fibrin monomer complexes, none of the more specific thrombin specific tests is readily available ~r useful for immediate clinical application in the diagnosis of clinical DIC. These tests include the FPA (fibrinopeptide A) test where FPA is measured by a specific RIA procedure, fibrin monomer assays, fibrinogen gel excluslon chromatography and tests for FPB
, 7~Z;~

(fibrinopeptide B~ or thrombin increasable FPB, Tests with biochemical nonspecificity for throm-bin action include the prothombin time (PT) thromboplastin tLme (A PTT) and thrombin clotting time (TCT) tests, Althoug~
S frequently useful in practice it must be recognized that information obtained from these tests is nonspecific in nature, acting as a measure of clotting factor depletion regardless of etiology.
Coagulation factor assays have also been found to be relatively non specific and these include assays for cofactors V and VIII as well as tests for fibrinogen levels.
Tests for fibrin-fibrinogen degradation products so far have not proved to be specific for the action of plasmin on fibrin and may yield positive results where there has been fibrinogenolysis without prior thrombin action on the fibrin-ogen molecule. These tests include tests for fragments D and E.
Tests for thrombin-mediated platelet interaction or release have been found to be nonspecific in nature. These include platelet count, platelet survival and tests of platelet release.
The use of radio labelled fibrinogen in relation to identifying clotting factors have also been attempted but found to be time consuming and difficult to perform.
Thus, in summary of the prior art, the efficacy of a diagnostic test lies in its ability to indicate the presence or a~sence of disease. There are well recognized essential design principles for studies determining the efficacy of a diagnostic test which enables the four 3~ indices of sensitivity, specificity, positive predictive value, and negative predictive value to be determined. The first requirement is the adoption of a suitable standard for diagnosis~ Ideally, this standard should be slightly more than a clinical definition and should be as specific as possible for the disease entity. An inherent difficulty ~7~;~2 in relation to ~IC is the absence of a comprehensive definition of this disorder~ The clinical picture is very nonspecific. Many of the routinely available laboratory tests also lack diagnostic specificity. A low platelet count supports the likelihood of DIC but may occur as an isolated findlng secondary to infection. Si~ilar limitations apply to many of the coagulation assays. Hypofibrinogenemia does not distinguish bekween primary fibxinolysis, due either to the action of plasmin or elastases, and secondary fibrinolysis following the thrombin-medicated conversion of fibrinogen to fibrin. Alternatively, sensitive tests of thrombin action are available, but there are obvious draw-backs with their clinical use. An example is the ~PA assay, which, although specific for thrombin action, is exquisitely sensitive and may detect localized intravascular coagulation yielding a positive result in uncomplicated venous thrombosis.
The clinical significance of an elevated FPA level~ even with a positive paracoagulation test, is then at issue, particularly if the platelet count, global clotting tests, and fibrinogen level are normal.
For these reasons, sensitivity, specificity, and predictive v~lues cannot be determined~in a standard fashion.
The clinical presentation of the disorder is complex and unpredictable~ The application of the available tests for diagnosis are therefore best considered in relation to the different clinical syndromes of intravascular coagulation.
It has also been proposed to assay for D dimer as a diagnostic test for DIC. However, this has necessitated the use of PAGE as described previously and this technique is far too cumbersome for routine clinical use. ~ntibodies have been raised to fibrin derived D~D-E fragments but in their current form these cross react with fibrinogen fragment D derivatives and as yet are unsuitable for clinical use.
A useful summary of DIC and conventional diagnostic tests will be found in Seminars in Thrombosis and Hemostasis Vol 8 No. 3(1982) and an article entitled DIC; The Application and Utility o~ Diagnostic Tests by Ockelford and Carter.
In the abovementioned Budzynski reference there i5 described the study o~ polyclonal anti-D dimer antibodies using two different antisera. In this test antibodies were raised against specific markers on the D dimer molecule, In the test antisera were obtained in chickens and rabbits against a mixture (1;1) of D2E complex and D dimer and against D dimer exposed to 3M area at pH 5.5. It was however stated in this reference that it was hoped by the authors that the results of this test could be applied to clinical situations, such as the distinction between disseminated intravascular coagulation and primary fibrinogenolysis, since circulating fragment D
dimer should be ~resent in the former conditions but not in the latter~ ~owever, such application ~ould require a much higher difference in reactiVity between fragments D dimer and D, since this and other relevant clinical states have high concentrations o~ circulating fibrinogen derivatives in aadition to the crosslinked fibrin fragments~ It was also

2~ considered that althou~h it was shown that the assays could be performed eVen in the presence of enormous concentrations of fibrinogen, ~urther development of the antibody specificity was needed before it can be reliably applied to the relevant clinical situations.
The abo~ementioned Graeff and Hafter article also points out that crosslinked fibrin derivatives in blood such as D dimer may be considered as a marker for DIC.
~owever, there is nothing in this article to demonstrate that a reliable di,agnostic'test for D~C could be devised based on crosslinked fibrin derivatives.
It is there~ore an object of the present invention to provide ~n ass~ pr~cedure for crosslinked ~brin derivat-ives which may be used on a clinical basis.
The in~ention pxovides a method of preparation

3~ of a monoclonal ,a,ntibody deri~ed ~rom a crosslinked fibrin derivative including the steps of (i) obtaining a crosslinked fibrin dexivative or extract containing same, and (ii) forming antibody to said derivative or said extract by cloning antibody producing cells from an animal ha~ing been administered thereto said derivative or said extract.
In step (i) a suitable antigenic extract could be obtained from plasmic degradation of fibrin clots or by simultaneous action of thrombin, Factor XIIIa and plasmin on fibrinogen with transient clot formation and subsequent clot lysis. In the latter method the fibrinogen is converted to fibrin by the action of thrombin and Factor XIIIa and subsequently digested with plasmin, It will of course be appreciated that the fibrin derivative or extract containing 1~ same may be obtained from ~ human or other suitable animal source, The aboye methods of obtaining the crude antigenic fraction are in vitro methods. A suitable in vivo method would be to obtain ser~ or other body fluid containing the crosslinked fibrin derivative from an animal including humans and subject the body 1uid to a PAGE process wherein substant-ially pure crosslinked fibrin derivative may be isolated.
Alternatively crosslinked fibrin derivatives could be purified from seru~ obtained from patients suffering severe thrombotic disorders based on a technique using gel filtration in combination with ion exchange chromatography as described in Willner et al Biochemistry 21 2687-2692 (1982) where human fibrinogen, purified fragments D, E and D dimer were prepared.
When using a pure crosslinked fibrin derivative such as D dimer, care must be taken in its preparation to not denature the ~olecule as it is susceptible to denaturation fairly easily, ~7~

