Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/745—Assays involving non-enzymic blood coagulation factors
  • G01N2333/75—Fibrin; Fibrinogen
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/972—Plasminogen activators
  • G01N2333/9726—Tissue plasminogen activator

Definitions

• the invention relates to a molecular film supported by a fibrin network in solid phase, fixed to the surface of a support, preferably covacently, to its use for the selective retention of tissue activator of plasminogen. and to the applications arising from this selective retention.
• the invention also relates to means for obtaining such supported molecular films.
• the activators of plasminogene are enzymes having for function the conversion of plasminogene, a proenzyme of ubiquitous distribution, into plasmin, the active proteolytic enzyme.
• Plasmin is a serine protease capable of lysing fibrin, its main substrate, and other proteins.
• Plasmin is therefore the effector enzyme of the fibrinolytic system which allows the dissolution of the batch.
• We can schematically summarize the main reactions involved in the fibrinolytic system as follows:
• PAs are secreted by a variety of cell types under the control of specific stimuli (Cash, 3.D., Progress in chemical fibri ⁇ olysis and thro bolysis, eds. 3.F. Davidson, MM Samama and PC Desnoyers (raven Press, NY ), p. 97, 1975); they are, on the other hand, freely secreted by neoplastic cells and by transformed cells (Christman 3.K. et al, Proteinase in mammalian cells and tissues, ed. Barrett (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 vis-à-vis conventional polyclonal antibodies.
• Activators of the uro inase (u-PA) type with a molecular weight of 54,000 are produced by epithelial cells and are capable of activating plasminog in both the liquid phase (plasma) and 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 plas inogen activator. Thromb. Diathes. Hae orrh. 28; 65-73, 1972) and their properties with regard to the activation of plasminogen can lead to the production of fibrinolysis and fibrinogenolysis simultaneously.
• R AP is the AP Original endothé- subsidiary or tissue activator (t-PA); it is a protein of molecular weight 70,000 which is considered to be the physiological AP of plasma fibrinolysis and thrombolysis. Lack of activity in plasma has been linked to the development of thrombosis
• the detection and the plasma dosage of the endothelial activator are therefore of paramount importance in the study of normal fibrinolysis' and pathological.
• the endothelial activator has a very high affinity for fibrin allowing it, on the one hand, its specific absorption on a clot and, on the other hand, to develop its enzymatic activity. In fact, this activator is only able to activate plasminogen if it is absorbed on a fibrin clot. T-PA therefore regulates true fibrinolysis without fibrinogenolysis. Under these conditions, the methods currently in common use, allowing the detection of this activator, are methods calling on the lysis of a clot (Kluft C. et al, Screening of fibrinolytic activity in plasma euglobulin fractions in the fibrin plate, Progress in chemical fibrinolysis and thrombolysis, 3.F.
• anti-t-PA monoclonal antibodies were forced to choose a test for inhibition of the enzymatic activity. These anti-activity tests make it possible to choose only antibodies directed against epitopes located at the level of the active site of the protein or situated very close to it. However, obtaining antibodies directed against other epitopes of the molecule could hardly have been envisaged to date.
• the invention aims to remedy the diffi ⁇ culties that have been mentioned above and particu ⁇ larly to provide means for both extract and assay quantitatively the tPA ini- tial ent contained in biological samples, in particular plasmas or fractions enriched with plasmas, such as euglobulins, and in addition to making available to specialists a material making it possible to practice in practice the properties, for example enzymatic or immunological of t-PA , even when it is only isolated or isolable in quantities tiny.
• the main means according to the invention consists of a molecular film supported by a network of fi ⁇ strands in solid phase, preferably fixed in a co-valent manner to the surface - or in certain regions of the surface - of a support.
• the film of the fibrin network is fixed to at least certain regions of the surface of the support by means of a polyglutaraldehyde network, itself fixed to this support.
• the support consists of a titration plate comprising one or more wells, the interior surfaces of these wells defining the regions on which mo ⁇ lecular films of the fibrin network according to the invention are formed.
• the titration plate can be made of any suitable material to which the polyglutaraldehyde can be fixed directly or indirectly, by covalent bonds or not, however stable. Such a result can be obtained more particularly with plastic supports, preferably polyvinyl chloride or polystyrene. It is in fact observed that it is possible to produce films which are fixed in an extremely stable manner to polyvinyl surfaces, for example by incubation of a glutaraldehyde solution in contact with these surfaces.
