EP3265230B1 - Electrodot: method for the analysis of biological samples in a dot matrix fixed by the action of an electrical current combined with a pressure difference - Google Patents

Description

Introduction

• extrait cellulaire,
• cellules en suspension,
• plasma sanguin,
• protéine et ou acide nucléique en suspension,
• molécule biologique en solution ou en suspension et
• plus généralement un matériel biologique, etc.

Matrix filtration or dot plot methods are conventional methods of analyzing a plurality of biological samples, such as:

• cell extract,
• cells in suspension,
• blood plasma,
• protein and or nucleic acid in suspension,
• biological molecule in solution or in suspension and
• more generally biological material, etc.

These techniques are used to produce on a support such as nitro cellulose membrane, vinyldene polyfluoride (PVDF), micro or nano structured membrane, filter polymer membrane, etc., a matrix of sample deposits where each sample forms a spot, such that each spot represents the capture of all or part of said sample.

The implementation of the dot plot filtration generally involves devices comprising a plate pierced with transverse wells, such as a biological sample in solution or suspension is placed in each well of the plate, said plate being arranged contiguously on a filtration membrane. A pressure difference ΔP, applied on both sides of the membrane, allows the filtration of the biological sample through said membrane so as to retain on the latter targets of interest of the samples at the same time as the other elements of the samples, each sample forming a spot of a spot matrix of mirror geometry to that of the well plate.

• des méthodes d'imagerie de fluorescence, de chimioluminescence, colorimétrie, lorsque les cibles d'intérêt sont marquées avant, durant ou après la filtration par un marqueur fluorescent, chimioluminescent ou colorimétrique, tel que par exemple le marqueur soit intégré à la structure de la cible ou interagit avec la structure de la cible, notamment par l'utilisation d'anticorps directement marqués ou marqués grâce à un anticorps secondaire ;
• des méthodes de spectroscopie telles que LIBS (spectroscopie sur plasma induit par laser), masse, infrarouge, etc. ;

ou toute autre méthode permettant de quantifier ou d'identifier la ou les cibles d'intérêts. De telles techniques sont connues, par exemple, de EP0776700 A1 et WO2005/035132 A2 .The targets of interest of each spot will then be analyzed, by different methods of analysis used in biology, such as:

• methods of fluorescence imaging, chemiluminescence, colorimetry, when the targets of interest are labeled before, during or after filtration by a fluorescent label, chemiluminescent or colorimetric, such as for example the marker is integrated into the structure of the targets or interacts with the target structure, including the use of antibodies directly labeled or labeled with a secondary antibody;
• spectroscopy methods such as LIBS (Laser Induced Plasma Spectroscopy), mass, infrared, etc. ;

or any other method that quantifies or identifies the interest target (s). Such techniques are known, for example, from EP0776700 A1 and WO2005 / 035132 A2 .

The use of dot blot technologies by filtration is largely limited by sample preparation methods. Indeed, the addition of chaos-tropes agents such as urea or detergents such as sodium dodecyl sulfate (SDS), tritone, etc., in the sample to extract interest targets, strongly penalizes the capture by the filtration membranes of many targets nucleic acid or protein.

These limitations are particularly observed in the analysis of nuclear proteins, in particular for the dot blot analysis of certain histones of certain cell types. For example, some quantitative histone extraction protocols of hair follicles, such as histones γH2AX, or H3 require the use of SDS, while SDS is incompatible with the quantitative adsorption by filtration of said histones on nitro membranes. cellulose or PVDF.

