In vivo micro-invasive investigation device including a metal guide

CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 1 IN VIVO MICRO-INVASIVE INVESTIGATION DEVICE INCLUDING A METAL GUIDE DESCRIPTION Technical field The present invention relates to the functionalization of a transparietal investigation device, a method ex vivo analysis of a substrate using a functionalized device of the invention, and the use of such a device for the manufacture of a tool intended for the diagnosis of cancer, of an infection, inflammation, neurodegenerative disease or transplant rejection in a patient.

STATE OF THE ART In the prior art, devices for investigation or treatment in vivo are known.

Such devices take the form of a rigid tube of the endoscope type or of a catheter consisting of a flexible tube which is inserted into the body, in particular by natural routes or vessels, and which makes it possible to reach an organ. or a specific tissue.

These devices allow in particular the elimination of blood clots or, when they are associated with optical fibers, the visualization and control in vivo of the state of a system, such as the digestive tract, or of an organ, like the colonist.

The trauma for the patient resulting from the use of such devices is then minimized but still remains to be improved.

However, it is not always possible to perform organ or tissue analysis in a patient with these devices. CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 2.

Such an impossibility may result from the reduced accessibility of said tissue or organ with regard to blood circulation or natural pathways, or else from the difficulty in making a reliable diagnosis without resorting to a detailed study of the cells of said organ or tissue.

In these cases, it is commonly used in the prior art and to date, the removal of a fragment of said tissue or organ (biopsies) in order to check ex vivo the morphology of these tissues or organs, or more precisely that cells which constitute them, in particular using fine needles (FNA or Fine Needle Aspiration:

Engelstein et al., Br. J.

Urol., 7: 210-213, 1994;

Rodrigues et al., J.

Am.

Acad.

Dermatol., 42: 735-740, 2000; Ariga et al., Am. J.

Surg., 184: 410-413, 2002;

Pérez-Guillermo et al., Diagn.

Cytopathol., 32: 315-320, 2005; Fernandez-Esparrach et al., Arch.

Bronconeomol., 43:

219-224, 2007).

In addition, it is also possible to analyze the expression status of a certain number of markers whose expression is correlated with a specific pathological state (cancer, inflammation, infection, neurodegenerative disease or graft rejection in particular).

However, these different methods, due to the taking of a biopsy or aspiration of cells in situ, sometimes cause significant trauma to said tissue or organ and, consequently, to the patient.

The organism of the latter can thus be severely tested due to hemorrhages or even scarring following the removal, in particular for certain organs (brain, pancreas, liver or lung).

There is therefore still today a need for the identification of new investigation methodologies making it possible to carry out a reliable and precise diagnosis, CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 3 in particular with regard to the expression of markers associated specifically with various pathologies such as in particular cancer, inflammation, infection or neurodegenerative disease, while limiting the trauma inflicted on the patient.

Patent EP 1 358 481 describes an in vivo analysis or treatment device comprising (i) a micro-system for investigating a substrate other than by analysis of a fluorescent signal, (ii) a flexible rod with a end of which is fixed said microsystem and the other end of which is intended for the operation of said microsystem, (iii) a medical instrument having an internal lumen in which said flexible rod can slide, (iv) a sliding system for the protection of the removable micro-system at the level of the substrate, and (v) at the level of said micro-system, a tissue or cell dilaceration system, optionally associated with one or more devices chosen from among a remote monitoring device by sensory receptors (tactile, optical, physicochemical and in particular electronic or digitized computer), biopsy, treatment, local injection of biological or chemical products.

In order to allow the breaking of blood vessels and the laceration of tissue or cells, the micro-system is then associated with another more rigid system ensuring this function, preferably in a distal position.

However, in the case of an in vivo investigation device, the investigation device is most often directed to the target organ or tissue using the endovascular or endocavitary route.

Coupling the microsystem with a macerating system then increases the diameter at the end of the CA 02716751 2015-06-22 \ Vo 2009/106295 PCT / EP2009 / 001308 4 non-negligibly way.

The latter then turns out to be of complex use in order to be correctly directed to its target site without simultaneously altering the blood circulation pathways.

At the same time, the coupling of multiple elements adversely affects the overall flexibility of the device, and therefore its correct guidance, as well as the obtaining of the rigidity necessary to allow the perforation of an organ or a tissue.

Description of the invention Following extensive research, the inventor has now succeeded in developing a device comprising a metal guide at a perforating end of which reactive groups, in particular antibodies or fragments of antibodies, are directly coupled. of a substrate to be tested, and the other end of which is intended for maneuvering said guide from the insertion site to the microanalysis and / or micro-sampling site of said substrate.

Said guide can be inserted into a removable protection system, for example a flexible catheter, thus making it possible to protect the functionalized end of said guide up to the site of micro-analysis and / or micro-sampling of the substrate to be tested constituting the tissue. , organ, or cells thereof.

In accordance with the present invention, this involves structuring the surface of the guide in order to define locations on the guide, wells, where the reactive groups will be deposited and where the biochemical interactions will take place.

Said wells can be produced by various methods such as for example by focused ion beam lithography (FIB or for Focused Ion beam: Xie et al., Nuclear Instruments &

Methods in Physics research Section B-beam Interactions with CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 Materials and Atoms, 211 (3): 363-368, 2003), by laser lithography followed by etching electrochemical and laser ablation.

Alternatively, the structuring of the metal guide 5 can be carried out with the aim of defining places on said guide, for example at least one groove in which is integrally associated at least one miniaturized linear, circular or ribbon biochip, where the reactive groups will be deposited and where the biochemical reactions will take place.

The places formed by the structuring of the surface of the guide, for example wells, do not form any relief on the surface of the guide, but grooves, or hollows.

The places formed by the structuring of the surface of the guide therefore do not increase the diameter of the guide.

Advantageously, the places formed by structuring the surface of the guide are located on the surface of the guide, which means that these places are directly in contact with the fluids, the tissues and the cells.

Said device can also be inserted into a medical instrument having an internal lumen in which said metal guide can slide, and in particular in a transparietal puncture needle, in particular transcutaneous or transmucosal, or an endoscope, including an endovascular navigation system.

Said device can also be associated with an optical fiber, or the metal guide of said device can be replaced by an optical fiber at a perforating end of which is associated a metal ring on which are directly coupled reactive groups, in particular antibodies or antibody fragments, specific for a substrate to be tested, thus allowing fine visualization CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 6 in situ, with a view to capturing in vivo the elements necessary for diagnosis and possibly for prognostic evaluation.

The properties of the optical fiber are used for location imaging and device placement from in situ visualization.

The device according to the invention due to its simplification has, while remaining micro-invasive, an improved flexibility or elasticity compared to the devices of the prior art.