Fo~ a mo~e complete description of the abovemention ed methods in preparing an antigenic extract which is usually of a crude nature reference may be made to the abovementioned Graeff and Hafter article. Suitable crosslinked fibrin derivatives for use in the invention may be any one of those previously described but preferably the derivative is D2E or D dimer and most suitably the derivative for use in the invention is D dimer.
In step ~ii) a suitable animal to which the derivative or extract thereof may be administered is a mouse or rat. A mouse is preferred. It is also preferred to administer crude extract one or more tLmes initially and follow this up with administration of pure or substantially pure crosslinked fi~rin derivative. This procedure is pr-eferred so that the task of obtaining monoclonal antibodies specific to the derivative is simplified.
After administration the mice which have had derivative or extract administered thereto are suitably killed and the spleens removed for subsequent processing to form a cell suspension. Further purification of the cell suspension may take place (eg, by centrifugation) to isolate spleen white blood cells or l~mphocytes which may be fused with mouse myeloma cells.
The cloning technique may be broadly based on the technique described in Galfre et al Nature 266 550-2 ~1977) where polyethylene glycol is used as the cell fusing agent to form a hybridoma cell which may then be cloned or recloned as desired suitably or the basis of limiting dilution using appropriate cell feeder layers.
Preferably for the cultivatio~ of hybridoma cells well plates are utilized wherein cell suspensions are placed in each well with appropriate cell cultivation media.
It is pxeferred to remove samples of cells for screening assays and these may be carried out as described hereinafter. A number of the strongest gro~h wells are ~ 7~2;i~

suitably chosen for maintenance on the basis of the screening assays. After the screening assays it is possible to choose a number of specific antibody producing clonotypes to produce monoclonal antibody secreting cell lines by limiting dilution.
On the basis of further screening assays carried out on samples taken from well plates incorporating the limiting dilution clonotypes a number of specific antibody produced clones may be chosen for expansion to mass culture.
A suitable screening assay for use in the above-mentioned process may comprise the steps of:
(a) coating a surface with antigen selected from cross-linked fibrin derivative or extract containing same or fibrinogen degradation product, (b) contacting tne antigen in step (i) with monoclonal antibody derived from fibrin crosslinked derivativ~
prepared as described ~bove, [c) sub~ectinq the complex formed in ~tep ~ii) to a signal amplification step.
Suitably in step (a) ~ well plate may be utilized in which crosslinked fibrin derivative such as D dimer and~or fibrinogen degradation product (preferably obtained from a procedure wherein fibrinogen was suitably digested with thrombin to obtain fragment D, fragment E and optionally 5 fragments X and Y) was applied to the individual wells.
Subsequently monoclonal antibody derived from a crosslinked fibrin derivati~e was then added to each well.
An appropriate signal amplification step which may be applied is an EIA step wherein an appropriate enzyme conjugate may be coupled to the complex and substrate subsequently added.
Alternatively RIA~ FIA, agglutination, adherance or chemil-uminescence may be used as ~ppropriate signal amplification st~ps.
The purpose of the screening assay procedure referred to above is to ensure that the cells being tested are producins antibody specific to the relevant crosslinked fibrin deri~ative.

10 .

~7~

There should be no reaction with fibrinogen or fibrinogen degradation products and a positive reaction with the derivative.
The invention also includes within its scope an assay procedure for detection of the presence of a cross linked fibrin derivative including the steps of:
(1) contacting monoclonal antibody prepared from a crosslinked fibrin derivative with a fluid sample suspected of containing an antigen derived from a crosslinked fibrin derivative or comprising a crosslinked fibrin derivative per se; and (2) subjecting the complex formed in step (1) to a signal amplification step~
In the abovementioned assay the crosslinked fibrin 15 derivative is suitably D dimer, DzE or any other derivative of a high molecular weight nature as described above. The monoclonal antibody is pxepared as described previously which is relevant to the parti~ular crosslinked fibrin dexiyative being assayed.
The single amplification step may be any one of those already described in relation to the screening assay procedure but is suitably EIA.
The presence of the crosslinked fibrin derivative may be used as a suitable diagnostic aid for prethrombotic, thrombotic or other conditions that involve the formation and lysis of fibrin.
The assay of the invention may also be used for monitoring lytic therapy such as streptokinese therapy and tissue plasminogen activator therapy (TPA). An example of a prethrombotic state is a stress condition, Examples of thrombotic states include DIC, pulmon~ry embolus, throm, bosis, invasive tumours and other thrombotic states describ-ed hereinafter.
The fluid sample may be obtained from any suitable body fluid such as lymph, serum, plasma or exudate.

~ t7~ ~

The assay procedure ~ay be performed using a tube, well plate or micro plate as described previously or may be carried out in any other suitable manner including convent-ional procedures. A n stick" procedure using a single elongate member may be utilized wherein Ag or Ab is initially coated thereon before application of steps (b) and (c).
In another embodiment of the invention the monoclonal antibodies can be covalently attached to small beads. Such beads enable a quick (e.g. 2-3 minute) test to be carried out in serum plasma or other body fluids for the presence of crosslinked fibrin derivatives. The beads may be formed from polystyrene, nylon, glass or other suitable material.
In relation to polystyrene, the MAb can be coupled thereto using the carbodiimide method as described in Molday et.
~5 al J.Cell Bio ~4 75 (1975). For nylon beads a suitable coupling procedure is described in Hendry and Herrman J.
Immun. Method 35 2B5 (1980) using glutaraldehyde. For glass beads a suitable coupling pr~cedure using silaning agents may be utilized as described in U.S. Patent 4210723. In these bead assays or latex assays when the beads which have already been coupled to MAbs are tested with t~st serum or plasma or other body fluid they may be checked for agglutinat-ion by use of a suitable calibration standard. In this embodiment latex particles or beads are prepared as uniform sphexes o~ known refracti~e index and used as calibration standards for light scattering magnification, shadow angle, thickness of shadow material, scanning and transmission electron microscopy or laser scattering~
It will of course be appreciated that assays 3~ based on capture-tag techniques whe~ein a ixst M~b is captured by an ~ntigen which is subsequentially tagged by a labelled second MAb (where the label may be ~uitably used in an EIA or RIA test)may be used in relation to this invention, 12.

