FR2561661A1 - MOLECULAR FILM SUPPORTED WITH A FIBRIN NETWORK FOR SELECTIVE SELECTIVE RETENTION OF THE TISSUE PLASMINOGEN ACTIVATOR AND APPLICATIONS THEREOF - Google Patents

Abstract

Molecular film supported from a fibrin network in solid phase formed at the surface of a support, more particularly at the surface of cups of a microplate. Said film is intended to all applications which implement the capacity of selective fixing of tissular activators of the plasminogene, optionally of the plasminogene itself, particularly to their respective quantitative dosings and to the detection of monoclonal antibodies specific of said tissular activators and of the plasminogene.

Description

Molecular film supported by a fibrin network allowing the selective retention, on its surface, of the tissue-associated activator and applications thereof
The invention relates to a molecular film supported on a solid phase fibrin network, fixed on the surface of a support, preferably covalently, to its implementation for the selective retention of tissue plasminogen activator and to applications resulting from this selective retention. The invention further relates to means for obtaining such supported molecular films.

 It is first recalled that plasminogen activators (PA) are enzymes whose function is the conversion of plasminogen, a proenzyme of ubiquitous distribution, into plasmin, the active proteolytic enzyme. Plasmin is a serine protease that can lyse fibrin, its main substrate, and other proteins.

Plasmin is therefore the effector enzyme of the fibrinolytic system which allows the dissolution of the clot. The main reactions involved in the fibrinolytic system can be schematically summarized as follows

<tb><SEP> fibrinogere <SEP> (soluble)
<tb><SEP> plamioge <SEP> --thllombine
<tb> activators <SEP>: <SEP> fibrin <SEP> clot <SEP>)
<tb><SEP> librine <SEP> (clot)
<tb><SEP> plasmin <SEP> 1.1 <SEP> lysis
<Tb>
PAs are secreted by a variety of cell types under the control of specific stimuli (Cash, JD, Progress in Fibrinolysis and Thrombolysis, JF Davidson, MM Samama and PC)

Desnoyers (Raven Press, N.Y.), p. 97, 1975); they are, on the other hand, freely secreted by neoplastic cells and by transformed cells (Christman J.K.

et al., Proteinase in mammalian cells and tissues, ed.

 Barrent (Elsevier / North Holland, Amsterdam), p. 91, 1977).

 PAs can be classified into two different types according to their molecular and functional characteristics and their reactivity with conventional polyclonal antibodies.

 Activators of urokinase type (u-PA) of molecular weight 54,000 are produced by the epithelial cells and are capable of activating plasminogen both in the liquid phase (plasma) and in the solid phase (clot). These enzymes have almost no affinity for fibrin (Thorsen S. et al., Differences in the binding to fibrin of urokinase and tissue plasminogen activator, Thromb, Diathes, Haemorrh, 28: 65-73, 1972) and their properties. With respect to plasminogen activation, the production of simultaneous fibrinolysis and fibrinogenolysis can be achieved.

The other type of AP is AP of endothelial origin or tissue activator (t-PA); it is a protein of molecular weight 70,000 which is considered the physiological PA of plasma fibrinolysis and thrombolysis. The absence of its activity in plasma has been linked to the development of thromboses (Johansson et al., Acta Meds Scand., 203: 477, 1978, and
Angles-Cano E. et al., J. Lab. Clin. Med. 94: 312, 1979).

 Detection and plasma determination of the endothelial activator are therefore of paramount importance in the study of normal and pathological fibrinolysis.

 The endothelial activator has a very high affinity for fibrin allowing, on the one hand, its specific absorption on a clot and, on the other hand, to develop its enzymatic activity. Indeed, this activator is capable of activating plasminogen only if it is absorbed on a fibrin clot. T-PA therefore regulates true fibrinolysis without fibrinogenolysis. Under these conditions, methods currently in common use, allowing the detection of this activator, are methods using the lysis of a clot (Kluft C. et al, Screening of fibrinolytic activity in euglobulin plasma fractions in the fibrin plate , Progress in chemical fibrinolysis and thrombolysis, JF Davidson, Samama MH, PCDesnoyers, Raven Press, New York, 1976, vol 2:57) or for the purpose of measuring lysis times of plasma euglobulins or lysis fibrin tray. These tests are qualitative, non-specific (due to the diffusion of t-PA and other proteins in the thickness of the fibrin trays used to date). They also require a very long production time (from 8 to 24 hours). A test allowing the specific and quantitative determination of the enzymatic activity of endothelial plasminogen activator is not yet available.

 During the last four years, the study of coagulation and fibrinolysis has been considerably modified by the development of synthetic substrates (Hemker H.C. et al, Handbook of Synthetic Substrates, Tartinas Nijhoff Publishers, The Hague, 1983).