The latter can then be used much more effectively for an investigation by the endocavitary route, in particular by the endovascular route, or by the transparietal route, in particular not by the transcutaneous or transmucosal route.

In addition, the device according to the invention, due to the use of a metal guide, one end of which is coupled to specific reagents, has sufficient rigidity at its functionalized end to efficiently perforate a tissue or an organ.

Finally, the diameter of the device according to the invention at its end is small enough to allow simplified navigation in the blood circulation or in natural cavities, and above all to minimize the trauma to the tissue or organ that it must. perforate.

In addition, and after removal of the device, it is then possible to identify ex vivo using conventional techniques, such as an immunoenzymatic assay or by immunofluorescence, for example on a solid support [ELISA technique, protein chip (ESPINA et al. ., J.

Immunol.

Methods, vol. 290, p: 121-133, 2004)], the presence and the relative concentration of said specific substrate (s) at the end of the device according to the invention and therefore at the level of the micro-analysis site and / or micro-sampling (organ or tissue).

CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 7 Finally, and due to the metallic nature of the guide, the device has an improved signal in imaging (arteriography, ultrasound, scanner, MRI, etc.) ).

This property makes it possible to considerably simplify the radio guidance of the device according to the invention during the intervention to the targeted tissue or organ.

The same is true for the coupling of said device with an optical fiber for marking imaging purposes, and which allows fine visualization in situ.

Consequently, a first object of the invention is a device for the analysis of a substrate, characterized in that it comprises a system for micro-invasive investigation and / or for micro-sampling of said substrate, said system being consisting of at least one metal guide at one end Ea of which is provided at least one series of wells to which is directly coupled at least one specific reactive group of said substrate, said functionalized end Ea being perforating, and the other end of which Em is intended for the operation of said metal guide and possibly associated with a suction system.

Advantageously, the surface of the Ea end can be structured so as to define at least one series of wells to which is directly coupled at least one specific reactive group of said substrate, said Ea end being perforating.

The device according to the invention, due to the low trauma it generates in the patient, makes it possible to carry out several micro-analyzes or micro-samples from a patient at regular intervals (for example, analysis and / or samples staged in the context of prostate).

Said analyzes and / or successive samples allow CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 8 thus, in addition to the diagnosis, to follow the evolution of cancer, inflammation, infection. , a neurodegenerative disease or the successful taking of an organ transplant in a patient.

Advantageously, the functionalized end Ea may have, over a length of approximately 0.5 to 2 cm, at least one series of 1 to 25 wells, preferably 2x25 wells, having an average diameter of approximately 30 to 80 km, of preferably about 40 to 60 km, and most preferably about 50 µm, a depth of about 20 to 30 km, preferably 25 km, said wells being spaced apart from each other by about 60 at 120 km.

Preferably, the wells have a smooth or rough wall, an oval or round shape, with a flat or concave bottom.

Advantageously, the functionalized end can have as high a number of wells as possible.

The number of wells can be such that the metal guide is of a rigidity allowing the perforation of the tissues or organs.

In this embodiment, the average diameter of the functionalized end may be about 0.3-3.5mm, preferably about 0.35mm.

Advantageously, the metal guide can be structured over a length of 0.5 to 2 cm, preferably about 1 cm, to form wells.

Advantageously, the diameter of the wells can be about 30 to 80 tm, preferably about 30 to 60 km, for example Km.

Advantageously, the depth of the wells can be approximately 20 to 30 km, preferably approximately 25 km.

The spacing of the wells can be 20 to 120 km, preferably 25 km.

Advantageously, the functionalized end Ea may be of cylindrical, planar, spiral or modified shape from CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 9 so as to increase the surface area of ​​the wells in contact with the them. fluids, tissues and cells.

According to a particular embodiment of the device according to the invention, said device further comprises a removable protection system at the functionalized end Ea.

According to yet another particular embodiment of the device according to the invention, said device further comprises a medical instrument having an internal lumen in which said at least one metal guide can slide.

By metal guide is meant for example a flexible solid metal rod or a rigid hollow metal rod, having a diameter ranging from 0.2 to 3.5 mm and a length ranging from 5x10-2 to 2 m, and can be inserted into a blood vessel, a small cavity or through an organ or tissue, so that it can be directed from the insertion site to the site of microanalysis and / or micro-sampling in situ.

In particular, a flexible solid metal rod may consist of an optical fiber at one end Ea of which is associated a metal ring provided with at least one series of wells to which is directly coupled at least one specific reactive group of said substrate, said substrate. functionalized end Ea being perforating.

Advantageously, said metal ring has a width of approximately 0.5 to 2 cm, and may have at least one series of 1 to 25 wells, preferably 2x25 wells, having an average diameter of approximately 30 to 80 km, preferably of about 30 or 40 to 60 km, and very preferably of about 50 km or 35 km, a depth of about 20 to 30 km, preferably CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 25 m, said wells being spaced from each other by about 20 to 120 m, for example about 60 to 120 m.

Preferably, the wells have a smooth or rough wall, an oval or round shape, with a flat or concave bottom.

5 By metal guide is also meant a flexible or hollow, rigid or flexible solid rod made entirely or in part of a metal alloy whose characteristics of flexibility, rigidity, oxidation and immunogenicity are compatible with such a: use in living beings and in particular in animals, and most particularly in humans.

Such biocompatible alloys can be identified simply by a person skilled in the art with regard to his general knowledge and in particular include stainless steels, alloys based on titanium, nickel, cobalt, or mixtures thereof.

The inventor has been able to demonstrate that a guide based on a titanium and nickel alloy (Nitinol alloy) exhibited particularly advantageous properties in terms of overall flexibility and rigidity at its end to be used effectively for endovascular or endocavitary routes and also to efficiently perforate a tissue or an organ while minimizing the trauma (the size of the perforation in said tissue or organ is for example of the order of 0.05 to 0.5 mm2, and preferably of the order of 0.07 mm2).

Advantageously, the metal guide is based on an alloy of nickel and titanium, preferably based on nitinol (metal guides marketed by the company Euroflex).

Said metal guide may be covered, à CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 11 except for the functionalized end Ea, with a hydrophilic polymer, preferably a hydrogel, or with a porous protective polymer layer, having a thickness ranging from about 0.1 to 51 m.

Advantageously, the protective polymer consists of a film of parylene, of TiO2 or of OptoDex® (Arrayon Biotechnology, Switzerland), and more preferably of a film of parylene.

The removable protection system in which the metal guide is inserted can take many forms, and in particular that of a flexible catheter, which can be determined simply by a person skilled in the art, for example forms which can be used for the endovascular or endocavitary route. , transparietal, and in particular transcutaneous.