• la géométrie des dispositifs classiquement utilisés pour la mise en oeuvre de la filtration par exemple :
  • ∘ pour permettre une meilleure sensibilité de détection, les puits des plaques de filtration doivent disposer d'un certain volume minimal permettant la détection des cibles en fonction de la sensibilité de détection, ce qui entraîne une dispersion et/ou un écartement des spots sur la membrane de filtration directement proportionnelle à la taille des puits de la plaque à puits. De ce fait la surface des membranes est importante par rapport au nombre de spots, et nécessite alors pour l'analyse des volumes importants de solutions de saturation et de révélation comportant des composés coûteux tels que des anticorps marqués, anticorps non marqués et anticorps secondaires marqués, etc. ;
  • ∘ la filtration n'est pas puits-indépendante, de ce fait lorsque les échantillons possèdent des viscosités différentes d'un puits à l'autre la vitesse de filtration varie d'un puits à l'autre. Les conditions et la vitesse de filtration n'est plus contrôlée dès qu'un puits de filtration est vidé, puisque ΔP appliqué à la membrane chute dès que le premier puits de la plaque est vide.
• le manque d'ergonomie dans la mise en oeuvre des procédés, d'une part pour la manipulation des dispositifs de filtration existant, concernant notamment les méthodes de montage et de fixation utilisées (vis) ;
• la durée de mise en oeuvre liée à la préparation des membranes de filtration notamment pour les méthodes utilisant un marquage par anticorps fluorescent ou chimioluminescent après l'étape de filtration ;
• les problèmes d'affinité croisée qui peuvent survenir pour les marquages par anticorps par exemple dirigés contre certaines formes de phosphorylation de protéine. En effet, sur un spot de la membrane il peut coexister plusieurs types de protéine phosphorylé. Or la plupart des anticorps dirigés spécifiquement contre la forme phosphorylé d'une protéine, présentera une affinité non négligeable pour le groupement phosphore d'autres protéines. Lorsqu'une protéine d'intérêt est en faible quantité sur un spot comportant d'autres protéines phosphorylées en plus grande quantité, l'anticorps spécifique de la protéine d'intérêt donnera un fort signal non spécifique en reconnaissant les autres fonctions phosphate présentes sur le spot.

We can also mention as a limitation in the use of dot blot techniques by filtration:

• the geometry of the devices conventionally used for the implementation of filtration, for example:
  • ∘ to allow a better detection sensitivity, the wells of the filter plates must have a certain minimum volume allowing the detection of the targets according to the sensibility of detection, which causes a dispersion and / or a spacing of the spots on the filtration membrane directly proportional to the well size of the well plate. As a result, the surface area of the membranes is large relative to the number of spots, and then requires for the analysis of large volumes of saturation and revealing solutions comprising expensive compounds such as labeled antibodies, unlabeled antibodies and labeled secondary antibodies. etc. ;
  • ∘ the filtration is not well-independent, therefore when the samples have different viscosities from one well to another, the filtration rate varies from well to well. The conditions and the rate of filtration are no longer controlled as soon as a filtration well is emptied, since ΔP applied to the membrane drops as soon as the first well of the plate is empty.
• the lack of ergonomics in the implementation of the processes, on the one hand for the manipulation of the existing filtration devices, in particular concerning the mounting and fastening methods used (screws);
• the duration of implementation related to the preparation of the filtration membranes, especially for the methods using fluorescent or chemiluminescent antibody labeling after the filtration step;
• the cross-affinity problems that may arise for antibody markings, for example directed against certain forms of protein phosphorylation. Indeed, on a spot of the membrane it can coexist several types of phosphorylated protein. However, most of the antibodies directed specifically against the phosphorylated form of a protein, will have a significant affinity for the phosphorus group of other proteins. When a protein of interest is in a small quantity on a spot containing other phosphorylated proteins in greater quantity, the antibody specific for the protein of interest will give a strong non-specific signal by recognizing the other phosphate functions present on the spot.

To circumvent these limiting problems, we propose a method and an implementation device involving an electric field perpendicular to the plane of the membrane and a simultaneous filtration or subsequent to the application of the electric field, in order to produce a matrix of samples, or spots, in the presence of chaos-tropes agents or detergent, while increasing the ergonomics of implementation, decreasing the labeling time and increasing the specificity of labeling for certain phosphorylated proteins.