These substrates allow, using a spectrophotometer, the quantitative determination of components of these systems.

 Assays using a synthetic substrate for liquid phase u-PA assay have been described (Drapier IC et al., Regulation of plasminogen activator secretion in mouse peritoneal macrophages, Biochemistry 61: 463-471, 1979, and Searls DB, An improved colorimetric assay for plasminogen activator, Analytical Biochem 107: 64-70, 1980); the principle used is not, however, applicable to the detection of t-PAs.

Significant efforts have been made by several researchers around the world to develop a test for the enzymatic detection of t-PA using a plasmin-sensitive chromogenic substrate (Ranby M. and Wallen P., A sensitive parabolic rate assay for the tissue plasminogen activator, Progress in Fibrinolysis, Davidson Jr. Nilson, IM, Astedt B. (eds),
Churchill Livingstone, Edinburgh, 5: 233-235, 1981, and
Wiman B. et al, Plasminogen activator release during venous statis and exercise as determined by a new spe- cific assay. Clin. Chim. Acta. 127: 279-288, 1983,
Eriksson B. et al, Tissue plasminogen activator activity after total hip replacement and its correlation to postoperative deep vein thrombosis. Thromb. Haemostas.

50:57, 1983, and Verheijen J.H. et al, A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to plasma measurements. Thromb. Haemostas. 48: 266-269, 1983). These tests use as stimulator t-PA compounds close to fibrin mimetizing its stimulatory action on t-PA: fibrin monomers in solution (articles already cited Ranby and Wallen, 1981, and Wiman et al, 1983) fragments of fibrinogen in solution (article already cited by Verheijen et al, 1983). In other cases (previously cited article by Eriksson et al., 1983), the stimulator selected (poly-L-lysine, Allen R. A., An enhancing effect of polylysine on the activation of plasminogen.

Thromb. Haemostas. 47: 41-45, 1982) has no structural link with fibrin. The detection of t-PA in the absence of a stimulator (Campbell E. E. et al., A colorimetric assay for releasable plasminogen activator Clin Chem.

28: 1125-1128, 1982) has also been reported. But these methods are not always simple to implement and do not always have the strict specificity required.

 In the absence of precise techniques for the quantitative determination of t-PA, there are difficulties in isolating it in sufficient quantity to study it. The acquisition of knowledge on its molecular structure, its tissue localization and on the structurefunctions ratio would therefore be of primary importance to understand its physiological role and its role in the development of thromboses; this type of information can be obtained using immunochemical methods. However, the polyclonal nature of conventional antibodies does not allow the realization of types of studies.

Over the past eight years, the development of the cell hybridization technique (Kohler
G. and Milstein, C., Nature (London), 256: 495, 1975) has opened up new possibilities for carrying out this type of fine immunopathy work: the production and use of monospecific monoclonal antibodies. In this type of technique, the limiting factor seems to be the screening test for the hybrids producing the antibodies of the desired specificity. Indeed, the immunization of the mice can be carried out with very small amounts of semi-purified antigen. particularly involving radioimmunological assays often referred to as expression
and the abbreviation "rsdioimmunoassay" (RIA),
calls for the use of much larger quantities of the purified antigen; the use of semi-purified products that can introduce "parasite" proteins, it can lead to the selection of desired nor antibodies. This is particularly problematic when the same preparation is used both for immunization and for the detection test.

Due to the difficulties encountered (technical difficulties and colts) to obtain purified t-PA in sufficient quantities for use in an RIA, the use of such a test for the detection of anti-t-PA monoclonal antibodies proved almost impossible.

This is how most authors (Kalil et al,
Prot. Biol. Fluids 28: 587, 1980, Kaltorft K. et al,
Proc. native. Acad. Sci. USA 79: 3720, 1982; and NIELSEN
LS et al., Embo D., 2, 1983) wishing to manufacture anti-tP A monoclonal antibodies were forced to choose a test for inhibition of enzymatic activity. these anti-activity tests make it possible to choose only antibodies directed against epitopes located at the active site of the protein or located very close to it. But obtaining antibodies directed against other epitopes of the molecule has hardly been considered until today. There are therefore hardly any techniques that could give access to the study of the structural configuration of the plasma molecule or of endothelial origin, to the realization of cytochemical studies, to the development of a "radioimmunoassay" for 1 ' vascular activator and activator detection probes in molecular biology and genetic engineering studies.

 The aim of the invention is to remedy the difficulties which have been mentioned above and more particularly to provide means making it possible both to extract and quantitatively assay t-PAs initially contained in biological samples, in particular plasmas or enriched fractions of plasmas, such as euglobulins, and further to provide the specialists with a material for practically implement the properties, for example enzymatic or immunological t-PA, even when it does not is isolated or isolatable in minute amounts.