Said removable protection system can be inserted endovascularly in particular to reach the vessels of the heart, the brain, the lung, the pancreas, the kidney and the liver.

Said removable protection system can be inserted by endocavitary access, in particular by means of an endoscope, orally, anal, urogenital and respiratory, or ENT by transmucosal route, or even transcutaneously by means of a puncture. at the level of the skin, for example to reach the mammary gland, and in particular up to the kidney, in a joint, in the spinal canal, by lumbar puncture or transparietally in the liver, lung or kidney, but also by transmucosal route, in particular for the digestive tract.

The inventor has thus demonstrated that the device according to the invention then makes it possible to reach tissues or organs, normally difficult to access, by the endovascular, endocavitary or transparietal routes, and CA 02716751 2010-08-23 WO 2009 / 106295 PCT / EP2009 / 001308 12 in particular transmucosal or transcutaneous, conventionally used.

More generally, the inventor has demonstrated that the device according to the invention makes it possible, because of its specific characteristics of overall flexibility and rigidity at its end, to reach and perforate by the transparietal route (transcutaneous, transmucosal), endovascular or endocavitary certain organs and tissues belonging to the digestive system from the oropharynx to the rectum (including liver and pancreas), to the urogenital system (including bladder, kidney, prostate, testis, ovary and mammary gland), the tracheobronchial system (including lung), ENT system (including ear and nasopharynx), osteoarticular system (including synovial cavities), endocrine system, neurocerebral system or integumentary system, making thus possible the realization of diagnoses on pathologies which until then required the realization of biopsies or even aggressive deep punctures such as trans-hepatic biopsies, including the collection of cells by aspiration using fine needles (a technique called fine needle aspiration or FNA).

Advantageously, said removable protection system in which the metal guide is inserted takes the form of a flexible catheter suitable for the endovascular or endocavitary route.

The device according to the invention is then particularly suitable for carrying out an investigation, for example, at the level of the arteries and veins, of the vessels of the heart, of the prostate, of the mammary gland, of the pancreas, of the kidney, of the heart muscle. , the central nervous system and its cavities or channels, the brain or the liver.

CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 13 According to a first particular embodiment of the device according to the invention, the removable protection system in which the metal guide is inserted is itself inserted into an endoscope.

The device according to the invention is then particularly suitable for administration by the endocavitary route.

The device according to the invention is then particularly suitable for carrying out an investigation, for example, at the level of the tracheobronchial system (including lung), of the digestive system from the pharynx to the rectum (including liver and pancreas), of the urogenital system (including bladder, kidney, prostate, testis, ovary and mammary gland), the ophthalmic system (tear ducts), the ENT system (including the ear and nasopharynx), the osteoarticular system, or at the level of the central nervous system, especially by endorachidian route or mammary gland by endogalactophoric route.

According to a second particular embodiment of the device according to the invention, the metal guide consisting of a fine needle for transparietal puncture, and in particular for transcutaneous or transmucosal puncture, can be inserted into a removable protection system, for example a flexible catheter. .

The device according to the invention is then particularly suitable for administration specifically by the transcutaneous or transmucosal route.

The device according to the invention is then particularly suitable for carrying out an investigation, for example, at the level of the integuments (skin, scalp, etc.), of the breast, of the kidney, of the lung, of the liver, of the muscle, of the the osteo-muscular or osteo-articular apparatus, of the central or peripheral nervous system, or of the endocrine glands (in particular thyroid, parathyroid, adrenals, testes, CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 14 glands mammaries or ovaries).

As regards the functionalized Ea end of the device according to the invention, it is provided with at least one series of wells to which reactive groups specific to a substrate to be tested are directly coupled.

By specific reactive groups is meant for example a nucleic acid sequence [DNA (amplified, gene fragment, EST, SNP) or RNA] complementary to a nucleic acid sequence to be detected, an antigen specific for an antibody in detecting or an antibody or an antibody fragment specific for an antigen to be detected, preferably an antibody or an antibody fragment.

Advantageously, said specific reactive groups are placed in the microwells of the functionalized end of the device according to the invention according to an increasing or decreasing range. Those skilled in the art can easily determine, using their general knowledge and routine experiments, said range as a function of the affinity of the reactive group for its substrate. By way of example, the range of the reactive group is of the order of 50 to 500 ltg / ml, preferably from 10 to 100 μg / ml, for a reagent, in particular an antibody, having an affinity for its substrate, in particular an antigen, of the order of 10-9.

The term “antibody” preferably means an immunoglobulin of a living being, of a mammal, in particular a human being, and particularly preferably an IgG.

By antibody fragments is meant antibody fragments capable of maintaining specific binding of their antigen. By way of example of such antibody fragments, mention may be made of the fragments Fab, Fab ', F (ab') 2 CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 or Fv.

Methods of coupling reagents, in particular proteins on a metal support are well known to those skilled in the art.

Such methods, due to the low chemical reactivity of amino acids, generally require the activation of the metal surface either by oxidation mechanisms or by covering the latter with at least one layer of binding molecules, most often polymers (this may for example have thiol groups, carboxylic acids and / or amines). By way of example of such methods, mention may be made of the adsorption on metal supports of functional molecules organizing themselves in self-assembled monolayer SAMs and in particular the alkanethiols (see in particular WITTSTOCK and SCHUHMANN, Anal.

Chem., Vol. 69, p: 5059-5066, 1997; and international application WO 03/006948) or pyrrole (By electrochemical polymerization of biotinylated pyrrole; DUPONTFILLIARD et al., Anal.

Chim.

Acta., Vol. 449, p: 45-50, 2001).

By coupling the antibody or antibody fragment to this layer of functional molecules is meant a covalent bond (such as disulfide bridges between the free thiol groups of alkanethiols) or a non-covalent bond (such as a streptavidin-biotin bond between biotin a layer of polymers of a pyroll / biotin complex and streptavidin of a streptavidin / antibody complex or antibody fragment) streptavidin).

According to a first preferred embodiment, the device according to the invention comprises at least one metal guide, one end of which Ea is coupled with at least one reactive group, preferably an antibody or a CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 16 antibody fragment, specific for a marker (antigen) for cancer, in particular breast, ovarian, prostate, colon, intra-abdominal, kidney liver cancer , lung, pancreas, the central or peripheral nervous system or an endocrine gland (especially thyroid, testes or ovaries).