However, in some cases assays based on use of a single MAb may be used such as the abovementioned bead or latex assays described above.
In relation to an assay for a specific crossllnked fibrin derivative it has been found that of all the MAbs tested a number were discovered to be panspecific tie. binding to epitopes or reactive sites on fibrin breakdown products as well as fibrinogen breakdown products) and others were monospecific (ie~ binding only to reactive sites on ~ dimer and other crosslinked fibrin derivatives)., When capture-tag experiments were carried out as discussed hereinafter in one type of assay a monospecific MAb was bound to a support sur~ace and ~as tested with serum or other bocly fluid suspected o~ containing crosslin~ed fibrin derivative. When tagged with a second antibody which was a panspeci~ic MAb attached to an appropriate label used in the signal ~mplification step,this ~rovided a precise ass~y for crosslinked fibrin derivatiye if the panspeci~ic MAb bound to an epitope in the sa~ple~
In a variation of this technique a panspecific MAb could be bound to a support surface and tested with body fluid suspected of containing the crosslinked fibrin derivat-ive, Subsequently a monospecific MAb could be tagged to the body fluid antigen having a suitable label attached thereto.
In another version it is also possible to bind a fi~st monospecific MAb to a support surface and test same with a sample of bod~ ~luid suspected of containing a cross-linked fibrin derivati~e. Subsequently a second monospecific MAb could be tagged to the body fluid antigen having a suitable label attached thereto.
In the following experi~ents human fibrinogen and puri,f,ied fragments D~ E and D dimer were prepared as described abo~e in the ~ilner reference. Flbrinogen degradation products were prepared as described in Thromb ~es 10 803~812, (19?7) Haverkate and Timan. Crosslinked fibrin which was necessary 2~

for the preparation of D dimer was prepared and digested with plasmin as described by Olexa and Budzynski Biochemistry 18 991 (1979).
Reference is made to the accompanying drawings, in which:
FIGURE 1 represents graphically the results of an experiment to determine binding of D dimer specific DD-386/22 with either D dimer fibrinogen degradation productus, fibrinogen, fibrin degradation products and D
monomer using perooxidase conjungated DD~4D2/108 as label.
FIGURE 2 represents graphically the results of an experiment to determine binding of panspecific DD-4D2/182 with either D dimer, fibrin degradation products, fibrinogen, fibrinogen degradation products or fragment D
lS using peroxidase conjugated DD-lC3/108 as label.
FIGURE 3 represents graphically the results of experiments to determine the efficacy of monoclonal antibody to recognize crosslinked fibrin derivatives in blood extracts of suspect test subjects.
FIGURE 4 represents the test results obtained when monoclonal ~D~3B6/22 is covalently attached to fixed latex beads and subjected to a solution containing crosslinked derivative.

14.
-~Z~7~zæ

EXPERIMENTALCell Fusion and Selection o Hybrlds Spleens were removed aseptically from 2 immunized mice killed by cervical dislGcation three days after an injection of D dimer. Previously the mice had been immunized with three injections of fibrin lysate digested with proteolytic enzymes thrombin and plasmin as reported in the aforementioned Graeff and Hafter reference. Two spleens were placed in a 60 mm Petri dish (Falcon, 3001, Oxnard, Calif.) containing 5 ml complete medium (85% RPMI
1640, 15% foetal calf serum, 100 I.U./ml penicillin, 100 ~g/ml streptomycin and 2X10 M Glutamine; Gibco, Grand Island, N.Y.). A cell suspension was prepared by decapsulating the spleen with 2X18 gauge needles attached to 3 ml disposable syringes with the last cm of the tip bent through an angle of 60. The cell suspension was then aspirated into a 10 ml syringe fitted with a 22 gauge needle and ejected with moderate pressure. This operation was performed twice before filtering the cells into a Falcon 2001 tube through a fine mesh stainless steel screen to remove larger cell clumps and debris.
The cell suspension was allowed to stand for 5 min~
utes at room temperature to allow smaller clumps and membrane fragments to settle before transferring the cell suspension to a fresh Falcon 2001 tube. The cells were centrifuged at 350G for 5 minutes at room temperature and the supernatant was decanted f rom the first cell pellet to a fresh tube and spun at 700G for five minutes to give a-second cell pellet and the two pellets were pooled and resuspended in 5 ml complete medium. The spleen white blood cells (SWBC) were then counted and their viability estimate~ by Turks and Txy-pan blue stains respectively, and 100Xl06 viable SWBC were placed in separate Falcon 2001 tubes in a total volume of 5 ml complete medium. The NS-l myeloma cells to be used for 35 fusion~ were washed once by centrifugation at 380G for 15 15.

7~22 minutes at room temperature and adjusted to 5X106 viable cells/ml in complete medium.
Twenty-five X 106 NS-l and lOOX10~ immune SW~C
were mixed and spun at 350G for 5 minutes a-t room temperature.
The supernatant was decanted, the remaining medium was care-fully removed with a Pasteur pipette and 2 ml of a 42% (w/v) solution of polyethylene glycol (PEG, MW1540) (Baker Chemical Co., New Jersey) In RPMI 1640 containing 15~ (v/v) dimethyl sulfoxide (DMSO) at 37C was added with a 5 ml glass dispos-able pipette (Corning Glass, Corning, N.Y.) and the cellswere resùspended with the same 5 ml pipette for 30 seconds with the aid of an electric pipetter (Pipet-aid Drummond 5cientific Co., Broomall, Pa.). The PEG-cell suspension was allowed to stand for a further 30 seconds at room temperature before adding 5 ml complete medium, dropwise, with a Pasteur pipette, over a period of 90 seconds with constant flicking of the tube, sufficient to ensure complete mixing with the viscous PEG solution. A further 5 ml complete medium was immediately added and mixed by inversion and the cell suspension was allowed to stand for a further 150 seconds at room temperature before centrifugation at 350G for 5 minutes at room temperature. The supernatant was decanted and the cell pellet was gently resuspended in 5 ml complete medium using a 5 ml pipette with the electric pipetter; extreme care was taken not to break up all cell clumps. Using a Tridak stepper (Bellco Glass Inc., Vineland, N~J.), 0.05 ml of the cell suspension was added to each well of 4 Costal~ 24 well plates (Costar 3524, Cambridge, Mass.) containing lXl06 normal BALB/c mouse SWBC as feeder cells in 1 ml complete medium containing 10 M ~ypoxanthine (Sigma), 4X10 M
Aminopterin (Sigma), 1.6X10 M Thymidine (Sigma) and 4X10- M
2-Mercaptoethanol (HAT me~ium), hereafter referred to as 1 fusion plates.
The 1 fusion plates were then placed in a humidif-ied 5% CO2 95% air atmosphere at 37 C. The cells were first * TRADE MARK

16.
.. ..