 The principal means according to the invention consists of a molecular film supported by a solid phase fibrin network, preferably covalently attached to the surface - or in certain regions. of the surface - of a support.

 In an advantageous form of the invention, the film of the fibrin network is attached to at least some regions of the surface of the support by means of a polyglutaraldehyde network, itself attached thereto.

 Advantageously, the support consists of a titration plate comprising one or more buckets, the inner surfaces of these buckets defining the regions on which are formed mo lecular films of the fibrin network according to the invention. The titration plate can be made of any suitable material to which the polyglutaraldehyde can be attached directly or indirectly, by covalent or non-stable bonds, however stable. Such a result can be obtained more particularly with plastic supports, preferably polyvinyl chloride or polystyrene. Indeed, it is possible to produce films which are extremely stable to polyvinyl surfaces by example by incubation of a solution of glutaraldehyde in contact with these surfaces. Fibrinogen can then be deposited on these surfaces or surface regions, in particular from a solution of fibrinogen which has been found to be able to react with the free functions of glutaraldehyde, in particular the aldehyde functions, without in any way losing its properties. In particular, the preservation of this characteristic is manifested by the ability of fibrinogen to be converted to fibrin by incubation in the presence of thrombin at the temperature and under the appropriate conditions to form a molecular film, or even monomolecular, in which are nevertheless reconstituted structural features of fibrin in the form of a network polymer (as found in clots).

 The supported molecular film of the invention makes use of the fundamental property of the endothelial plasminogen activator, to be adsorbed on a fibrin network allowing it to express its enzymatic activity. This allows, under the appropriate conditions to detect only the endothelial activator to the exclusion of other activators not having this physiological property. In addition, this molecular film can be implemented in tests carried out in phase. solid on a molecular network of fibrin and not in liquid phase, which will not detect urokinase activators. It has indeed been found that the fibrin network of the film or mloléculaire layer is in all respects comparable to the fibrin generated in the plasma by activation of coagulation. It is, moreover, the ability of this fibrin film to selectively bind the t-PA which attests to the typical fibrin network structure of said film.

 This selective binding is also quantitative in that it is detectable in its entirety by its ability to be practically entirely involved in the activation of plasminogen that can be brought into contact with it, in particular inside a microplate cup, the amount of activated plasminogen can be detected in situ, for example using any synthetic substrate convertible by plasmin.

 The invention completely overcomes the aforementioned disadvantage which resides in the in-t-PA diffusion in the thickness of the fibrin plates that have been used to date. It is furthermore the totality of the t-PA adsorbed on the inner surfaces limited by the buckets of the titration plates which is involved in the subsequent reaction with a plasminogen solution, so that the proportion of plasminogen then activated (measurable by a synthetic substrate) is directly correlable to the amount of t-PA retained in the bucket concerned.

 As already follows from the foregoing, the film according to the invention is molecular, even monomolecular. In particular, it comprises from about 50 to about 500 ng of fibrin per cm 2 of surface, preferably from 120 to 180, for example from 145 to 150 ng of fibrin per cm 2 of surface. It will be appreciated that lower fibrin ratios than the lower limits of the above-mentioned limits could result in the absence of the "lattice" format, which would be manifested by the absence of possible adsorption of the PA, under the conditions indicated above or in those which will be illustrated below as examples.

 The maximum levels of fibrin per unit area will generally be limited by the number of attachment points, especially via functional groups available on the surface of the support and which are involved in the fixing of fibrinogen molecules brought into contact with this material. surface, through complementary functional groups carried by fibrinogen.

 The film fibrin networks of the invention can be used for the assay of t-PA (even unpurified) from the most diverse sources: cultured endothelial cell supernatant, isolated glomeruli supernatant, and euglobulin suspensions . Complete human plasma can also be used, which until now was hardly possible.

 The t-PA absorbed on the solid-phase fibrin can, after washing out contaminants and inhibitors, then be detected by activation of plasminogen using a synthetic substrate sensitive to plasmin (indirect method); the use of a synthetic substrate sensitive to t-PA would allow direct dosing.

Spectrophotometric reading of the reaction (release of paranitroanilide from synthetic substrates such as HD-isoleucyl-L prolyl-L-arginine-p-nitroanilide dihydrochloride (substrate S-2288) or HD-valyl-L-leucyl dihydrochloride L-lysine nitroanilide (substrate 5-2251), both marketed by
AB KABI) is carried out at 405 nm wavelength. These readings may be converted to fibrinolytic units with reference to an international t-PA standard.