As an example of a breast cancer marker, mention may be made of the CA 15-3 marker (Carcinoma-Associated Antigen 15-3; Duffy MJ, Shering S, Sherry F, McDermott E, O'Higgins N, Int J Biol Markers, 2000 Oct-Dec; 15 (4): 330-3), CA 2729 (Carcinoma-Associated Antigen 27-29; Kaohsiung J, J Med Sci, 1999 Sep; 15 (9): 520-8), CEA ( Carcinoembryonic antigen; Soletormos G, Nielsen D, Schioler V, Mouridsen H, Dombernowsky P, Eur J Cancer, 2004 Mar; 40 (4): 481-6);

TPA (Tissue Polypeptide Antigen); TPS (Tissue Polypeptide Specific Antigen; Given M, Scott M, Mc Grath JP, Given HF, Breast, 2000 Oct; 9 (5): 277-80), HER2 (Fehm T, Jager W, Kramer S, Sohn C, Solomayer E, Wallwiener D, Gebauer G., Anticancer Research, 2004 May-Jun; 24 (3b): 1987-92), ER (Estrogene Receptor; Platet N, Cathiard AM, Gleizes M, Garcia M., Crit Rev Oncol Hematol, 2004 Jul; 51 (1): 5567), PR (Progesterone Receptor; Duffy MJ, Clin Chem, 2005 Mar; 51 (3): 494-503.

Epub 2005 Jan 6.), Ki-67 (cell proliferation-associated antigen of antibody Ki-67;

Schlüter C, Duchrow M, Wohlenberg C, Becker MH, Key G, Flad HD, Gerdes J, J Cell Biol, 1993 Nov; 123 (3): 513-22) and UPA (Urokinase Plasmogen Activator; Duffy MJ, Crit Rev Clin Lab Sci, 2001 Jun; 38 (3): 225-62).

As an example of a marker for ovarian cancer, mention may be made of the marker CA125 (Carcinom Antigen 125; Moss EL, Hollingworth J, Reynolds TM, J Clin Pathol, 2005 Mar;

58 (3): 308-12), CA 15-3 and CEA (Valenzuela P, Mateos S, CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 17 Tello E, Lopez-Bueno MJ, Garrido N , Gaspar MJ, Eur J Gyn Oncol, 2003; 24 (1): 60-2).

As an example of a prostate cancer marker, mention may be made of the PSA marker (Prostate-Specific Antigen;

Gray MA, Clin Lab., 2005; 51 (3-4): 127-33); PMSA (ProstateSpecific Membrane Antigen) and AR (Androgen Receptor; Birtle AJ, Freeman A, Masters JR, Payne HA, Harland SJ, BJU Int, 2005 Aug; 96 (3): 303-7).

As an example of a colon cancer marker, mention may be made of the CEA marker (Duffy MJ, Clin Chem, 2001 Apr;

47 (4): 624-30), CA 19-9 (Carcinom Antigen 19-9), CA242 (Carcinom Antigen 242), CA 72-4 (Carcinom Antigen 72-4), TPA, TPS (Duffy MJ, van Dalen A, Haglund C, Hansson L, Klapdor R, Lamerz R, Nilsson 0, Sturgeon C, Topolcan 0, Eur J Cancer, 2003 Apr; 39 (6): 718-27).

As an example of an intraabdominal cancer marker, mention may be made of the CEA or CA 19-9 marker (Coban E, Samur M, Bozcuk H, Ozdogan M, Int J Biol Markers, 2003 Jul-Sep;

18(3) : 177-81).

As an example of a marker for pancreatic cancer, mention may be made of the marker TA90-IC (a 90-kDa immugenic Tumorassociated Antigen), CA-19-9 (Chung MH, Gupta RK, Bilchik AJ, Ye W, Yee R , Morton DL, Curr Surg, 2002 MarchApril; 59 (2): 194-198), TPS, HCG beta (hCG beta, Human Chorionic Gonadotropin beta), CA 72-4, CEA, CA 19-9, CA 242 (Louhimo J, Alfthan H, Stenman UH, Haglund C, Oncology, 2004; 66 (2): 126-31).

As an example of a marker for liver cancer, mention may be made of the alpha-fetoprotein marker.

As an example of a lung cancer marker, CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 18 can cite the marker Cyfra A41 (Cytokeratin fragment 41), SCC (Squamous Cell Carcinoma antigen), ACE (Angiotensin Converting Enzyme), CA 19-9, CA 125, NSE (Neuron Specific Enolase), chromogranin A, CYFRA 21-1 (Cytokeratin fragment 21-1) CA 15-3.

According to a second preferred embodiment, the device according to the invention comprises at least one metal guide, one end of which Ea is coupled with at least one reagent, preferably an antibody or an antibody fragment, specific for a specific marker d. 'inflammation, especially rheumatoid arthritis.

By way of example of a marker for rheumatoid arthritis, mention may in particular be made of IL-13, IL-1Ra, IL-2, IL-2R, IL-4, IL -5, IL-6, IL7, IL8, IL10, IL12p40P70, IL-13, IL-15, IL-17, TNFa, IFNa, IFNγ, GMCSF, MIP-1, IP-10, MIG, Eotaxine, RANTES and MCP-1 (COCKRUM et al., Lab Automation, BTi, October 2005, p: 19- 21).

According to a third preferred embodiment, the device according to the invention comprises at least one metal guide, one end of which Ea is coupled with at least one reagent, preferably an antibody or an antibody fragment, specific for a specific marker d. an infection, in particular a viral, bacterial or parasitic infection.

Many infectious markers are known to those skilled in the art and the latter will be able to identify without difficulty the specific marker or markers associated with a given infection.

According to a fourth preferred embodiment, the device according to the invention comprises at least one metallic guide CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 19, one end Ea of which is coupled with at least one reagent, of preferably an antibody or an antibody fragment, specific for a specific marker of transplant rejection.

Many markers of graft rejection are known to those skilled in the art. By way of example, mention may be made of MIP-113 and VE-cadherin for heart grafting (ROUSSOULIERES et al., Circulation, vol.111 (20), p: 2636-2644, 2005).

With regard to the specific markers which are described above, a person skilled in the art with regard to his general knowledge will be able to identify easily and without excessive experimentation the antibodies or fragments of specific antibodies which can be used in the device according to the invention. By way of example, such antibodies, there may be mentioned the antibodies available from TEBU or AXXORA. Those skilled in the art will also be able to obtain such antibodies by well known immunization methods.

Likewise, a person skilled in the art will be able to identify without difficulty the suitable specific nucleic acids which can be used in the device according to the invention.

According to a fifth preferred embodiment, the device according to the invention comprises at least one metal guide, one end of which Ea is coupled with at least one reagent, preferably an antibody or an antibody fragment, specific for a specific marker or d. 'a set of specific markers for neurodegenerative pathologies, such as, for example, Alzheimer's disease (AD), Parkinson's syndrome, amyotrophic lateral sclerosis (ALS), this list not being exhaustive.

CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 Several markers are known and used by those skilled in the art for the study of these pathologies. By way of example, there may be mentioned for dementia or predemential states, the total Tau protein (MAPT-Microtubule Associated Protein 5 Tau), the Amyloid peptide ABETA1-42, the hyperphosphorylated Tau proteins (P Tau phosphorylated in 128), described ar example by Waldemar G., Dubois B., Emre M. et al, Eur.J. Neurol., 2007,14, pp 1-26; Dubois B., Feldmann HH, Jacova C., Dekosky ST et al, Lancet Neuro1.2007,6, pp 10 734-746; Krolak-Salmon P. et al., Towards a biological diagnosis of Alzheimer's disease and syndromes related, The Journal of Internal Medicine (2008), doi: 10.1016 / J. revmed. 2008.01.029.

These markers can be assayed by an ELISA.INNOTEST B-Amyloid type Antigen-Antibody assay technique (1-42), INNOTEST hTAUAg, INNOTEST PHOSPHOTAU (181p); Innogenetics, Ghent, Belgium.

Other markers also make it possible to test the state of degradation of the brain and in particular the Visin-like protein (VLP6 or VILIP-1 or VSNL), as described for example by Ref: Lee JM et al, Clin Chem , 2008,54, pp 1617-1623.

These markers can be used in the practice of the present invention.

For Parkinson's disease and multisystem atrophies (synucleopathies), mention may be made of the marker, Alpha-SYNUCLEINE (Mollenhauer B., Cullen V., Khan I., Experimental Neurology, 2008,213, pp 315-325).

Finally, there are also non-specific markers of conditions affecting the central nervous system (CNS), which can be used in the context of the present invention.

These are proteins from the CNS such as the GFAP protein, myelin, neuropeptides and neurotransmitters.

The BNDF protein (brain derived neurotrophic CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 21 factor), more particularly described in the document University of California San Diego Medicine health / diseases February 200, is of particular interest 9 .

In addition, immune response proteins, e.g. IgG, Albumin, Complement Proteins, Reactive C Protein, as well as inflammation proteins, such as e.g. Transferrin, Haptoglobin, Ceruloplasmin, Lysozyme, Enolase, can be used. in the implementation of the invention.

Whatever the embodiment of the invention, several different markers, that is to say markers for different pathologies, can be placed in wells of the same metal guide.

In this case, the markers can be placed in wells such that the different markers do not interact with each other.

Advantageously, the device according to the invention, at least in its functionalized end part in contact with the substrate to be analyzed, has a sterility assurance level (SAL for Sterility Assurance Level) of the order of 10-6.

Different alternatives are possible in order to achieve this level of sterility.

One possibility is to sterilize the device in the absence of reactive groups specific for the substrate to be detected and then to add the latter under sterile conditions.

Another possibility is to sterilize the device after the addition of the reactive groups, which requires the use of sterilization techniques that do not significantly reduce the activity of said reactive groups (for example sterilization by ethylene oxide or radiation) .

A second object of the invention is a method of CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 22 ex-vivo detection of a substrate present in a tissue or organ, characterized in that it comprises the following steps:

a) incubation of the functionalized end Ea of a device according to the invention, with a solution comprising at least one detection agent specific to said substrate, after said end has been brought into contact with said tissue or organ to be examined.

b) detecting said substrate.

The incubation step is carried out for a time sufficient for the detection agent in solution, in particular an antibody, to be able to bind specifically to the substrate (label, antigen, antibody, etc.), in particular u antigen, optionally. present at the end of the device. Those skilled in the art can simply determine, using their general knowledge and routine experiments, this incubation time as a function of the affinity of the detection agent in solution, in particular an antibody, for its substrate. , in particular an antigen.

This incubation time is also a function of the temperature of the solution during this incubation. by way of example, the incubation time is of the order of 1 minute to 2 hours, preferably 5 minutes to 1 hour, and particularly preferably 10 to 30 minutes, for a temperature between 20 ° C. (room temperature) and 37 C.

Advantageously, the detection agent in solution is different from the specific reagent coupled to the functionalized end of the device according to the invention.

Preferably, the detection agent is an antibody.

Advantageously, the antibody in solution and the antibody coupled to the functionalized end of the device according to CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 23 the invention are each a polyclonal antibody, preferably said antibodies are the same.

Advantageously, the antibody in solution and the antibody coupled to the functionalized end of the device according to the invention are each a monoclonal antibody, preferably said antibodies are different.

Advantageously, the antibody in solution is labeled, and in particular is coupled to an enzyme, for example to peroxidase or to alkaline phosphatase.

According to a first particular embodiment of the method according to the invention, the method further comprises a step a ′) of incubating said end in a solution comprising at least one detection agent specific for the detection agent of the. step a), inserted between steps a) and b).

Those skilled in the art will be able to identify, simply and using their general knowledge, the antibodies suitable for the process according to the invention. By way of example, it is possible to use in this second step an antibody which specifically recognizes mouse immunoglobulins, if such mouse immunoglobulins directed specifically against the substrate (marker, antigen, antibody, etc.) to be identified are used in step a).

According to a second particular embodiment of the method according to the invention, the method according to the invention comprises a washing step following the incubation step a), and optionally from step a ′), which step The wash eliminates antibodies that have not specifically bound to the marker (the antigen).

The protocol for such a washing step is again part of general knowledge or can be determined simply by routine experiments. by way of example, such a step is carried out with a solution comprising a more or less high concentration of detergent (from 0.05 to 1%), such as TRITON noce or TWEEN 20 @, and this depending on the affinity of the antibody in solution for its specific antigen.

The detection step is carried out by demonstrating an activity, in particular enzymatic, coupled to the antibody used in step a) or, optionally in step a ′).

The protocol used for this detection step depends on the label used, and in particular on the enzyme used, for example peroxidase and alkaline phosphatase, and is part of the general knowledge of those skilled in the art.

This detection step makes it possible to deduce the quantity of specific substrate (for example antigen) attached to the functionalized end of the device and finally the quantity of specific substrate present at the level of the organ or tissue where (e ) micro-analysis and / or micro-sampling.

Finally, the various reactive groups that can be used to carry out the method according to the invention are well known to those skilled in the art and include, in particular, the reagents used for the immunoenzymatic assay technique or by immunofluorescence, for example on a solid support [ELISA technique , protein chip (ESPINA et al., cited above, 2004)].

A third object of the invention consists in the use of a device according to the invention, for CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 manufacture of a tool intended for the diagnosis of a cancer, inflammation, infection, transplant rejection or neurodegenerative disease in a patient.

According to a particular embodiment of the invention, said diagnostic tool may comprise at least one metal guide inserted into a flexible catheter inserted into an endoscope.