• Fibrinogen can then be deposited on these surfaces or surface regions, in particular from a fibrinogen solution, the capacity of which has been found to react with the free functions of glutaraldehyde, in particular the aldehyde functions, without in any way losing its fibrinoformation properties.
• the conservation of this characteristic is manifested by the ability of fibrinogen to be transformed into fibrin by incubation in the presence of thrombin at the temperature and in the suitable conditions for forming a molecular or even monomolecular film in which structural features of fibrin are nonetheless reconstituted in the form of a polymer constituting a network (as found in cail ⁇ lots).
• the molecular film supported by the invention takes advantage of the fundamental property of the endothelial activator of plasminogen, of being 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.
• this molecular film can be used in tests carried out in the solid phase on a molecular fibrin network and not in the liquid phase, which will not detect activators of the urokinase type. It has in fact been observed that the fibrin network of the film or lolecular 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 fix t-PA which attests to the network structure typical of fibrin that said film possesses.
• This "selective fixation" is also quantitative in that it is detectable in its entirety by its ability to be practically entirely involved in the activation of plasminogen which can be brought into contact with it, in particular inside a bucket. of mi ⁇ croplaque, the amount of activated plasminogen being able to be detected in situ, for example using any synthetic substrate transformable by plasmin.
• the invention completely overcomes the drawback mentioned above which resided in diffusion reverse of t-PA in the thickness of fibrin plaques that have been used to date.
• the film according to the invention is molecular, even monomolecular.
• 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 fi ⁇ strand per cm 2 of surface.
• proportions of fibrin lower than the lower values of the above-mentioned limits could result in the absence of the formation of a "network", an absence which would manifest itself in the absence of possible adsorption.
• t-PA under the above-mentioned conditions or under those which will be illustrated below by way of examples.
• the maximum fibrin levels per unit area will generally be limited by the number of attachment points, in particular by means of functional groups available on the surface of the support and which are involved in the attachment of the fibrinogen molecules brought into contact. of this surface, by means of complementary functional groups carried by fibrinogen.
• the dandruff fibrin networks of the invention can be used for the assay of t-PA (even unpurified) from the most diverse sources: supernatant of endothelial cells in culture, supernatant of isolated glomeruli, and euglobulin suspensions. It is also possible to use complete human plasma, which until now was hardly possible•
• the t-PA absorbed on fibrin in the solid phase can, after removal of contaminants and inhibitors by washing, be then 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 assay.
• Spectrophotometric reading of the reaction release of paranitroanilide from synthetic substrates such as HD-isoleucyl-L-prolyl-L-arginine-p-nitroanilide dihydrochloride (substrate 5-2288) or HD-valyl-L dihydrochloride -leucyl-L-lysine nitro-anilide (substrate 5-2251), both marketed by AB KABI) is carried out at 405 nm of wavelength. These readings can be transformed into fibrino- lytic units by referring to an international standard of t-PA.
• the dandruff fibrin networks according to the invention allow not only quantitative assays of t-PA, for example under the above-mentioned conditions, but also and conversely the quantitative assay of plasminogen contained in similar biological environments. It is in fact observed that the fibrin film according to the invention can also adsorb plasminogen, in particular by retention on the fi ⁇ strand, at binding sites distinct from those of the t-PA (s). The assay of the adsorbed plasminogen can then intervene by activation of the latter, via one of its activators, whether it is a suitable t-PA or a non-adsorbed activator, such as than urokinase or streptokinase.
• the dosage of the plasminogen gene thus activated can bring into play the aforementioned substrates.
• the fibrin network according to the invention not only allows rigorously selective retention of t-PA (or inogenic plas), for their assay, but generally makes the latter accessible to reagents capable of activating them or, more generally still, of reacting with them.
• the invention therefore also relates to the material obtained as such, namely the film formed by the fibrin network, retaining by selective adsorption a tissue activator of plasminogen (or the film comprising a fibrin-t- complex PA).
• the level of purity reached is such that the entire material can, for example, be used as a reagent for the specific detection of anti-t-PA antibodies.