2~

fed either on days 5 or 7 and thereafter when necessary, with 0.5 ml fresh HAT medium, Gener~lly~ on day 10, O.5 ml of the medium was removed for the screenin~
assay from each well showing hybridoma growth and 0.5 ml fre~h 5 XAT medium was replaced. A number of the strongest growth wells were chosen for maintenance on the basis of the screen-ing assay. The chosen wells were allowed to grow to conflu-ency in the original well (1 well)l then ea'ch was split in half and transferred to a fresh well (2 well) of a 24 well 10 Costar plate (2 plate). The wells were checked daily and expanded to a 2nd, 3rd or 4th well of the 2 24-well Costar plate when necessary. From days 14-28, cells were fed with ~T ~edium. When there was strong growth in at least 2 wells of the 2 plate, supernatant from one well of each clonotype 15 was chosen for rescreening and a number of specific antibody producing clonotypes were chosen from the results of the second screening assay to produce monoclonal antibody secret-ing cell lines by limiting dilution.
Clonin~ of'Hy~'r'id'omas One 2 well of each chosen clonotype was resuspended and the number of viable cells per well was estimated by Try-pan blue exclusion. Immediately before plating each clono-type, the relevant series of dilutions were made in HT medium or complete medium (if the c~lls were older than 28 days post 25 fusion) to give a frequency of 0.5 cells/0.05 ml. This volum~ was then added with a Tridak stepper to each well of a 96 well flat bottomed tissue culture plate (Flow Laboratories, Mississauga, Ontario, Canada)' (L D plate) containing lX10 normal mouse spleen feeder cells in 0.1 ml HT or complete 30 medium The LD plates were~then placed in a 37~C humidified 5% CO2, 95% air atmosphere and screened for clonal growth 7-10 days later~ From each positive growth well, 0.1 ml supernatant was remo~ed for screening and these wells ~ere fed for the first time with 0.1~0.15 ml HT or complete 35 medium. On the basis of the LD screening assay, a minimum of 2 of the 'better' specific antibody-producing clones were finally selected for expansion to mass culture.

17.

zz Alternatively if it was desired to obtain a large amou~t of ~b, female BALB/c mice were given an intraperitoneal injection of 0,5 ml 2, 5, 10, 14, tetramethyl-pentadecane (Pristane* Aldrich Chemical Corp., Milwaukee, Wisconsin) 14 days prior to the injection of 2X106 viable hybridoma cells and ascites fluids were collected from the mice 12 to 14 days after injection of the cells. The ascitic fluid was clarified by centrifugation and MAb were recovered by precipitation with 45~ ammonium sulphate and stored at either 4C or -70C in phosphate buffered saline (PBS) containing 0.01~ sodium azide.
Monoclonal antibody screening assay The wells of a 96 well U bottomed microtest plate (Disposable Products Pty. Ltd., Adelaide, South Australia) were coated by adding 50~1 of either D dimer (5 ~g/ml) or Fibrinogen degradation products (5 ~g/ml in PBS for one hour at room temperature (25C). Excess antigen was removed by inverting and tapping the plate and the plate was then washed three times with PBS containing 0.05~ Tween*20 (Sigma Chemical Corp., St Louis, Missouri). Clones secreting MAb to D dimer or Fibrinogen degradation products were then detected by adding 50 ~1 of tissue culture supernatant to each well and incubating for one hour at room temperature.
Unbound MAB was removed by inversion and tapping and the plate was washed three times with PBS/Tween. One hundred ~1 of a /,000 dilution of peroxidase conjugated rabbit anti-mouse immunoglobulin (Dakopatts, Copenhagen, Denmark) in PBS/Tween was added and allowed to incubate a further one hour at room temperature. The plate was again inverted and washed three times with PBS/Tween and 100 ~l of activated substrate (immediately before use 10 ~1 of a 3% solution of hydrogen peroxide was added to 10 ml of a substrate solution contain-ing 50 mM citrate, 2.5 mM of 0-tolidine dihydrochloride (0-tolidine, Sigma Chemical Co. recrystallized from dilute HCl) 0.025 mM EDTA pH 4.5)was added to each well. The colour * TRA~E MARK

18.

~2~ 2 reaction was stopped after 10 minutes by the addition of 50 ~1 of 3M HCl which caused a colour change from blue to yellow and the absorbance was recorded at 450 nm on a Titertek multiskan.
Peroxidase conjugation Conjugation of the D dimer monoclonal antibodies was carried out by a modiEication of the method of Nakane and Kaiwoi, J. of Histochem and Cytochem 22 1084-91, (1974) with periodate oxidized peroxidase. 5 mg/ml peroxidase in distilled water was mixed with a 1/5 volume of O.lM sodium periodate for 20 minutes at room temperature and unreacted periodate was removed by gel filtration on a column of Sephadex*G25 equilibrated with O.OOlM citrat~ p~ 4.5.
Monoclonal antibody (in PBS) was added in a ratio of 2 mg antibody per mg peroxidase and the pH was immediately adjusted to pH 9.0 - 9.5 by the addition of lM sodium carbon-ate, pH 9.5. The reaction was allowed to proceed for 2-3 hours at room temperature with occasional mixing and stopped by the addition of l/lOth volume 2.OM ethanolamine pH 9.5 Barbour, H.M. J of Immunol Meth. 11, 15-23, (1976). After sitting overnight at 4C, ethanolamine was removed by gel filtration on a Sephadex G25 column equilibrated with PBS
and the enzyme conjugate was storèd at 4C in the presence of 0.01~ methiolate.
Protein Detennination Protein determination was carried out by the method of Rylatt and Parish Analytical Biochem, 121 213-214 (1982).
Capture/Tag Experiments and D dimer assa~
Antigen capture/tag experiments were perEormed by incubating each well of a 96 well microtitre plate with 50 ~1 (10 ~g/ml) of each of the relevant MAb in PBS for 1 hour at room temperature. Unbound MAb was removed by inversion and tapping the plate followed by washing with PBS/Tween as described for the screening assay. Antigen capture was then achieved by adding 50 ~1 of each antigen (0-1 mg/ml) in PBS/Tween to the MAb coated wells for 1 hour ;~ -* TRADE: MARK
19 .

z~

at room temperature. The wells were washed as previously described. Captured antigen was then ta~ged with peroxidase conjugated MAb by adding 50 pl (1 ~g/ml~ of the various peroxidase conjugated MAb in PsS/Tween to each well for one hour at room temperature. After washing, the presence of bound conjugate was determined by the addition of 100 yl substrate as described in the screening assay. For the determination of the presence of crosslinked derivatives in plasma or serum, 50 ~1 of a l/5 dilution of plasma or serum in PBS/Tween was incubated instead of antigen at the second step.