According to another particular embodiment of the invention, said diagnostic tool may comprise at least one metal guide consisting of a needle for transparietal puncture, and in particular for transcutaneous or transmucosal puncture, which can be inserted into a removable protection system, for example a flexible catheter.

In addition, the removable protection system and the metal guide cooperate so as to allow the functionalized end Ea of said guide to be brought into contact with the microanalysis and / or micro-sampling site.

In these two particular embodiments of the invention, said at least one metal guide can be associated over at least part of its length with an optical fiber with a view to marking and positioning.

Advantageously, said diagnostic tool is administered by the endocavitary route.

Said tool thus makes it possible to perform a micro-analysis and / or a micro-sample at the level of the digestive system from the pharynx to the rectum (including liver and pancreas), of the urogenital system (including bladder, urethra, kidney, prostate), the tracheobronchial system (including lung), ENT system (including ear and nasopharynx), osteoarticular system (including synovial cavities).

CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 26 Preferably, said diagnostic tool is administered by the transparietal route, in particular by the transmucosal or transcutaneous route.

Such a diagnostic tool thus makes it possible to analyze tissues or organs that are difficult to reach by the endocavitary or endovascular route usually used.

Such a diagnostic tool thus makes it possible to carry out a microanalysis and / or a micro-sample by the transparietal route from the skin, testes, prostate, ovary or mammary glands, but also from the kidney or the breast. liver, the peripheral nervous system as well as the central nervous system, in particular by the endo-spinal route as well as the endocrine system (for example the thyroid).

The examples which follow make it possible to illustrate the invention and are given without limitation.

Brief Description of the Figures - Figure 1 shows different possibilities for structuring the metal guides.

- Figure 2 represents scanning electron microscopy photographs of various holes made by FIB (the high surface roughness due to etching inhomogeneities can be observed).

- Figure 3 shows an explanation and an observation of the shading effect of the fluorite-assisted milling technique.

- Figure 4 shows the continuation of the micro-forming operations.

- Figure 5 represents (a) hemispherical holes observed by optical microscope (b) series of holes CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 27 observed on a Nitinol-based guide by electron microscopy scanning (c) detail of a cavity (d) comparison of the surface roughness in the cavity with the roughness of the surface of the guide.

- Figure 6 represents scanning electron microscopy photos showing the same hemispherical hole as in Figure 5 after the electrochemical polishing treatment.

- Figure 7 represents a diagram of the device of the invention for the immunocapture with a first monoclonal antibody (mAb 1) and the revelation of the ACE antigen with a second monoclonal antibody (mAb 2).

- Figure 8 represents the results of ELISA with the capture antibody 5910 and the revelation with the antibody 5909 (absorbance obtained with a serum positive for ACE antigen) - Figure 9 represents the results of ELISA with the antibody 5910 capture and revelation with 5909 antibody (absorbance obtained with serum negative for ACE antigen) - Figure 10 represents the results of ELISA with 5905 capture antibody and revelation with 5909 antibody (absorbance obtained with serum positive for ACE antigen) - Figure 11 represents the results of ELISA with capture antibody 5905 and revelation with = antibody 5909 (absorbance obtained with serum negative for ACE antigen) CA 02716751 2010-08 -23 WO 2009/106295 PCT / EP2009 / 001308 28 - Figure 12 represents the results of ELISA of the rigid plastic rods with the capture antibody 5910 and the revelation with the antibody 5909 (absorbance obtained with a serum positive for antigen ACE) These figures are illustrations of the sensitivity and specificity of the method used.

= EXAMPLE 1: PREPARATION OF A METAL GUIDE BASED ON NITINOL AND ITS ACTIVATION The surface of a metal guide based on Nitinol (Euroflex) is structured in order to define places, for example wells, where the reactive groups will be deposited and where the biochemical interactions will take place (Figure 1).

Said wells can be produced by various methods such as for example by focused ion beam lithography (FIB or for Focused Ion beam: Xie et al., Nuclear Instruments & Methods in Physics research Section B-beam Interactions with Materials and Atoms, 211 (3): 363-368, 2003), by laser lithography followed by electrochemical etching and laser ablation.

With the FIB technique, the machine creates an ion beam, which is focused on the surface to be structured.

Under the mechanical action of the ion beam, atoms of the surface material are removed from the surface.

Holes with a diameter of 20 one can be formed with the FIB technique within a reasonable time with a bite factor of 8 µm3s-1 under a beam current of 20 nA.

Figure 2 shows holes with diameters of 5, 20 and 40 µm with a depth of 10 and 20 µm.

The surface of the bottom of the hole is rough due to the re-deposition of the material sprayed during the attack.

The bite factor was measured at CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 29,200 nm min-1 over a circular area of ​​40 μm in diameter and with a beam angle of 20 nA.

This gives a bite factor of 0.2 [1m3 nC-1 (approximately 5 111rt3s-1), which corresponds to a procedure time of 20 min to make a hole 20 pri in diameter and 20 wrt in depth.

In order to improve the roughness of the surface, a fluorite-assisted milling technique (XeF2) was used; a very low surface roughness was then obtained, but as the source of XeF2 was not exactly in the axis of the etching beam, a shading effect was observed (Figure 3).

The technique of laser lithography and electrochemical etching consists in a first step in covering the surface with a layer of polymer.

In a second step, the polymer layer is shaped using laser ablation.

In a third step, the surface is etched using isotropic electrochemical etching through the opening made in the polymer layer (Figure 4).

FIG. 5 shows the results of various structuring tests on metallic guides based on Nitinol.

Moreover, the metallic guides based on Nitinol which are used in vivo are usually treated by electrochemical polishing, which replaces the layer of native NiTi oxide with a biocompatible TiO2 layer.

Guides shaped with holes are subjected to this process in order to assess the influence of the process on the structure of the holes (Figure 6).

Another way to prepare the cavities on the surfaces of the Nitinol-based guides is through laser ablation. The use of short laser pulses allows local evaporation of the metal without affecting the surrounding metal CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 due to the heat generated.

The smallest reported dimensions are of the order of 20 mrt.

If the three methods described above allow the production of wells, it is the method by electrochemical etching which gives the best results.

EXAMPLE 2: TRAUMA CONSECUTIVE TO THE IN VIVO INSERTION OF A METAL GUIDE IN A PARTICULAR BODY For these experiments, metal microguides (MTI 0.012 "Silver speed) were used, which metal guides were inserted into microguides. catheters.

The device was introduced into pigs, under general anesthesia, at the level of a puncture and then at the level of the scarpa to the kidney by the endovascular route (via the femoral artery).

This guidance was ensured by monitoring said device in the femoral artery by arteriography.

Once positioned at the entrance to the kidney, the device was introduced into the kidney by endoarterial invasion.