• the invention therefore opens up a whole field of investigation, that of the detection and study of various t-PA epitopes, whether or not they are active sites of the enzyme.
• the fibrin-t-PA complexes supported by the invention can therefore be used in carrying out specific, very sensitive and rapid tests, for the detection of monoclonal anti-t antibodies.
• -PA directed against the various epitopes of this molecule and not only against the active site.
• This as in the physiological fibrin-t-PA interaction, provides a molecular structure which retains its antigenic and functional characteristics.
• This "im u ⁇ oassay” is based on the ability of the fibrin-t-PA complex to detect anti-t-PA antibodies present either in serum or in culture supernatants of hybriomas.
• the invention also relates to the process for making a molecular film of fibrin in a network, this process comprising the steps which consist in bringing a fibrinogen solution into contact with glutaraldehyde adsorbed or fixed to the surface of a support, in particular plastic and preferably vinyl chloride, then reacting the retained fibrinogen film with thrombin or the like under conditions conducive to the in situ activation of fibrinogen to insoluble fibrin, for forming the above fibrin film in a network.
• the quantities of glutaraldehyde initially fixed or adsorbed on the regions or surfaces of the support and the concentrations or quantities of fibrinogen contained in the solutions brought into contact with said regions or surfaces are adjusted so as to ultimately obtain a concentration of 120 to 180, preferably 145 to 150 nanograms of fibrin per cm of surface.
• the invention also relates to the supported films of fibrinogen themselves, these films preferably comprising doses per cm 2 of fibrinogen equivalent to those which have been indicated for fibrin.
• the coupling of fibrinogen to a surface carrying polyglutaraldehyde groups is preferably carried out at a temperature between 0 ° C and room temperature, preferably at a temperature below 10 ° C, for example at 4 ° C, contact being maintained during the time required for coupling. It is advantageous, in order to deposit the fibrinogen, to have recourse to 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.
• the pH has little effect on the level of fibrinogen coupling. However, it has been found that the most effective t-PA adsorptions were obtained when the initial fixation of fibrinogen on said surfaces was carried out with solutions having a slightly basic pH, in particular between 7 and 9.5, preferably of around 8.5.
• polyglutaraldehyde to achieve bridging between the support surface and the fibrinogen film is particularly advantageous. Any known method can be used to deposit polyglutaraldehyde on the surfaces in question. By way of example, mention will be made of that described by MOZAN et al, 1975, Biochemistry 57, 1281.
• Glutaraldehyde allows the chemical fixation of fibrinogen, via imine bonds stabilized by resonance with ethylenic bonds.
• the distribution of the aldehyde groups on the polymer and the flexibility of the latter promote the covalent bonds via the aine groups of fibrinogen, without substantially disturbing the molecular structure of the latter.
• any surface carrying enough reactive free functions with fibrinogen can be used to retain sufficient quantities to form a molecular film under the conditions which have been described above.
• - fig. 1 is a curve representative of the variations in absorbance (ordinate axis on the right) and of the corresponding fixed t-PA rates (in mU on the axis of the right or ⁇ data) as a function of the t-PA contents of solutions brought into 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 represents the different steps involved in the fixation on a fibrin film according to the invention of t-PA, of antibodies formed against t-PA and in the detection of these antibodies;
• - fig. 3 is representative under similar conditions of a test aimed at distinguishing the nature of the monoclonal antibodies capable of being retained on a film according to the invention.
• Solid phase microtitration plates in polyvinyl chloride (CPV), 96 U-shaped cups (DYNATECH LABS).
• the plate is ready and it can be stored at this stage by adding 100 microliters of buffer F containing 0.01% sodium azide in each well, c) Carrying out the test
• the plate is emptied and three washes are carried out with buffer E. 2.
• a dilution of the euglobulins or of the source of t-PA to be tested is carried out in buffer E.
• the source of t-PA is, for example , consisting of venous plasma taken from a patient, after stimulation of endothelial cells, in particular as a result of the placement of a tourniquet (before carrying out the sampling). 100 microliters of this solution are added to the wells and incubated for 1 hour at 37 ° C. This dilution takes into account the possible adsorption capacities of t-PA by the fibrin film used.