• un extrait cellulaire,
• des cellules en suspension,
• plasma sanguin,
• des protéines et/ou acide nucléique en suspension,
• des molécules biologiques en solution ou en suspension et

toute combinaison de ces éléments et plus généralement tout produit biologique.We mean by sample:

• a cell extract,
• cells in suspension,
• blood plasma,
• proteins and / or nucleic acid in suspension,
• biological molecules in solution or in suspension and

any combination of these elements and more generally any biological product.

• les membranes de nitro cellulose
• les membranes de PVDF,
• les membranes micro ou nano structurées,
• les membranes de polymère
• les filtres

plus généralement tout substrat de fixation d'échantillon biologique.Fixation membrane means any membrane or substrate such as:

• nitro cellulose membranes
• PVDF membranes,
• the micro or nano structured membranes,
• the polymer membranes
• the filters

more generally any biological sample attachment substrate.

The present invention relates to a method for fixing on a fixing membrane, in spot matrix, all or part of a plurality of biological samples such that each sample is placed in a well. Fig1.1 a plate with transverse wells F1.2, and such that is positioned under said well plate the F1.3 attachment membrane, so as to subject each sample to the simultaneous or sequential action of an electric field Fig1.4 and a pressure Fig1.5 such that the electric field has a direction perpendicular to the fixing membrane, and a potential differential between the two faces of the fixing membrane between 1 milivolt and 1 kilovolt (preferably between 10mV and 100V, and more preferably between 100mV and 10V), and such that the pressure has a differential pressure ΔP between the two faces of the fixing membrane of between 0.1 millibar and 100 bar (preferably between 1mb and 10b , and more preferably between 10mb and 10b).

In this embodiment the fixation of the molecules of interest on the attachment membrane is by the double action of the electric field and the filtration. The action of the electric field allowing a better fixation of the targets of interest on the membrane even in the presence of a detergent in the medium such as the SDS, the triton or other chaos-trope agents, or any combination of these agents and detergents.

The electric field is obtained thanks to two electrodes of conductive materials, such as conductive polymer, conductive composite, metal, gold, platinum, tantalum, etc., disposed above Fig1.6 and below Fig1.7 of the well plate. , the fixing membrane being interposed between the lower face of the well plate Fig2.14 and the lower electrode Fig1.7.

In an even more particular embodiment, at least one of the electrodes will be a sheet of an electrical conductor such as gold, platinum, copper, tantalum, etc., optionally deposited on a polymer such as kapton, silicone, PMMA, PLA. ...

In other embodiments, the metal electrode sheet will comprise at least one sticky structured surface, obtained for example by means of a repositioning adhesive, or at least one joined structural surface FIG. 9.9, for example made of rubber, silicone, or any other polymer, such as the tacky surface, leaves the surfaces of the spot-shaped electrode Fif1.10 free from the wells of the well plate, so as to have an array of electrodes exposed metal. The spots of the electrode left free by the contiguous or sticky surface will comprise at least for the lower electrode pores for letting a liquid pass.

In some embodiments, at least one of the faces of the fixing diaphragm FIG. 1, 3 will comprise a joined or sticky structured surface 9 leaving free a matrix of fixing membrane spots FIG. 11 in relation to the well matrix.

In some embodiments, a porous paper, forming a pad Fig. 1.8, for example 0.1 to 5 mm thick such as wattman paper, laid paper etc., is arranged against at least one of the electrodes, in order to make a buffer between the electrode and the membrane, or the electrode and the well plate. In some embodiments, at least one side of the paper will be structured by a glue or a joined polymer to leave free a paper spot matrix Fig1.12 vis-à-vis the well matrix of the well plate.

In some embodiments, the well plate is made such that the bottom face Fig2.14 of the well plate as well as the lower diameter of the wells (face of the well plate receiving the binding membrane) are reduced for example by a homothety of a factor K and / or k 'relative to the size of the upper face Fig1.13 of the well plate and the upper diameter of the wells. This reduction of a factor K and / or k 'makes it possible to reduce the spot size by a combination factor K and k' but also, the distance between the spots of the same combination factor K + k 'and makes it possible to achieve reduced binding membranes of K-factor relative to the upper surface of the well plate. This makes it possible to reduce the reaction volume for the antibody revelation steps, as well as to reduce the areas to be analyzed for the imaging or spectroscopy methods.