This penetration into the tissue was made to a depth of a few millimeters and said device was kept there for about ten minutes.

Finally, the device was then removed.

The animals were then euthanized and the kidneys of the latter were removed to assess the state of the latter after penetration of the device according to the invention.

25 The results showed that no significant kidney bleeding was associated with the break-in.

The largest lesion observed had a dimension of 3 x 1 mm at the site of the breach.

The device according to the invention therefore makes it possible to access an organ while being very slightly invasive.

EXAMPLE 3: MICRO-TRAUMATISM CONSECUTIVE TO THE INSERTION OF THE DEVICE AT THE LEVEL OF THE LIVER Metal micro-guides (MTI 0.012 "Silver speed) were used, which metal micro-guides were placed in a fiberscope unlike the example. 1.

The device was introduced in pigs, under general anesthesia, at the level of a puncture at the level of the scarpa and then to the liver by endoarterial navigation (via the femoral artery).

This guidance was provided by monitoring said device in the femoral artery by arteriography.

Once positioned near the liver, the device was introduced into it.

This penetration into the tissue was made to a depth of a few millimeters and said device was kept there again for about ten minutes.

Finally, the device was then removed.

As before, the liver sample after the operation made it possible to judge the aggressiveness of the intervention on the organ.

No macroscopically visible lesions were observed on the surface of the liver. On slice, the presence of two intra-parenchymal hemorrhagic foci of subcapsular site of 1.5x0.4 cm and 1.8x0.5 cm were observed.

Histologically, the hepatic architecture is preserved at all points with congestion of the sinusoids, portal venules and centrilobular veins without any other notable abnormality.

Conclusion CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 32 The results showed that the hemorrhagic lesions are minimal: two minor macroscopic lesions could be observed without any destruction of the parenchymal cells and with simple congestion of the capillaries and centrilobular veins.

The use of a metal guide therefore makes it possible to obtain a minor trauma and, in all cases, considerably less than that resulting from a biopsy.

EXAMPLE 4: STUDY OF THE PARAMETERS FOR THE DESIGN AND REALIZATION OF A DEVICE ALLOWING IMMUNOCAPTURE AND DETECTION OF ACE IN VITRO ANTIGEN ON SOLID SUPPORTS The device uses the principle of the ELISA technique to highlight the antigen ACE.

Two monoclonal antibodies recognizing different epitopes on this antigen were used for the capture (mAb1) and the revelation of the ACE antigen (mAb2).

These monoclonal antibodies having the same isotype (IgG1), the revelation of the ACE antigen was carried out using a monoclonal antibody coupled to biotin and a streptavidne-peroxidase complex (Figure 7).

Two types of support were used, either plates for ELISA, or rigid plastic rods.

Plates for ELISA (Greiger) 100 tl of a monoclonal antibody directed against the ACE antigen (clone 5910 or clone 5905, produced in mice and marketed by Medix Biochemical) diluted (1/5000 to 1/128000) in carbonate buffer / bicarbonate were CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 33 deposited per well, and the plate was placed for 1 hour at 37 C. A negative control was performed by replacing the antibody with carbonate / bicarbonate buffer.

After three washes with 250 L per well of PBS the free sites of the plate were saturated with 200 ml of 3% PBSBSA (bovine serum albumin) for 2 hours at 37 C.

The wells were then washed three times with 250 1 of PBS-0.5% Tween before the addition of 100 4 per well of a positive serum in ACE antigen diluted to 1/10, 1/100, 1/1000 in PBS-Tween, and the plate was incubated for 1 hour at 37 C.

Three washes of 250 4 per well were carried out in PBS-Tween before the addition of 100 4 per well of a monoclonal antibody directed against the ACE antigen (clone 5909 produced in mice and marketed by Medix Biochemical, which differs from previous capture antibodies used by its affinity constant and by the recognized epitopes) biotinylated at 1/500 in PBS-Tween, and the plate was again incubated for 1 hour at 37 C.

After three washes with PBS-Tween, 100 4 of streptavidin conjugate coupled to peroxidase diluted to 1/2000 were added to each well and incubated for 1 hour at 37 C.

After three washes with PBS-Tween, the visualization was carried out by adding 200 4 per well of the substrate (H202) and chromogen (OPD, Sigma) mixture in citrate-phosphate buffer (pH 5).

In parallel, the same operation was carried out using as antigen, a serum from a normal patient (negative control with a dosage of ACE <5 IU / ml).

CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 34 The reaction was then stopped by adding 50 µl of 1M sulfuric acid per well. The absorbance was read at 492 nm on a plate reader (ref: ELX.800 UV).

The results obtained using the 5910 monoclonal antibody for the immunocapture (diluted to 1/500 then half and half to 1/128000) and the revelation by the biotinylated monoclonal antibody 5909 are presented in Figure 8 for the serum positive for ACE antigen, and in Figure 9 for serum negative for ACE.

The results obtained using the monoclonal antibody 5905 for the immunocapture (diluted to 1/100, 1/200, 1/500 then half and half until 1/32000) and the revelation by the biotinylated monoclonal antibody 5909 are shown in Figure 10 for the serum positive for ACE antigen, and in Figure 11 for the serum negative for ACE.

Legend for Figures 8 to 11:

Ordinate: absorbance (OD) at 492 min Abscissa: dilutions of the capture antibody (5910 or 5905) = = dilution of positive serum in ACE at 1/10 = = dilution of positive serum in ACE at 1/100 = dilution of positive serum in ACE at 1/1000 X = no serum The results show that serum positive for ACE antigen at 1/10 gives an absorbance (OD) greater than 0.5 when the capture monoclonal antibody is used at 1/500 (Figure 8).

Under the same conditions, the serum CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 negative for ACE antigen gives an OD of less than 0.15 (FIG. 9).

However, it should be noted that better results were obtained with the pair monoclonal antibody 5905 of capture and monoclonal antibody 5909 of detection (Figures 10 and 11) than with the pair monoclonal antibody 5910 of capture and monoclonal antibody 5909 of detection. (Figures 8 and 9).

Indeed, an OD of 1 was observed with the serum positive for ACE antigen diluted to 1/10 (Figure 10) while the serum negative for ACE antigen gives under the same conditions an OD of 0.1 (Figure 11). .

These results were confirmed using monoclonal capture antibody 5910 at various dilutions (data not shown).

Rigid Plastic Supports In a first step, rigid plastic supports in the form of rods 2-3 cm in length and 0.5-1 mm in diameter were activated.

