EP1604206A2 - Method of detecting and analysing organelles immobilised on biochips and applications thereof - Google Patents

Abstract

The invention relates to a method of detecting and analysing organelles, such as synaptic vesicles, which are immobilised on biochips. The invention also relates to a method of detecting a linkage between an analyte and a cell-wall constituent of an organelle. The invention is particularly suitable for: the detection of neurotoxins, such as botulinum toxins or tetanus toxin; the functional characterisation of organelles; the detection of tumour markers; or the screening of novel molecules which interact with specific cell-wall constituents of organelles.

Description

 METHOD FOR DETECTION AND ANALYSIS OF ORGANELLA

BIOPUCE FIXED ASSETS AND THEIR APPLICATIONS

The present invention relates to a method for detecting and analyzing organelles, in particular synaptic vesicles, immobilized on biochips. The invention also relates to a method for detecting a bond between an analyte and a membrane constituent of an organelle. The applications of the present invention relate in particular to the detection of neurotoxins, such as botulinum toxins or tetanus toxin, the functional characterization of organelles, the detection of tumor markers or the screening of new molecules interacting with specific membrane constituents of organelles .

The present invention relates in particular to the fields of medical diagnosis, pharmacology and toxicology.

The eukaryotic cell contains different structures delimited by a membrane forming intracellular compartments, called organelles.

These organelles can either remain localized in the cells or, for physiological or physiopathological reasons, be secreted or released in the extracellular media. Examples of organelles that may be mentioned include mitochondria, chloroplasts, vesicles or secretory granules, lysosornes, peroxisomes, exosomes, dexosomes, or synaptic vesicles.

The structural characteristics and composition of organelles strongly depend on their function in the cell, but also on the cell types in which they are found and on the physiological state of the cell. The assay or analysis of the structural and functional characteristics of specific organelles obtained from cells in culture or from cells taken from a biological organism is a means of obtaining information on these cells and in some cases identify physiological or pathological states of these cells.

An example of organelle whose structural characteristics are dependent or cell type analyzed and the physiological state of the cell is the synaptic vesicle. Synaptic vesicles are organelles found in large quantities in the nerve endings of neurons. Related vesicles, known as SLivlV (synaptic like microvesicle), are found in lesser amounts in endocrine and neuroendocrine tissues and in certain types of tumors. In the nervous system, these synaptic vesicles carry neuromediators and can be considered as real functional units of communication. They are around 50 nm in size and have between 20 and 40 proteins on their surface, some of which are specific to these organelles (Femandez-Chacon and Sudhôf, 1999). These specific proteins, such as for example synaptophysin or VAMP, constitute markers of choice for identifying differentiated neurons within a tissue, and are studied in fields as varied as neuronal degeneration / regeneration, synaptic plasticity or oncology. Some of these proteins are also the targets of neurotoxins, and in particular botulinum and tetanus toxins.

The detection of proteins in synaptic vesicles is commonly carried out by conventional methods in immunopathology (immunohistochemistry). More rarely, these proteins have been analyzed after tissue solubilization, qualitatively and quantitatively, by techniques such as Western Blot or ELISA (Schlaf et al., 1996). An example of a method for analyzing proteins of synaptic vesicles by Western blotting after purification is for example described by Lévêque et al., 2000 or even Quetglas et al. 2000. The result of such analyzes is generally only obtained after 24 hours and requires the use of several types of apparatus not suitable for metering applications in routine. In addition, the constraints linked to the preparation of samples containing membrane proteins, in particular the solubilization and centrifugation steps, make the ELISA technique difficult to access for routine assay.

The present invention provides a method for purifying, detecting and analyzing on a biochip, in a single step, organelles such as synaptic vesicles extracted from neuronal cells.