RESULTS
Specificity Several hundred hybridoma clones secreting MAb against human D dimer were initially identified by enzyme immunoassay and ~wo different classes of MAb were obtained (TABLE 1).
The first group which contained the vast majority of positive clones (examples of which were B44.7.4D2/182 tDD-4D2/182), ~0 B44.7.2Cl/13 (DD-2Cl/l9), B41.7.2D5/38 (DD-2D5/38)), produced MAb that bound to epitopes present on intact fibrinogen, an extract containing fibrinogen degradation products, fragment - D and D dimer. However, the abovementioned first group did not bind to fragment E. The second and much smaller group 25 (examples of which were B42.7.3B6/22 (DD-3B6/22) and B41.7. lC3/108 (DD lC3/108)), react~d with determinants present on D dLmer but not on fragmen~ D.
No cross reaction was found with purified intact fibrinogen or fibrinogen degredation products.
D dimer monoclon~l~ as capture antibodies In order to establish whether the Various MAb were reacting with the same or distinct sites on D dimer r capture/tag experiments were carried out. The wells of ~ 96 ~ell micro plate were coated with e~ch ~Ab and incubated ~ith 20.

either fibrinogen bxeakdown pxoducts or D dimer. After washing away unbound pxotein,peroxidase conjug~ted MAb were added and after washing the presence of bound conjugate was determined by the addition of activated substrate (TAsLE
2).
DD-2Cl~l9 This MAb was able to combine with the monospecific MAb DD-IC3/108 or DD-3B6/22 only when D dimer was ~tilized as antigen and the panspecific ~Ab DD-2D5/38 when either fibrinogen as degradation products of D dimer antigens were used. It was unable to combine with the other panspecific MAb DD-4D2/182 with either antigen. These results suggest that DD-2Cl/l9 binds close to the site recognized by DD-4D2/
182 but to epitopes quite distinct to those recognized by DD-2D5/38, DD-IC3/108 or DD-3B6/22.

The panspecific MAb DD-4D2/182 had a specificity patt-ern analo~ous to DD-2Cl/l9. The results suggest that DD-4D2/
182 and DD-2Cl/l9 may have very close or overlapping binding sites.

The panspecific MAb DD-2D5~38 was also able to combine with DD-lC3/108 and DD-3B6~22 only when D dimer was used but was capable of combining with both of the other panspecific 25 MAb DD-4D2/182 and DD-2Cl/l9 with either D dimer or Fibrinogen degradation products as antigens. This monoclonal was the only one of this series which was capable of combination with itself, suggesting the presence of at least two binding sites per D dimer molecule. However, it is clear that these binding sites must be distinct from the sites recognized by the other four monoclonals.
DD-3B6/22 and DD~lC3/108 The D d~mer specific MAb DD-3B6/22 was capable of combining with any of the panspecific monoclonals DD-4D2/182, 35 DD-2Cl/l9 or DD-2D5/38 when D dimer was the captured antigen.

~2~ 2~

MAb DD-lC3/108 had a similar speciEicity pattern, however it performed relatively poorly as the capture MAb. Ovarall the resul-ts suggest that this set of monoclonals binds to three distinct areas on -tha D dimer molecule, a uni~ue site recognized by DD-2D5/38, another sharad by DD-4D2/182 and DD-2Cl/19 and a D
dimer specific site shared by DD-lC3/108 and DD-3B6/22.
A specific assay for D dimer The results above suggested that several combinations of these MAb might prove useful in developing a specific assay for D dimer and perhaps lead to a general assay for fibrinolysis.
In the first type of combination, the monospscific MAb DD-3B6/22 was used as a capture ~Ab and antigen was tagged with either of the panspecific MAb DD-4D2/182 or DD-2D5/38. An assay using peroxidase conjuga-ted DD-4D2/182 as a tag MAb had a sensitivity of 10-20 ng/ml of D dimer (Fig. 1). There was a strong reaction with fibrin degradation products but no reaction was seen with fibrinoyen degradation products or fragment D. Essentially identical results were obtained using peroxidase conjugated DD-2D5/38 as the tag MAb (not shown). In another type of combination the panspecific monoclonal DD-4D2/182 was used as the capture MAb and the antigen was tagged with peroxidase conjugated DD-lC3/108 (Fig. 2). In this case 10-20 ng/ml concentrations of both D dimer and fibrin d~gradation products produced clear signals but there was no detectable cross-reaction wi-th either intact fibrinogen, fibrinogen degradation produc-ts or fragment D.
Qualitatively similar resul-ts were obtainad capturing with either DD-2C1/19 or DD-2D5/38 (not shown).
Assays based on both these monoclonal combinations were investigated for their ability to detect D dimer and other crosslinked derivatives in blood. Serum or plasma diluted 1/5 in PBS/Tween from either normal healthy volunteers (control was 19, 20, 23) or from patients with clinically diagnosed DIC were incubated with micro plates coated with 7~