 The film fibrin networks according to the invention allow not only quantitative assays of t-PA, for example under the aforementioned conditions, but also and inversely the quantitative determination of plasminogen contained in similar biological media. It is in fact observed that the fibrin film according to the invention can also adsorb plasminogen, in particular by retention on the fibrin, at binding sites that are distinct from those of the t-PA (s). The adsorbed plasminogen assay can then be used. by activation of the latter, through one of its activators, whether it is a suitable t-PA or a non-adsorbed activator, such as urokinasa or streptokinase. the determination of the plasminogen thus activated may involve the substrates mentioned above.

 The fibrin network according to the invention not only allows the rigorously selective retention of t-PAs (or plasminogens), for the purpose of their determination, but generally renders them accessible to reagents capable of activating them or more generally to react with them. In this respect, the invention therefore also relates to the material obtained as such, namely the film which forms the fibrin network, retaining by selective adsorption a tissue plasminogen activator (or the film comprising a fibrin-t-complex). PA). The level of purity achieved is such that the entire material is for example usable as a reagent for the specific detection of anti-t-PA antibodies.

It is such a property that is at the basis of one of the preferred applications of the material according to the invention, namely the detection of monoclonal antibodies strictly specific for t-PA. The invention therefore opens a whole field of investigation, that of the detection and the study of various epitopes of t-PA, whether or not active sites of the enzyme.

 The fibrin-t-PA supported complexes of the invention can therefore be used in carrying out specific tests, very sensitive and rapid realization, for the detection of anti-t-PA monoclonal antibodies directed against the various epitopes of this molecule and not only against the site-active.

These tests can be carried out in the form of "radioimmunoassay" (RIA) using a second antibody (usually immunoglobulin of sheep anti-mouse immunoglobulin: GamIg, of the English "goat-antimouse immunoglobulin") labeled with iodine 125 , or in the form of an enzyme-linked immunosorbent assay (ELISA) using
GamIg conjugated to an enzyme, for example to peroxidase or alkaline phosphatase. It goes without saying that the invention is not limited to the nature of the marker used.

 The sensitivity of the assay is related to the selective and very high affinity uptake of t-PA on a solid phase fibrin support. This, as in the physiological interaction fibrin-t-PA, provides a molecular structure that retains its antigenic and functional characteristics. This "immunoassay" is based on the ability of the fibrin-t-PA complex to detect anti-t-PA antibodies present either in serum or in hybridoma culture supernatants.

It goes without saying that it will be ensured in all cases of the absence prior to any possible contaminant adsorbed nonspecifically, such as albumin or certain coagulation factors, they can be removed by washing the The surfaces supporting the films of the fibrin-t-PA complex with the appropriate buffers. If appropriate, the surfaces will also be treated with a lysine solution, the ability of which is known to replace any traces of plasminogen or plasmid. adsorbed via lysine groups on said surfaces
The invention also relates to the method for producing a network fibrin molecular film, which method comprises the steps of contacting a solution of fibrinogen with glutaraldehyde adsorbed or attached to the surface of a support, particularly plastic material and, preferably, vinyl chloride, and then reacting the retained fibrinogen film with thrombin or the like under conditions conducive to the in situ activation of fibrinogen to insoluble fibrin to form the aforesaid fibrin film in network.

 Advantageously, the amounts of glutaraldehyde initially fixed or adsorbed on the regions or surfaces of the support and the concentrations or amounts of fibrinogen contained in the solutions brought into contact with said regions or surfaces are adjusted so as ultimately to obtain a concentration of 120 to 180. preferably from 145 to 150 nanograms of fibrin per cm 2 of surface. In this respect, the invention also relates to the fibrinogen-supported films themselves, these films preferably comprising doses per cm.sup.2 of fibrinogen equivalent to those indicated for fibrin.

 Coupling fibrinogen to a surface carrying polyglutaraldehyde groups is preferably carried out at a temperature between OOC and room temperature, preferably at a temperature below 10 0C, for example at 4 0C, the contact being maintained for the required time for coupling. It is advantageous, in order to achieve the deposition of fibrinogen, to use solutions which will allow the "saturation" of the surface, for example solutions containing from 50 to 200 micrograms, advantageously 100 micrograms, per milliliter of fibrinogen. pH has little effect on the coupling of fibrinogen. however, it has been found that the most effective adsorptions of t-PA are obtained when the initial fixation of fibrinogen on said surfaces has been carried out with solutions having a slightly basic pH, ranging from 7 to 9.5, preferably of the order of 8.5.

The use of polyglutaraldehyde for bridging the support surface with the fibrinogen film is particularly advantageous. Any method known to produce polyglutaraldehyde deposits on the surfaces in question may be used. By way of example, mention will be made of the one described by MONZAN and
al. 1975, Biochemistry 57, 1281. Glutaraldehyde allows
the chemical fixation of fibrinogen, via
resonance-stabilized amine bonds with ethylenic bonds. The distribution of the aldehyde groups on the polymer and the flexibility of the latter favor the downstream bonds via the amine groups of fibrinogen, without substantially disturbing the molecular structure of the latter.