In a second step, the supports thus activated were placed in 1 ml hemolysis microtubes (Fisher) 20 and were functionalized with a monoclonal antibody directed against the ACE antigen (clone 5910 produced in mice and marketed by Medix Biochemical ) and diluted to 1/50, 1/100, 1/250, 1/500 in carbonate / bicarbonate buffer (250 1 / tube) for 1 hour at 37 C.

A negative control was performed by replacing the monoclonal antibody with carbonate / bicarbonate buffer.

After fixing and washing, saturation was carried out with 500 4 of 3% PBS-BSA overnight at +4 C.

The supports were then incubated with 250 4 of 30 positive serum in ACE antigen diluted to 1/10, 1/100 in PBS or CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 36 with serum from a healthy subject (ACE antigen negative control) at the same dilution for 1 hour at 37 C.

A monoclonal antibody directed against the ACE antigen (clone 5909 produced in mice and marketed by Medix Biochemical, which differs from clone 5910 by its affinity constant and by the recognized epitopes) purified, at 1 mg / ml, was dialyzed. overnight at 4 ° C. against 0.1 M borate buffer pH 8.8.

A solution of biotin at 10 mg / ml in DMSO was then added at a rate of 50 μg / mg of antibody.

After incubation for 4 hours at room temperature and with stirring, 1 M ammonium chloride was added, at a rate of 20 t11 / 250 1.1 g of biotin, and the solution obtained was incubated again for 1 minute at room temperature. .

After blocking the reaction, the labeled antibody was dialyzed for 24 hours at +4 C against PBS and this labeled antibody was stored as aliquots at -20 C.

After 3 washes in PBS-Tween, the supports were incubated with 250 μl of the biotinylated 5909 antibody and diluted 1/500 in PBS-Tween for 1 hour at 37 C.

The detection of biotin (ester of 6biotinamidocaproylamido-caproic acid and Nhydroxysuccinimide, Sigma) was demonstrated using a streptavidin-peroxidase complex (Amersham Biosciences) diluted to 1/2000 in PBS for 1 hour at 37 C.

The enzymatic activity was revealed by addition of 750 III per tube of the substrate (H202) and chromogen (OPD, Sigma) mixture in citrate-phosphate buffer (pH 5).

The reaction was then stopped by adding 1M sulfuric acid. The absorbance was read at 492 nm.

CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 37 The results of ELISA on plastic rods are presented in Figure 12.

Legend for Figure 12:

Ordinate: absorbance (OD) at 492 nm Abscissa: dilutions of the 5910 capture antibody = = dilution of ACE antigen to 1/10 = = dilution of ACE antigen to 1/100 = dilution of negative control to 1/10 X = dilution of negative control at 1/100 In general, the results show that the ODs are 7 to 10 times higher with serum positive for ACE antigen than those obtained with serum negative for ACE antigen.

The best results were obtained with the rigid plastic supports on which the monoclonal capture antibody 5910 diluted to 1/50 or 1/100 was fixed.

The concentration of ACE antigen having been the best detected, corresponds to the patient's serum diluted to 1/100, i.e. 6 IU / m1 (close to the level considered to be normal:

<5U1 / ml) and when the dilution of the 5909 detection monoclonal antibody is 1/500.

The use of plastic supports makes it possible to validate the specificity and the sensitivity of the immunocapture processes on a functionalized metal rod according to the protocol described above.

Conclusion CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 38 The good results obtained for the detection of the ACE antigen with the techniques of immunocapture and in vitro revelation, validate the evaluation of the functionalized rod devices allowing the in vivo capture of the ACE antigen followed by ex vivo revelation.

EXAMPLE 5 IDENTIFICATION OF THE EXPRESSION OF THE ACE MARKER IN A BREAST TUMOR FOR EXAMPLE UNDER THE CONTROL OF IMAGING TECHNIQUES, IN PARTICULAR RADIOLOGICAL According to the protocol described in PCT application WO 03/006948, in a first step, a layer of alkanethiol is adsorbed on one of the ends of metal guides based on Nitinol (Euroflex) in a first step.

In a second step, the free thiol functions of this layer allow the formation of disulfide bridges with a monoclonal antibody directed against the ACE antigen.

The metal guide obtained is then introduced into a biopsy needle suitable for use in animals or humans.

An extemporaneous anatomopathological examination is carried out using this device on an operative specimen (mammary tumor), after its removal in a patient suffering from breast cancer.

Alternatively, when the ethical medical conditions are met, a micro-incision in the breast is performed under local or general anesthesia in a patient suffering from breast cancer.

The needle, in which is inserted the metal guide coupled to the antibody directed against the ACE antigen, is introduced into the tumor or through the micro-incision and then directed to the tumor by following its progression by imaging, and in particular by ultrasound.

CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 39 Said micro-invasive guidance system then makes it possible to exit the end of the metal guide coupled to the antibody directed against the ACE antigen. The end of the metal guide is then introduced into the tumor (by perforation) to a depth of the order of a few millimeters.

After a short waiting time, of the order of ten minutes, which allows the immunocapture of the ACE antigen possibly expressed by the tumor, the device is withdrawn.

The micro-sampling is limited to an immunocapture of the analyte in vivo and does not require a biopsy.

Finally, the device is removed and then an ELISA assay of the ACE marker is carried out on the end of the device with a monoclonal antibody directed against the ACE antigen which differs from the capture antibody by its affinity constant vis-à-vis. screw of the ACE antigen and by the recognized epitopes, and which is coupled to biotin.

The revelation of the enzymatic activity using a streptavidin-peroxidase complex makes it possible to conclude on the expression of the ACE marker by the tumor and consequently to modulate the therapy to be used to best treat the patient.

EXAMPLE 6 SKIN CANCER According to the protocol described in PCT application WO 03/006948, an alkanethiol layer was adsorbed on the end of a metal guide based on Nitinol (Euroflex) in a first step.

In a second step, the free thiol functions of this layer allowed the formation of disulfide bridges with a monoclonal antibody directed against the FAP marker (Fibroblast-activation protein; RETTIG et al., Proc.

Natl.

Acad.

Sci. USA, vol. 85, CA 02716751 2010-08-23 WO 2009/106295 PCT / EP2009 / 001308 p: 3110, 1988).

The metal guide obtained is then introduced at the level of a skin tumor in animals or in humans when the ethical medical conditions are met, or again at the level of a skin tumor in a patient suffering from cancer of the skin after its ablation for a classic or extemporaneous anatomopathological examination.

The micro-sampling is limited to an in vivo immunocapture and again does not require a specific biopsy.

10 Finally, the device is removed and an ELISA assay of the FAP marker is performed on the end of the device with a monoclonal antibody directed against the FAP marker coupled to peroxidase.

The revelation of the peroxidase activity makes it possible to conclude that the FAP marker is expressed by the tumor and consequently to modulate the therapy to be used to best treat the patient.