either DD-3B6/22 or DD-4D2/82 and after incubation for one hour at room temperature the presence of bound D dimer or crosslinked derivative was established by addition of the relevant conjugated MAb (Table 3). Assays based on DD-3B6/22 gave positive results with both serum and plasma whereas those based on DD-lC3/108 gave positive results with serum only.
IATEX BEAD TEST
Latex particles are polystyrene beads approximately 1 micron in diameter to which has been covalently attached the mono-clonal antibody DD-3B6/22.
TESTIMG ~PROC~DURE
1. 0.02 ml beads were mixed on a slide (shaken before use) with 0.01 ml of serum or diluted ~ample under test.
The slide was rocked gently for 2 minutes and the presence or absence of agglutination was noted.
ESTIMATION OF LEVEL OF CROSSLINKED FIBRIN DERI~ATIVES
Positive ayglutination was obt~ined with samples containing >200 ~g/ml crosslinked deriyati~ More accurate estLmates of higher levels of crosslinked derivatives 2~ in a particular sample were obtained by serial dilutions of the sample in PBS bu~fer as illustrated in Fig 4.
FURTHE~ ~INICAL TRIALS
Sub~ects The groups studies were (~) 45 he~lthy laboratory volunteers as controls; (b) 10 patients with venographically proven deep venous thrombosis and/or arterial thrombosis;
(c) 6 patients with pulmonary embolism and (d~ 30 patients with laboratory evidence of consumption coagulopathy and diagnose~ characteristic~lly associated-with disseminated intravascular coagulation, The patients in group (d) all ulfilled the criteri~ for disseminated i-~trav~scular coagul~tion ~s described in Whaun and Oski Can. Med. Assc.J.
`107 963-66 (1972). Two ml o~ blood was clotted with thrombin (20 iu) in the presence of soy bean trypsin inhibitor (Becton and Dickinson 3.67 n,~. units~ and the serum was used 23.

~7~Z~

to assay soluble crosslinked fibrin deriyatives using the capture/tag method pre~iously described, ResultS in terms of D2 ratio (~atio of samplP compared to blank) are given in Table 4 for qroup (a), Table S for group (d), Table 6 for group (b) and Table 7 for group ~c). The results are also plotted graphically in Fig 3.
Previous attempts to obtain specific antibody probes to discriminate crosslinked fibrin derivatives have been hampered by the nature of the polyclonal antibody response iO to the antigens used for immunization. Several antibody preparations have already been described with a marked preference for the crosslinked derivative - 50 fold greater reactivity for D di~er compared to D (see the aforementioned Budzynski re~erence) or 100 ~old for c~osslinked r-Y chains com~ared to non~crosslinked and peptide (Purves et al Biochem-istry 19 4051~5B 1980) or 8 ~old for D di~er compared to fibrinog~n or fibrinogen degradation products (Lahiri et al - Thromb Res 23 103-112 1981). The degree of cross-reaction with non~crosslinked fragments has been Still enough to 2~ preclude their value as diagnostic reag~nts~
The problem of producing diagnostic reagents could not ~e overcome until monospecific MAb were produc~d (DD-3B6/22 and DD~lC3!108) that reacted only with crosslinked fibrin derivatives. These MAb have been employed to produce diagnostic assays of which capture/tag t~pe systems are preferred, However conventional binding inhibition assays employing labelled monospeci~ic DD~3B6j22, DD~lC3/108 or labelled crosslinked fibrin derivative could also be used.
In a capture/tag assay, the antigen in question is reacted with two antibodies With specificity for different regions of the ~ame molecule. Usually a capture antibody is attached onto a solid phase and after addition of antigen to allow binding to occur, the presence of bound antigen can be detected after washing by the addition of the second labelled antibody.

2~.

;L,~ æ

MAb DD~lC3/108 although ~uite specific for D dimer, performed quite poorly ~s a capture ~Ab, yet the peroxidase conjugated MAb was a good tag. On the other hand, the other specific monoclonal DD-3B6/22 w~s a good capture MAb but a relatively poor tag.
Assays based on DD-lC3/108 as a tag bound D dimer and fibrin breakdown products equally, whereas assays using DD-3B6/22 as a capture MAb bound D dimer approximately 100 fold better. Similarly a signal can be generated with high concentratIons of fibrinogen with the DD-3B6/22 assay ~ut not DD-lC3/108. Each of these monoclonals shows a similar degree of cross-reaction With both these antigens in the standard enzyme immunoassay tTable 1)~
Both o~ these as~ays can detect low levels of D dimer or other crosslinked derivatives present in serum from patients with DIC but is is not surprising that the high levels of fibrinogen present in plas~a (approx, 3000 ug/ml) would prevent assays based on MAb DD~lC3/108 as a tagging antibody from giving positive results with plasma~ The capture antibody in this case DD-4D2~182 has a stro~g reaction with both fibrinogen and D dimer and the relatively high concentration of fibrinogen in plasma might be expected to swamp the capture monoclonal on the solid phase~ On the other hand the plasma assa~ based on the monospecific DD-3B6/
22 will selectively capture crosslinked derivatives even in the presence of several orders of magnitude higher concentrat-ions of fibrinogen (Fig 2~ and is therefore more efficient in relation to assay of crosslinked fibrin derivatives.
The latex bead assay results as shown in Fig 4 correl~ted with the other experimental results obtained by EIA referred to previously. The latex bead ~ssay therefore o~fers rapid diagnostic test potential.
The abovementioned ass~y ~rocedures may be carried out between 4-40C but more suitably at room te~perature.
The contact between the test sample and the relevant MAb may be carried out at a pH of 5-9 with a suitable upper limit of ionic strength being IM.
~5, ~L~47C~22 T~LE 1 Specificity of D dimer monoclonal antibodies : cross reaction with fibrinogen and fibrinogen deg,adation products I Antigen I Fibrinogen Fibrin Monoclonal Fibrinogen Degradation Degradation D E D Dimer Titre Products Products _ _ (1) (2) DD-4~2/t82 255 71 35 23 0 100 2,5v~

DD-2Cl/l9 156 68 35 28 0 100 ~Sxl~6 DD-2DS/38 423 111 ~0 37 0 100 2.5x106 DD-3B6/22 ¦ 4 3 45 0 0 12.5x106 DD-lC3/108 1 2 0 31 0 0 100 (1) Optical density for the reaction asainst D dimer for each monoclo~al W2S taken to be 100'~. The v31ues then represent cross-reactions as determined by the relative optical density obtained with the other antigens.
(2) The titre is the lowest dilution which gives a.reading using D dimer as the antigen of A450.,~ 0.1-f~

~l ~ coo coi' ~c ~ o ~ o o o r >
~ E
a~ r~J ~ . . Q) C~
' C~ o O o o C- ~ o ~ t ~> ~
C~ C~ ~ o ~ ~
O O O O O C

O G O O C:> r~ O U

r-- r~~ S_ u o O o o O O o E c ~ g C o~ 4~-~
J E ~) O O O O O O ~ V C
r V C r S t~l~ . I ~Oc~l E e ~ O O o o O

~5 ~o o o O O O -u~

Uo~ ~ - ~ C
~- o o o o o o ~ C

~ O O O O O ~ ~

r~ O
N ~t N rl _ _C O

~:`,...