 It goes without saying that the formation of a network of fibrinogen first, and then of fibrin into a thin film, can be carried out using any other agent, itself fixed and retained on a support, and then allowing the coupling or bridging, direct or indirect, with fibrinogen, since this coupling involves chemical functions carried by fibrinogen that do not alter the major property that it has to be subsequently activated in insoluble fibrin, under action of thrombin or any other agent likely to lead to the same result. In general, any surface carrying sufficient free functions reactive with fibrinogen can be used to retain sufficient quantities to form a molecular film under the conditions which have been described above. For example, it is also possible to use conventional covalent binding techniques involving cyanogen bromide. It is also possible to envisage the use of surfaces which have been pre-adsorbed or fixed with proteins under conditions ensuring the presence thus formed of carboxylic groups, which can then be chemically combined with any amine group of fibrinogen via agents. coupling of the type used in protein synthesis techniques to form CO-NH bonds. It is clear from the foregoing that the invention lies essentially in the formation of the aforesaid fibrinogen and fibrin films having the characteristics which have been defined above.

 Additional features of the invention will become apparent in the light of the following examples of the manufacture of network-supported fibrin films and the conditions under which they may be misused. we will first present the materials and materials that are used. Reference will also be made hereinafter to the drawings in which - FIG. 1 is a curve representative of the absorbance variations (ordinate axis on the right) and the corresponding rates of fixed t-PA (in mU on the ordinate axis of the right) as a function of the t-PA contents of misas solutions in contact with a film according to the invention formed in the wells of a microplate, as a function of the t-PA content of the solutions used (in IU / ml on the abscissa axis), - fig. 2 schematically shows the various steps involved in binding to fibrin film according to the invention of t-PA, antibodies formed against t-PA and in the detection of these antibodies; - fig. 3 is representative under similar conditions of a test for distinguishing the nature of the monoclonal antibodies that may be retained on a film according to the invention.

EXAMPLE I: Application to the quantitative determination of t-PA

a) Material 1. Solid phase: chloride microtiter plates
of polyvinyl (CPV), 96 U-shaped buckets (DYNATECH LABS).

2. Human fibrinogen purified from fibrinogen
Human grade L (KABI LABS at FLOW LABS), per meadow
precipitation with methanol and by chromatography on
lysine-sepharose and gelatin-sepharose.

3. Bovine thrombin (HUFFMANN-LA ROCHE).

4. Glutaraldehyde (TAAB LABS.) * 5. Human Plasminogen (KABI LABS.).

6. Synthetic Substrates S-2251 (KABI LABS); CBS. 3308
(Diagnostica stago) 7. tampons
A. 0.1 M Carbonate / Bicarbonate pH 9.5
B. Carbonate / Bicarbonate 0.1 M pH 8.5
C. Phosphate 5 m 6.8 containing 0.5 M NaCl
D. Phosphate 5 mM pH 6.8
E. Phosphate 50 m pH 6.8
F. PBS-BSA (1 mg / ml) -Tween 20 (0.05%)
C. PBS-BSA-Tween 20-EDTA 1 mM.

8. Sources of t-PA
at. Supernatants of cell cultures
b. Plasma euglobulins
c. Plasma
d. Other sources of tissue activator.

b) Manufacture of the solid phase fibrin network 1. 200 microliters of buffer A are deposited in each
bucket of the CPV plate. Incubate for 30 minutes
at room temperature (220C). The plate is
emptied.

2. 100 microliters of 2.5 S glutaraldehyde in
buffer A are deposited in each well and incubated
for two hours at 22oC. The plate is emptied and
rinsed three times with distilled water.

3. 100 microliters of a solution in buffer B of
100 micrograms of fibrinogen per milliliter
holding 2 mM CaCl2 are deposited in each well.

 Incubation for 18 hours at +4 C.

4. The unbound fibrinogen is removed and the plate is
washed once with buffer F. 100 microliters
of thrombin solution NIH 10 units per
milliliter in G buffer and containing 2 mM CaCl2
are deposited in each bucket. Incubation 30 mi
at 370C.

5. Three washes are done with buffer C with one
Rinse time of 5 minutes between each wash.

6. Three washes are done with buffer D and one der
deny washing with the F buffer.

 The plate is ready and can be preserved at this stage by adding 100 microliters of buffer F containing 0.01 m of sodium azide to each well.

c) Performing the test 1. The plate is emptied and three washes are carried out with
E buffer

2. A dilution of the euglobulins or the
source of t-PA to be tested, in buffer E. The source
t-PA is, for example, constituted by a plasma
venous sample taken from a patient after stimulation
endothelial cells, especially as a result of
placement of a tourniquet (prior to completion
sampling). 100 microliters of this
solution in the cups and incubate for 1
hour at 37 C. This dilution takes into account the capaci
very likely adsorption of t-PA by the film
of fibrin implemented.