In some embodiments, the well plate has a liquid ion electrode, gel or impregnated sponge, connecting the upper face to the lower face of the well plate. This electrode makes it possible to impose a potential difference between the two faces of the membrane whatever the level of sample in the wells of the matrix.

In some embodiments, the upper electrode will include Fig 1.6, the upper face of the well plate, and a portion of the well surface.

In some embodiments, a Fig2.15 structure forming an electrode such as a cross or any other shape connected to the upper electrode, will be disposed in a transverse plane of the wells of the well plate.

In some embodiments, the pressure difference ΔP is obtained by suction below the attachment membrane through the lower electrode. The stack of all or part of the elements described above for example is arranged on a suction grid Fig3.16 below which is applied a vacuum.

In an even more preferred embodiment, the grid Fig3.16 is structured by an adhesive or a joined material, so as to delimit tight spots between them where said grid appears free to form a matrix of grid spots vis-à- screw wells of the well plate.

In some embodiments, the suction grid Fig3.16 and the lower electrode are combined, the grid then comprises at least one electrically conductive element, for applying an electric field, in each well of the well plate, through the fixing membrane. The electrically conductive element may be a plating or a metallic deposit such as gold, copper, tantalum, platinum, etc.

In a particular embodiment, the pressure difference ΔP is obtained by a pressure applied above the membrane through the wells of the well plate and the upper electrode.

In some embodiments, the biological samples are introduced into the wells 1 of the well plate by means of a plate plate Fig3.18 forming a matrix of wells arranged in a manner homologous to the well of the well matrix. The samples are introduced into the plate of boxes kept horizontal, such as for example a sample per box. Then the well plate comprising any combination of the stacks described is clipped Fig3.19 on the plate plate remained horizontal, the upper face of the well plate being close to the openings of the plate of boxes.

In some embodiments, both well plates and well plates may be guided and held against each other by magnet and magnetic or ferrous platinum systems, introduced for example at the four corners of the plates.

In a particular embodiment, the box plate comprises in each box a movable piston Fig3.20, for example flexible polymer such as silicone, rubber etc. Behind each piston is disposed in the box plate, a pore Fig3.21 allowing, by displacement of the piston, to compensate the pressure or to apply pressure, depending on whether the device is used in overpressure or depression. The movable piston guarantees a homogeneous application of the pressure or of the depression, during the process as soon as a well is emptied it is obstructed by its piston which prevents the fall of ΔP.

In a particular embodiment, the various electrodes and membranes used in the device will comprise tabs 22 allowing rapid recovery of the fixing membrane and the different surfaces of the well plates, plate of boxes, electrode, paper pad etc. are structured 9 by a sticky material, joined to define a matrix of spots in the image of the well matrix such that each spot is isolated from other spots by said material.

In a particular embodiment, the device will comprise a rapid revelation kit. The kit will comprise for each fixing membrane, a plastication complexing bag of sufficient size to receive the membrane, the bag will be provided with a rapid closure device such as closing slide or closing slider. One of the faces of the bag will comprise a fast attachment device to a support, for example the velvet part of a fastening or self-gripping strip, the hook portion of said strip being arranged on the support receiving the bag for example in a incubation oven. The bag will preferably be opaque protecting light, for example black and opaque to ultraviolet, so that the light can not alter the properties of the reagents and biological material disposed in said bag. The bag will be pre-filled with a solution or with a powder that makes it possible to produce a solution for saturating the membrane. For example, the bag will contain from 0.1 to 10g of lyophilized milk powder. The kit will include, for each attachment membrane, a rapidly opening hermetically sealed capsule made of aluminum, plastics material, etc., containing freeze-dried labeling reagents such as lyophilized antibodies and saturation elements such as lyophilized milk. The contents of the capsule will preferably be compressed to obtain an aggregated body. The kit will also comprise a stirring furnace thermostatted between 4 ° C and 50 ° C, preferably 37 ° C, the oven will include elements to fix the revealing bag and allow it to stir. The kit may be formed by a combination of all or part of the aforementioned elements.