~4'~

The determination of the presence of cro5slinked fibrin derivatives in blood by enzym)e im~unoassay ( 1 ) . . .
Sample DIC Score DD-3B6/22 DD-lC3110S D dimerCapture Tag Gel Serum Plasma Serum Plasma _ __ _ (2) (3) MS 8 8 0.223 0.627 0.452 0.007 MS 9 7 0.704 1.153 0.584 0.013 +
MS11 gJ, 0.323 0.393 0.S87 0.004 +
MS14 9 0.272 0.296 0.227 0.000 ~FC 19 ~ !~. 0 047 0.051 0.013 _ O.C07 0.037 1 0.081 0.013 23 0.014 0.0~0 1 O.Og2 0.013 ~ _ (1) DiC Score The patients were diagnosed as having Disseminated Intravascular Coagulation according to the scoring system of Whaun and Oski, ~an. ~led. Assoc. J. 1077 963-66 (1972) (2) The values are expressed as change in A relative to a control - experiment in ~hich PBS/Tween was incub~8am with capturing MAb instead of th relevar.t antigen.
(3) The presence of D dimer by polyacrylamide gel electrophoresis was established according to the method of Lane D.A. et al, Throm~. Res., 9, 191-~00 (1976).

`` ~ z~ .

'rA81.E q l~rmal healthy Volunteers _ j D2 Ratlo I t~o. D2 Ratio 1 - 0.7 24 ~ 1 .2 2 0.7 2~ 1 .5 3 0.7 26 1.2

4 1.2 27 0.8 1 .1 28 1.1 6 0.9 29 0.3 7 0.7 30 0.7 8 0.6 31 0.7 9 0.5 32 1.0 0.5 33 1 ,0 11 1 .2 3~ 1.0 12 1 .2 35 1 .0 13 0.8 35 . 1.0 4 0.6 37 1 .0 0.9 3~ 1.0 1~ 0.5 39 1.0 17 0.6 40 1.0 18 0.7 41 1.5 19 0.8 ~2 0.5 0,6 43 0.5 21 0.7 44 1.0 22 0.4 45 1.. 0 23 1.0 _ _ _ .

tl3 Dz Ratio derived from A4so test sample 450 contro n = 45 x = O.g SD = 0.3 r7 ~ ~ 2 .
.
TA~LE S
Disseminated Intravascular Coagulation Patients ~1) ~ -- j~Page ~ 3) No. Clinical 6or)dition DIC Score D, Ra~io Analysis 1 Disseminated Breast Carcinoma, Pulmonary Embolus and Bleeding 8 13.3 +
2 Lacerated Placenta and Post Partum Haemorrhage 7 14.8 3 ni SS eminated Carcinoma, Venous Thrombosis and post Surgery Bleeding 7~ 15.7 4 L~mphoma, Ascites, Le Veen Shunt 7 18.7 +

5 hlcoholic Liver Disease, Septicemia and Bleeding 8 12 2 .

6 Haemorrhagic Pancreatitis and Septicemia 7 14.7 +

7 Acute Renal Failure, Septicemia, and Bleeding 9~29 . 2 +
~ Bleedir.g Oesophdgeal Varices 9 3.7 9 Analgesic Nephropathy and Septicemi d 7~ 20.3 +
10 Severe Pre-eclamptic Toxemia and Hae;nophtysis 7~i 13.8 +
11 Mult;ple Injuries and Oozing Blood~2 14-3 +
12 Alcoholic Liver DiseasP, Cirrhosis, ~leeding, and Thrombosis 9 ¦ 4.8 +
13 Chronic Ren~l Failure and Septicemia 7~i 12.7 +
14 Hcdg~ins Disease and Bleeding 10 11.2 +
lS Meningoc.occol Septicemia and Petechiae 9 16 0 +
16 l~elaena, Haematuria and Purpura 7 11.6 17 Carcinoma o~ the Prostate 8 9.5 18 Disseminated Carcir.oma and Microangiopathic H~emolytic Anemia 8 11,3 +
19 Acute Pancreatitis and Disseminated Thrombosis 10 4.4 20 Acute Pancreatitis, Acidosis and Bleeding 10 2.9 21 Alcoholic Liver Disease 7 7.2 +
~2 Septicemia, Acute Respiratory Disease Syndrome and Bleeding7~ 7.5 +
23 Subacute Bacterial Endocarditis 8~ 6.0 24 Severe Pre-eclamptic Toxemia 7 6.~ +
25 Chronic Renal Failure and Liver Disease 8 2.9 26 Septicemia 8 2.0 +
27 Severe Pre-eclamptic Toxemia 8 4.5 +
28 Promyelocytic Leukemia (M3) 7 7.0 29 Acute Pancreatitis and 31eeding 7~ 10.0 30 Perin3tal HVDOXj a 9 8.4 +
r (1) DIC diagnosed according to hhaun and Oski, Can.~ed Assc.J. 107, (2) D2 Rat~o derived from ~
(3~ The presence of D dimer or high molecular weight Fibrin degradation products established accordin3 to Lane et al, Thrombosis Res., 9, 191-200 (lg76) 3p ~2~7~2~

Deep vellnus throlnbGsis or arterial thrombosis patients . . _ ............... . . ._ ( 1 ) No. Clinical Condition D2 Ratio .. _ . . ___ . .......... .. _.. _ 1 Deep Ver,ous Thrombosis and Pulmonary Embolus 24.9 2 Cerebral Artery Thrombos;s (Mitral Stenosis) 9.6 3 Left Femoral Artery Thrombosis 2.6 4 Recurrent Deep Venous Thrombosis 5.0 Right Deep Venous Thrombosis 9.2 6 Left Deep Venous Thrombosis 3.6 7 Le~t Deep Venous Thrombosis 2.0 Right neep Yenous Thrombo-sis 6.6 9 Left Deep Venous Thrombosis 3.7 Severe Chronic Liver Disease, Probable Thrombosis in the Inferior Vena cava11.2 _ . _ ........... .. __. ~
(1) D2 Ratio derived from A450 test sample A450 Cor~tr Pulmonary Embolus Patients ... _ .
No. Clinical Condition D~ Ratio(l) - . _ _ . .
1 Deep Venous Thrombosis and Pulmonary Embolus 24.9 2 Pulmonary Embolus (Post operative) ~ 5.4 3 High Probability of Pulmonary Embolus by Lung Scan 1.0 4 Low Probability of Pulmonary E~bolus by Lung Scan 7.4 Possible Pulmonary Embolus 1.2 6 Pulmonary Embolus and Le Veen Shunt 9.9 . . ,.. ___ , ~. .. ... ..
~1) D Ratio deri~ed ~rom A4so test Sample ~450 control

Claims

1. A method of preparation of monoclonal antibody derived from a crosslinked fibrin derivative including the steps of:
(i) obtaining a crosslinked fibrin derivative or extract containing same;
(ii) isolating antibody producing cells from an animal immunized with said derivative or extract to form antibodies to said derivative or extract;
(iii) hybridizing said antibody producing cells with myeloma cells; and (iv) cloning said antibody producing cells.