3. The plate is emptied and washed three times with tam
pon F.

4. The reactants of the reaction are added in the following order
at. 50 microliters buffer 50 mM Tris, 12 mM NaCl pH
7.4
b. 25 microliters plasminogen (60 micrograms /
milliliter) in Tris-NaCl buffer,
c. 25 microliters 2mM S-2251 or CBS-330B.

5. The plate is protected with an adhesive plastic and
it is maintained at 370 ° C. for 1 hour.

6. The reaction is then read at 405 nM using a
micro-ELISA reader.

7 The results are reported in absorbance units at
405 na and we compare them to readings previously
obtained with normal witnesses and with
solutions that contained known amounts of t-PA.

The results can also be reported in
fibrinolytic units versus a curve ef
performed with an international standard of t-PA.

 Fig. 1 is representative of the results that can be obtained with a typical fibrin film contacted with typical solutions of t-PA, under the conditions which have been indicated above. It also results from this curve that, starting from a certain dose of t-PA, a saturation of the fibrin films formed in each of the wells of the microplate can be observed, hence the possibly necessary dilution of the sample. test, as mentioned above in paragraph c) 2. For example, these dilutions are adjusted so that they can a priori be considered to be between 1 and 10 I.U./ml.

EXAMPLE II: Application to the detection of antibodies
monoclonals directed against epitopes
t-PA.

a) Equipment 1. Microtiter plates made of polyvinyl chloride
(CPV), 96 U-shaped buckets (DYNATECH LABS).

2. Purified human fibrinogen. Starting material
human fibrinogen KABI (FLOW LABS), purified by
ethanol precipitation, chromatography on ly
sine sepharose and gelatin-sepharose.

3. Bovine thrombin (HOFFMANN-LA ROCHE).

4. Glutaraldehyde (TAABS LABS).

5. buffers.

 A. 0.1M Carbonate / Bicarbonate pH 9.5.

 B. 0.1 M Carbonate / Bicarbonate pH 8.5.

 C. Phosphate 5 m pH 6.8 containing 0.5 M NaCl.

 D. 5 mM phosphate pH 6.8.

 E. Phosphate 50 ml pH 6.8.

 F. PES-BSA (1 mg / ml) -Tween 20 (0.05%) EDTA 1 m.

6. t-PA of different origins, either unpurified or
semi-purified, either in purified form.

7. Immune globulin sheep anti-immunoglobulin
mice (Camîg) labeled with iodine 125 (MERSHAM)) or
peroxidase (AMERSHAM).

b) Incorporation of the t-PA-fibrin molecular complex
on a support (solid phase).

1. 200 microliters of buffer A are deposited in each
buckets the CPV plate. After 30 minutes at 220C,
the plate is emptied.

2. 100 microliters of a solution of glutaraldehyde
2.5 S in buffer A are deposited in each scoop
and incubated at room temperature (220 C)
2 hours. The plate is emptied and rinsed three
once with distilled water.

3. 100 microliters of 100 fibrinogen solution
micrograms per milliliter (in buffer B)
2 mM CaCl 2 are deposited in each well. Incu
18 hours at + 40C.

4. The unbound fibrinogen is removed, the plate washed
once with buffer F and 50 microliters of a
thrombin solution 10 NIH units per milliliter
in buffer F containing 2 mM CaCl2 are deposited
in each bucket. Incubation 30 minutes at 37 C.

5. Three washes are done with buffer C (time of
rinse 5 minutes between each wash).

6. Wash three times with buffer D and one
deny washing with F buffer. This fibrin carrier
solid phase can be preserved at this stage in
adding 100 microliters of F buffer containing 0.0lu
of sodium azide in each of the cups. It is
recommended to form the just fibrin-t-PA complex
before use for the RIA.

7. Wash with Buffer E and add
50 microliters of a solution of t-PA in the tom
pon E, containing 10 IU per milliliter of activator.

Preferably, the solution also contains ly
sine (for example at a concentration of 20 mM) for
prevent any absorption of plasminogen. We incubate
during the night (18 hours) at + 40C.

8. Remove loose t-PA and wash three times
with the F buffer

 The plate is ready for immunoassay.

c) Immunoassay (RIA or ELISA).

It is possible to operate, in particular as follows, on a t-PA-fibrin-CPV support plate: 1. In each scoop of the plate, one adds 50 micro
liters of a selected dilution of mouse serum
immunized with t-PA or 50 microliters of one over
culture of a hybridoma obtained by fusion
of a mouse myeloma line with spline
nymphs of a mouse immunized with t-PA. On in
cube for 1 hour at 370C or 18 hours at + 40C.