In a particular embodiment, the stirring system of the furnace may for example comprise a rotating axis with a diameter of between 0.5 cm and 60 cm, said axis having means for fixing the revelation bag, for example the hook part of a Velcro tape.

In a particular embodiment, the fast opening hermetically closed capsule will have at least one antibody directed against a target of interest, said antibody being labeled with at least one fluorophore, chromophore, bioluminescent agent or chemiluminescent, such that each antibody directed against a given target is labeled with a fluorophore, a chromophore, a given bioluminescent or chemiluminescent agent.

In a particular embodiment, the rapid-opening hermetically sealed capsule will have at least one primary antibody directed against a target of interest and a secondary antibody directed against the at least one primary antibody such that the secondary antibody is labeled with less a fluorophore, a chromophore, a bioluminescent or chemiluminescent agent.

In a particular embodiment, the fast opening hermetically closed capsule will comprise at least one unlabeled saturation antibody, neither directly nor by a secondary antibody, making it possible to compete with the parasitic aspecific bonds of a given primary antibody for other targets than its target of interest. For example, the capsule will comprise primary antibodies labeled, directly or by a secondary antibody, directed against phosphorylated proteins of interest such as histone γH2AX, Phospho KU 70, phospho ATM, etc., and at least one of the unmarked saturation antibodies. directed at phospho-serine, phospho-treonine, phosphothyrosine, etc., such that the aspecific binding of the primary antibodies to phosphorylated targets different from their target of interest is competed by the binding of the saturation antibodies.

• Solubilisation de l'agent de saturation contenu dans le sac de complexation par l'ajout d'une dose de tampon par exemple du 50 ml de TBS pour 2,5g de lait.
• Introduction de la membrane de fixation dans le sac de complexation et fermeture du sac.
• Incubation du sac de complexation entre 4 et 40°C dans le four à agitation thermostaté par accrochage sur l'axe tournant du four grâce aux bandes auto agrippantes.
• récupération du sac du four et ajout du contenue d'une capsule hermétiquement fermée à ouverture rapide dans le sac de complexation dilué à la concentration attendue par un tampon par exemple par du TBS.
• Incubation du sac entre 4 et 40°C dans le four à agitation thermostaté.
• récupération du sac du four rinçage de la membrane et lecture de la membrane.

The implementation of the kit will consist of six main steps:

• Solubilization of the saturation agent contained in the complexing bag by adding a dose of buffer, for example 50 ml of TBS per 2.5 g of milk.
• Introduction of the fixation membrane in the complexation bag and closure of the bag.
• Incubation of the complexing bag between 4 and 40 ° C in the stirring furnace thermostatically by hanging on the rotating axis of the oven thanks to the self-gripping strips.
• recovery of the oven bag and addition of the contents of a fast-opening hermetically closed capsule in the complexing bag diluted to the concentration expected by a buffer, for example by TBS.
• Incubation of the bag between 4 and 40 ° C in the thermostatically controlled oven.
• recovery of the oven bag rinsing of the membrane and reading of the membrane.

Examples:

• Fig4.23 Example of extraction of the biological material, for HEK cells, by different extraction buffers 25, 26, 27, 28, in dot-blot filtration then labeling with a primary anti-histone antibody γH2AX and a secondary chemo antibody -Luminescent directed against the primary antibody.
• Fig4.24 Example of extraction of biological material, for hair follicles, by various extraction buffers 25, 26, 27, 28, in dot-blot filtration then labeling with a primary anti-histone antibody γH2AX and a chemiluminescent secondary antibody directed against the primary antibody.
• Fig4.29 Example of extraction of the biological material, for HEK cells, with a 5% SDS extraction buffer, in electrodot, then simultaneous labeling with a primary anti-histone antibody γH2AX and a secondary chemiluminescent antibody directed against the primary antibody, the labeling products being lyophilized in an aluminum capsule.
• Fig4.30 Extraction example of the biological material, for hair follicles, by a 5% SDS extraction buffer, in electrodot, then simultaneous labeling with a primary anti-histone γH2AX antibody and a secondary chemiluminescent antibody directed against primary antibody, the labeling products being lyophilized in an aluminum capsule of the freeze-dried skimmed milk.
• Fig4.31 Example of extraction of the biological material for HEK cells, with a 5% SDS extraction buffer, in electrodot, then simultaneous labeling with a primary antibody anti-histone γH2AX labeled with a fluorophore at 667 nm and a primary antibody anti-histone H2AX labeled with a fluorophore at 488 nm, the labeling products being lyophilized in an aluminum capsule containing freeze-dried skimmed milk.

LEGENDS OF ALL FIGURES

1. 1) Well
2. 2) cross-well and through-hole plate
3. 3) fixing membrane
4. 4) electric field
5. 5) pressure gradient
6. 6) upper electrode
7. 7) lower electrode
8. 8) porous paper, forming a buffer
9. 9) Attached or sticky structured surface
10. 10) spot-shaped electrode
11. 11) spot membrane fixation
12. 12) spot buffer paper
13. 13) top plate well plate
14. 14) lower face well plate
15. 15) electrode in a transverse plane of the wells
16. 16) suction grille
17. 17) suction
18. 18) plate of lodges
19. 19) clips
20. 20) movable piston
21. 21) pore
22. 22) tabs
23. 23) HEK dot blot cell
24. 24) Hair follicle dot blot
25. 25) Basic lysis buffer extraction
26. 26) SDS Extraction 10%
27. 27) SDS extraction 1%
28. 28) RIPA Buffer Extraction
29. 29) HEK electrodot cell
30. 30) Electrodot hair follicle
31. 31) HEK cell electrodot extraction SDS 5%

Claims (15)