2. A method as claimed in Claim 1 wherein said extract is obtained from plasmic degradation of fibrin clots or by simultaneous action of thrombin, Factor XIII and plasmin or fibrinogen with transient clot formation and subsequent clot lysis.

3. A method as claimed in Claim 1 wherein said extract is prepared by subjecting animal body fluid containing said crosslinked fibrin derivative to an electrophoresis, chromatographic or other fractionation technique.

4. A method claim in any one of Claims 1, 2 or 3 wherein in step (ii) antigenic extract containing said crosslinked fibrin derivative is administered to a mouse which is subsequently killed and the spleen subsequently removed for further purification to isolate lymphocytic or spleen white blood cells which are subsequently fused to mouse myeloma cells to form a hybridoma cell which may be cloned or recloned as desired.

5. A method claim in any one of Claims 1, 2 or 3 wherein in step (ii) antigenic extract containing said crosslinked fibrin derivative is administered to a mouse which is subsequently killed and the spleen subsequently removed for further purification to isolate lymphocytic or spleen white blood cells which are subsequently fused to mouse myeloma cells to form a hybridoma cell which may be cloned or recloned as desired and wherein said hybridoma cells are cultivated in well plates whereby hybridoma cell suspensions are placed in each well with cell cultivation media.

6. A screening assay for determining whether cells are producing antibody specific to a crosslinked fibrin derivative including the steps of:
(A) coating a surface with antigen selected from (i) crosslinked fibrin derivative;
(ii) extract containing said derivative; or (iii) fibrinogen degradation product;
(B) contacting said antigen with monoclonal antibody reactive only with crosslinked fibrin derivative to form a complex;
and (C) detecting said complex formed in step (B).

7. An assay as claimed in Claim 6 wherein in step (A) a well plate is utilized in which antigen is added to each well prior to said monoalonal antibody.

8. An assay procedure for detection of the presence of crosslinked fibrin derivative in a fluid including the steps of:
(i) contacting monoclonal antibody reactive only with crosslinked fibrin derivative, with a fluid sample suspected of containing an antigen derived from a crosslinked fibrin derivative or a crosslinked fibrin derivative per se, to form a complex; and (ii) detecting said complex formed in step (i).

9. An assay procedure as claimed in Claim 8 wherein the crosslinked fibrin derivative is D-dimer.

10. An assay procedure as claimed in Claim 8 wherein a first monoclonal antibody in step (i) is bound or captured to an antigen in said body fluid which is subsequently tagged in step (ii) by a labelled second monoclonal antibody.

11. An assay procedure as claimed in Claim 10 wherein said first monoclonal antibody is monospecific and said second monoclonal antibody is pan specific.

12. An assay procedure as claimed in Claim 10 wherein said first monoclonal antibody is monospecific and said second monoclonal antibody is monospecific.

13. An assay procedure as claimed in Claim 10 wherein said first monoclonal antibody is pan specific and said second monoclonal antibody is monospecific.

14. An assay procedure as claimed in any one of Claims 10 to 12 wherein the second monoclonal antibody is labelled with an enzyme which is subsequently reacted with a substrate for said enzyme.

15. An assay procedure as claimed in Claim 13 wherein the second monoclonal antibody is labelled with an enzyme which is subsequently reacted with a substrate for said enzyme.

16. An assay procedure as claimed in any of Claims 10 to 12 wherein the second monoclonal antibody is labelled with a radioactive species.

17. An assay procedure as claimed in Claim 13 wherein the second monoclonal antibody is labelled with a radioactive species.

18. An assay procedure as claimed in Claim 8 or 9 wherein the monoclonal antibody in step (i) is initially coupled to latex beads before being contacted with said fluid sample and subsequently being checked for agglutination in step (ii).

19. A method of detection of crosslinked fibrin derivative in a body fluid including the steps of:
(i) immunizing an animal with a crosslinked fibrin derivative or extract containing same;
(ii) removing a spleen from the animal;
(iii) treating the spleen to form a call suspension;
(iv) purifying the cell suspension to isolate spleen white blood cells or lymphocytes;
(v) forming hybridoma cells containing as one component said spleen white blood cells or lymphocytes;
(vi) cloning or re-cloning said hybridoma cells using appropriate cell feeder layers;
(vii) carrying out screening assays with antigen selected from crosslinked fibrin derivative or extract containing same or fibrinogen degradation product so as to isolate hybridoma cells which produce monoclonal antibody reactive only with crosslinked fibrin derivative, (viii) contacting a fluid sample suspected of containing crosslinked fibrin derivative or antigen derived therefrom with monoclonal antibody prepared from hybridoma cells isolated after step (vii) to form a complex, and detecting said complex formed in step (viii).

20. A method of diagnosis of disseminated intravascular coagulation (DIC) and other thrombotic states including the steps of:
(i) isolating a monoelonal antibody reacting with crosslinked fibrin derivative but not fragment D, fragment E, fibrinogen or fibrinogen degradation products;
(ii) contacting said monoelonal antibody with a fluid sample from a patient suspected of suffering from DIC or other thrombotic state; and (iii) analyzing for presence of crosslinked fibrin derivative in said sample to determine if the crosslinked fibrin derivative reacted with said monoelonal antibody.

21. A method of diagnosis of disseminatad intravascular coagulation (DIC) and other thrombotic states including the steps of:
(i) isolating a monoclonal antibody reacting only with crosslinked fibrin derivative;
(ii) contacting said monoclonal antibody with a fluid sample from a patient suspected of suffering from DIC or other thrombotic state; and (iii) analyzing for presence of crosslinked fibrin derivative in said sample to determine if the crosslinked fibrin derivative reacted with said monoclonal antibody.

22. The method as in claim 20 wherein said crosslinked fibrin derivative is a D-dimer.