2. Wash three times with buffer F.

 The detection of the monoclonal antibodies which may be fixed may be carried out according to any one of the following methods.

3. For the RIA
at. 50 microliters (50,000 cpm) of a diluent
GamIg labeled with 125 Iodine.
1 hour at 370C and remove the Gamlg no
fixed.

 b. Six washes are carried out with buffer F.

c. We empty and dry the plates and, after
cut the buckets, measure the radioactivity
linked to the solid support.

 The bound radioactivity is proportional to the titre of the snti-t-PA antibody present in the serum or in the culture supernatants of the hybridomas. A blank is performed with F buffer or cupping supernatant from the parental myeloma line.

4. For the ELISA:
at. 50 microliters of a dilution is added to the required
of a conjugate (usually 1: 800) of Gsm;
of peroxidase. Incubate 1 hour at 37 C.

b. We empty the plate and wash three times with
buffer F.

c. 200 microliters of a 1 mM ABTS solution are added
(2,2'-azino-di-3-éthylbenz-fhiazoline-6-sulphonate)
and incubated for 1 hour at 370C.

d. The reaction is read at 405 m using a reader
for ELISA reactions.

 Fig. 2 shows the main steps of the selection of monoclonal antibodies under the conditions that have been indicated above.

 There is shown in (a) the glutaraldehyde-activated surface of a microplate cup (PVC-GA) on which a film of fibrin F. has previously been formed.

 In (b) was schematized a molecule of t-PA carrying an antigenic site SA. After contacting a solution containing t-PA with the fibrin film, it can be seen that the t-PA is fixed on this film, as shown schematically in (c) of FIG. 2.

By contacting a hybridoma supernatant containing a monoclonal antibody MoAb with the t-PA fixed on the fibrin dermal network, the binding of the monoclonal antibody to the t-PA is obtained as the scheme outlined in (b). Fixation of the monoclonal antibody is finally detected by a second mAb antibody carrying a radioactive or enzymatic label. The result of the fixation of the second antibody is shown schematically in (e) of FIG. 2. After elimination of the excess of the antibody mAb, the possible monoclonal antibody MoAb is found to be fixed after the cutting of the scoop and
introduction of it into the tank of a detector of
gamma or spectrophotometer radiation, depending on the nature of the label used. The specificity of the monoclonal antibody MoAb can be subject to various verifications.

 In particular, it can be verified that it does not bind to a fibrin film formed on a PVC-GA surface free of t-PA or on a surface onto which other proteins have been absorbed (specifically or otherwise). plasminogen, plasmin, serum albumin or coagulation factors in place of t-PA.

 As indicated above, the t-PA retaining fibrin network films allow the study of distinct and specific monoclonal antibodies of distinctly separate antigenic sites carried by t-PA. The distinction of the specific monoclonal antibodies involved in the activity of t-PA and of distinct sites can naturally be carried out under the following conditions, reference being then made to the schematic diagram of FIG. 3.

 A hybridoma supernatant containing the monoclonal antibody MoAb isolated according to the above-mentioned procedure is brought into contact with a Gamin antibody, itself fixed on a support, for example on the PVC CA medium (before any fixation of fibrinogen on the -this).

This step is shown schematically in (f) in FIG. 3. After saturation of the plate with serum albumin, the plaque retaining the fixed monoclonal antibodies was incubated with a solution of t-PA (containing for example 100 units / ml of t-PA) for 1 hour at 370 ° C. The operation corresponding to this step is shown schematically in (g) in FIG. 3. The result of this operation is shown schematically in (h) of FIG. 3.

 The plaque retaining the monoclonal antibodies is then incubated with a plasminogen (Pg) solution of fibrin (Fn) and a substrate of plasmin (for example S-2251) under the conditions which have been indicated above. These monoclonal antibodies obtained, all active against t-PA, are then considered as specific or non-specific for the active site of t-PA, depending on whether there is an activation or absence of activation of the miocene plasme brought into contact with the plaque.

EXAMPLE III Application to Quantitative Dosage
plasminoqènes.

The solid phase fibrin network as obtained by the manufacturing method described in
Example I may also be used for the determination of plasminogen contained in a biological sample, in particular in a blood serum. This is particularly likely to be obtained from plasma (supernatant
after separation of all cells) and
after separation of its fibrinogen content, preferably
in the form of fibrin (eg after addition
a calcium salt or thrombin to this plasma). The se
Rum can also be obtained directly from a blood sample, after separation of the clots, once these have occurred spontaneously and possibly other cells.