1. Method for fixing on a fixing membrane in a dot matrix, a plurality of biological samples of all or part thereof, such that each sample is placed in a well (1) of a microplate (2) with traversing and open wells, and such that the fixing membrane (3) is positioned under said microplate plate, characterised in that it consists of subjecting each sample to the simultaneous or sequential action of an electric field (4) and of a pressure (5) such that the electric field has a direction perpendicular to the fixing membrane, and a potential differential between the two faces of the fixing membrane of between 1 millivolt and 1 kilovolt, and such that the pressure has a pressure differential ΔP between the two faces of the fixing membrane of between 0.1 millibar and 100 bar.
2. Device for implementing the method according to claim 1 comprising the microplate (2) with traversing and open wells, as well as the fixing membrane, characterised in that it comprises the means to subject each sample to the simultaneous, sequential action of an electric field (4) and a pressure, these means comprising two electrodes supplying the electric field, said electrodes being made of conductive materials, such as for example a conducting polymer, a conducting compound, gold, platinum, or tantalum, arranged above (6) and underneath (7) the microplate (2), the fixing membrane being inserted between the lower face of the microplate and the lower electrode, and characterised in that at least one of the electrodes is a sheet of an electrical conductor which comprises at least one joined and adhesive structured surface (9), the structured surface leaving free surfaces of the electrode in the shape of dots (10) facing the wells of the microplate, so as to have a matrix of visible metal electrodes, the dots (10) of the electrode left free by the joined or adhesive surface comprising, at least for the lower electrode, pores making it possible for the passage of a liquid.
3. Device according to claim 2, characterised in that a porous paper forming a buffer (8) with a thickness of 0.1 to 3mm, such as for example a Whatman paper, or laid drawing paper, is arranged against at least one of the electrodes and in that at least one face of the paper is structured (9) by a glue or a joined polymer so as to leave free a matrix of paper dots (12) facing the matrix of wells of the microplate.
4. Device according to any one of claims 2 and 3, characterised in that the lower face (14) of the microplate, as well as the lower diameters of the wells, defined as the face of the microplate receiving the fixing membrane, are reduced in size by homothetic transformation, such as a homothetic transformation by a factor K, or by a combination of a factor K and k', with respect to the size of the upper face (13) of the microplate and of the upper diameter of the wells, said reduction making it possible to proportionally reduce the size of the dots and the distance between dots on the fixing membrane.
5. Device according to any one of claims 2 to 4, characterised in that the microplate comprises a liquid-based, gel-based or soaked-sponge ionic electrode connecting the upper face to the lower face of the microplate.
6. Device according to any one of claims 2 to 5, characterised in that the upper electrode comprises, on the upper face of the microplate, a portion of the surface of the wells, and optionally a structure (15) arranged in a transversal plane of the wells of the microplate.
7. Device according to any one of claims 2 to 6, characterised in that the suction strainer (16) and the lower electrode (7) are merged, such that said strainer comprises at least one electrically-conducting element, making it possible for the application of an electric field in each well of the microplate, through the fixing membrane.
8. Device according to any one of claims 2 to 7, characterised in that the pressure difference ΔP is achieved by suction (17) underneath the fixing membrane through the lower electrode of the suction strainer, underneath of which a low pressure is applied.
9. Device according to any one of claims 2 to 8, characterised in that the pressure difference ΔP is achieved by applying pressure above the membrane, through the wells of the microplate and the upper electrode.
10. Device according to any one of claims 2 to 9, characterised in that a plate housing (18) forming a matrix of housings arranged in a similar fashion to the wells of the matrix of wells, is used to introduce biological samples into the wells of the microplate, and comprises in each housing a mobile piston (20) preferably made of a flexible polymer, such as, for example, silicone rubber, so that behind each piston is arranged, in the plate housing, a pore (21) configured to compensate the pressure by moving the piston.
11. Device according to any one of claims 2 to 10, characterised in that the electrodes, membranes and buffer-paper used for the device comprise strips (22) making it possible for the quick recovery of the fixing membrane and in that the various surfaces of the microplate, plate housing, electrodes, buffer-paper are structured by an adhesive and joined material defining a dot matrix reflecting the matrix of wells, such that each dot is isolated from the other dots by said material.
12. Device according to any one of claims 2 to 11, characterised in that a quick revelation kit comprises for each fixing membrane, a complexation bag made of a plastic material and sufficiently large to receive the membrane, said bag being provided with a quick closing system such as a zip or slide fastener, one of the faces of the bag comprising a quick fixing means to a support, such as the velvet portion of a self-gripping strip, said bag is preferably opaque to protect from the light and prefilled with a powder so as to achieve a saturation solution of the fixing membrane, such as 0.1 to10g of lyophilised powder milk, the kit also comprises for each fixing membrane, a sealed capsule with a quick opening mechanism, made of aluminium, plastic material, which contains lyophilised marking and saturation reagents such as antibodies, saturation elements of the fixing membrane, fluorophore, chromophore, bioluminescent or chemiluminescent agents, the content of the capsule being preferably compressed so as to obtain an aggregated body.
13. Device according to claim 12, characterised in that a stirring system of a complexation oven comprises a rotating axis with a diameter ranging from 0.5 to 60cm, said axis having means for fixing the revelation bag, such as for example the hooks of a self-gripping strip.
14. Device according to claim 12 or 13, characterised in that the sealed capsule with a quick opening mechanism comprises at least one primary antibody directed against a target of interest, and at least one secondary antibody directed against said at least one primary antibody, such that said secondary antibody is marked by at least one fluorophore, chromophore, bioluminescent or chemiluminescent agent.
15. Device according to one of claims 12 to 14, characterised in that the sealed capsule with a quick opening mechanism comprises at least one saturation antibody, making it possible for competition with non-specific parasitic bonds of a primary antibody for targets other than the target of interest.

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