• Reagents for the reaction are added in the following order: a. 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 2 mM S-2251 or CBS-3308. 5. The plate is protected with an adhesive plastic and kept at 37 ° C for 1 hour.
• reaction is then "read” at 405 nM using a micro-ELISA reader.
• results can also be reported in fibrinolytic units compared to a curve performed with an international standard of t-PA.
• Fig. 1 is representative of the results which can be obtained with a standard fibrin film placed in contact with standard t-PA solutions, under the conditions which have been indicated above. It also follows from this curve that from a certain dose of t-PA, saturation of the fibrin films formed in each of the wells of the microplate can be observed, hence the possible dilution ne ⁇ of the sample to be tested, as indicated 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.
• t-PA from different origins either unpurified, semi-purified, or in purified form.
• the plate is ready for 1 * i muno-assay. c) Realization of the immuno-assay (RIA or EL15A).
• the ; detection of monoclonal antibodies, if any, can be carried out according to any one of the following methods. 3.
• the bound radioactivity is proportional to the titer of the anti-t-PA antibody present in the serum or in the culture supernatants of the hybridomas.
• a blank is made with buffer F or eu- supernatant ture of the parental yelomatous line. 4.
• ELISA a. 50 microliters of an appropriate dilution of a conjugate (usually 1: 800) of Gamig and peroxidase are added. Incubate for 1 hour at 37 ° C. b. The plate is emptied and washed three times with buffer F. c.
• Fig. 2 shows the main steps in the selection of monoclonal antibodies under the conditions which have been indicated above.
• the binding of the monoclonal antibody is finally detected by a second mAb antibody carrying a radioactive or enzymatic marker.
• the result of the attachment of the second antibody is shown diagrammatically in (e) of FIG. 2.
• the possible fixation of the desired MoAb monoclonal antibody is observed after cutting the bucket and introducing it into the tank of a gamma radiation or a spectrophotometer, depending on the nature of the marker used.
• the specificity of the MoAb monoclonal antibody can be the subject of various verifications.
• the films of the fibrin network retaining t-PA allow the study of distinct and specific monoclonal antibodies of also distinct antigenic sites carried by t-PA.
• the distinction of the specific monoclonal antibodies intervening in the activity of t-PA and of distinct sites can naturally be carried out under the conditions which follow, 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 Gamig antibody, itself fixed on a support, for example on the PVC-GA support (before any fibrinogen fixation on this one).
• This step is shown diagrammatically in (f) in FIG. 3.
• the plate retaining the fixed monoclonal antibodies is incubated with a t-PA solution (containing for example 100 units / ml of t-PA) for 1 hour at 37 ° C. .
• the operation corresponding to this step is shown diagrammatically in (g) in FIG. 3.
• the result of this operation is sché ⁇ matisé in (h) of fig. 3.
• the plate retaining the monoclonal antibodies is then incubated with a plasminogen solution. (Pg), fibrin (Fn) and a plasmin substrate (eg S-2251) under the conditions which have been indicated above.
• a plasminogen solution Pg
• fibrin Fn
• a plasmin substrate eg S-2251
• phase fibrin network "solid as obtained by the manufacturing method described in Example I can also be used for the determination of plasminogen contained in a biological sample, in particular in blood serum. This is particularly likely to be obtained from plasma (blood supernatant after separation of all cells) and after separation of its fibrinogen content, preferably in the form of fibrin (for example after addition of a calcium or thrombin salt This plasma 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 37 ° C., in contact with the fibrin pellets formed in the cups of the microplate.
• the absorbed plasminogen is then activated by contacting with a solution of a suitable activator, such as urokinase or streptokinase.
• a suitable activator such as urokinase or streptokinase.
• the plasmin produced in situ is then dosed by introduction into the cups of the plate as of a specific substrate for the enzyme, under the conditions which have already been described above. It is also possible to add the activator and the substrate simultaneously and then perform the reading under the conditions that have been indicated.
• the fibrin network films in accordance with the invention can, as in the previous case, also be used for the selection of antibodies specific to plasminogen, more particularly specific monoclonal antibodies.

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• 1984
  • 1984-03-23 FR FR8404587A patent/FR2561661B1/fr not_active Expired
• 1985
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  • 1985-03-22 WO PCT/FR1985/000059 patent/WO1985004425A1/fr not_active Ceased

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