 The serum sample, optionally diluted in buffer E, is incubated for 1 hour, in particular at a temperature of 370 ° C., in contact with the fibrin pellets formed in the wells of the microplate. The absorbed plasminogen is then activated by contacting with a solution of a suitable activator, such as urokinase or streptokinase. After washing the plate, the plasmin produced in situ is then assayed by introducing into the wells of the plate of a substrate specific for the enzyme, under the conditions which have already been described above. The activator can also be added. and the substrate simultaneously and then perform the reading under the conditions that have been indicated. It may be advantageous to evaluate the results of these assays with regard to the results obtained under similar conditions in the buckets of a separate plate under the same conditions as above, except that we omit the activating step by the external activator such as urokinase or streptokinase. This last measurement makes it possible to assess the possible background noise produced by the traces of t-PA which could possibly be contained in the tested serum. The results of the first assay can then be corrected in the light of those obtained from the fact that possible partial activation of the plasminogen tested by the traces of endogenous t-PA that it could initially contain.

 The fibrin network films in accordance with the invention can, as in the previous case, also be used for the selection of plasminogen-specific antibodies, more particularly of specific monoclonal antibodies.

 The specialist of these techniques is naturally able to appreciate the modifications that must be made to the techniques which have been described in Example II, in order to make them applicable to the detection and isolation of monoclonal antibodies. plasminogen specific.

 It goes without saying that the techniques which have been described apply equally well to enzymes (fibrinogen, fibrin, tissue plasminogen activator, plasminogen, plasmin, etc.) of human or animal origin.

 As it goes without saying and as it follows already from the above, the invention is in no way limited to those of its modes of application and of realization which have been more especially envisaged; on the contrary, it embraces all variants.

Claims (14)

 1 - Supported molecular film of a solid phase fibrin network formed on the surface of a support and capable of retaining a tissue plasminogen activator on its surface.  2 - supported film according to claim 1, characterized in that it is also capable of retaining on its surface plasminogen.  3. Supported film according to claim 1 or claim 2, characterized in that it comprises from about 50 to about 500 ng / cm 2, preferably from 120 to 180, and particularly advantageously from 145 to 150 ng / c 2 fibrin.  4 - supported film according to claim 3, characterized in that it is covalently attached to at least some regions of the surface of the support, in particular via a polyglutaraldehyde network, itself attached to the surface of this support.  5 - supported film according to any one of claims 1 to 4, characterized in that the support consists of a plastic titration plate, in particular vinyl chloride or polystyrene, comprising one or more buckets and in that the Molecular film of the fibrin network is formed on the inner surfaces of the buckets.  6 - supported film according to any one of claims 1 to 5, characterized by the selective attachment to its surface of the tissue plasminogen activator, especially in the form of a fibrin-tissue activator complex.  7 - supported film according to any one of claims 1 to 5, characterized by the selective attachment to its surface of plasminogen, in particular in the form of a fibrin-plasminogen complex.  8 - Application of the supported film according to any one of claims 1 to 5 to the specific and quantitative assay of the enzymatic activity of a tissue plasminogen activator contained in a biological sample, this application comprising contacting said film fibrin support with the biological sample containing the tissue activator to be assayed and, after washing the support with an appropriate buffered solution, contacting the plasminogen bound activator with the plasminogen and the plasmin assay resulting from activation of plasmin-activated plasminogen by the tissue activator then attached to the film, in particular via a natural or synthetic substrate specific for plasmin.  9 - Application of the supported film according to any one of claims 1 to 5 to the specific and quantitative assay of the plasminogen contained in a biological sample, in particular a blood serum, this application containing contacting said supported fibrin film with the biological sample containing the plasminogen to be assayed, after washing the support with an appropriate buffered solution, contacting the plasminogen fixed on the film with a plasminogen activator and the assay of the released plasmin resulting from activation of the plasminogen previously fixed on the film, in particular via a specific substrate of plasmin.  10 - Application of the supported film according to claim 6 to the detection of anti-tissue plasminogen activator antibodies comprising contacting said film with a solution containing said antibodies and detecting antibodies attached to the surface of said film.  11 - Application according to claim 10, charscterérisé in that the aforesaid film is applied to the detection of monoclonal antibodies anti-plasminogen activators, by contacting said film with the supernatant of a hybridoma culture secreting such antibody.  12 - Application of the supported film according to claim 7, for the detection of antibodies, in particular of anti-plasminogen monoclonal antibodies, bringing the said film into contact with a solution containing the anti-plasminogen antibodies, in particular of a supernatant of a hybridoma culture secreting such antibodies, and detecting antibodies attached to the surface of said film.  13. Fibrillatively supported molecular film for the manufacture of the carrier according to any one of claims 1 to 7.  14. The fibrinogen-supported molecular film according to claim 13, characterized in that it comprises from about 50 to about 500 ng / cm 2, preferably from 120 to 180, and particularly advantageously from 145 to 150 ng / cm 2. fibrinogen.