Biochips are tools that allow chemical or biochemical analysis of molecules previously immobilized. When the molecules immobilized beforehand are proteins, the immobilization method chosen must be appropriate to allow the maintenance of the conformation of the proteins immobilized on the biochip and their stability. For the biochemical analysis of immobilized molecules, various detection methods are known using sensors of the spectrometer, photometer, fluorimeter or luminometer type detecting molecular interactions revealed by enzymatic reaction or fluorescent labeling of a potential ligand, pharmacological, natural or synthetic. As an example, a method coupling a biochip to a biosensor capable of detecting a fluorescent signal will be cited, the method developed by the company Zyomix (Hayward CA) (Nat. Biotechnol., 2002, 20: 225-229).

When they are coupled to a sensor detecting variations in the optical signal resulting from variations in mass or molecular conformation on their surface, they allow the analysis of interactions between immobilized molecules and pharmacological ligands, natural or synthetic. Various optical detection methods capable of detecting variations in mass or molecular conformation of an analyte immobilizing on a biochip are known and include piezoelectric, optical, thermo-optical, surface acoustic wave (surface acoustic wave) methods. SAW), as well as electrochemical methods (voltametric, conductometric, potentiometric, amperometric, ...). Among the optical detection methods, we can retain, without being exhaustive, those which detect variations in mass or molecular conformation on the surface of biochips such as optical reflection methods such as ellipsometry or spectroscopy of evanescent waves. The latter method includes methods based on surface plasmon resonance, also called SPR methods. Evanescent wave spectroscopy methods have given rise to several types of biosensors on the market, including those based on the use of surface plasmon resonance and produced by different companies including

BIACORE AB (such as Biacore X, Biacore 3000), Texas Instruments (such as the Spreeta biosensor), Windsor Scientific IBIS. Or Quantec DP, and those based on frustrated angle reflection (FTR) and produced by IASYS. These techniques are described in detail in the state of the art (Biotechniques, 1991, 11: 620-627;

Methods, 2000, 22: 77-91; Nature Biotechnology, 2002, 20: 225-229).

In the following text, unless specifically stated, the term “optical detection” or “detection of variation of optical signal” means any method allowing the detection of an optical signal, including variation of chemiluminescence, of fluorescence , or even the detection of a variation in mass or molecular conformation by optical reflection, for example by surface plasmon resonance (SPR).

By the term "biochip" is meant any solid surface suitable for coupling with a biochemical molecule, and in particular a DNA molecule or a polypeptide, said solid surface coupled to a biological molecule, being suitable for its use in a method optical detection method, said optical detection method aimed in particular at detecting an interaction between the molecule coupled to a biochip and an analyte. By the term "biochip for biosensors by optical reflection" is meant a biochip usable in a device for detecting mass variation or molecular conformation by optical reflection.

To date, the development of biochips usable in an optical detection method, in particular by optical reflection, essentially concerned the analysis of simple molecules such as ADi, proteins, even lipids or lipid structures such as liposomes.

The inventors have now found that the organelles, and in particular the synaptic vesicles, are particularly robust structures, remaining intact for long periods (several weeks), even after having undergone various physicochemical treatments (freezing, sonication, lyophilization ) and biochemical. This stability is also observed for the vast majority of their membrane constituents. It follows from these observations that it may in particular be envisaged to carry out an assay of the synaptic vesicles and their constituents from normal or pathological tissues, using an optical detection method.

To the knowledge of the applicant, the development of biochips containing organelles immobilized on their surface has never been described. In particular, the development of biochips for biosensors by optical reflection, containing organelles immobilized on their surface, has never been described.

Furthermore, to the knowledge of the applicant, the routine use of organelles such as synaptic vesicles as a laboratory reagent, for example, as an enzyme substrate, has never been suggested. The invention provides in particular a method of preparing organelles which can be used as an enzyme substrate, capable of being incubated over a long period, more than 2 hours, or even more than 24 hours and up to at least 30 hours. incubation. The invention further provides a method for storing organelle for long-term use. The protein constituents of organelles, when analyzed directly on the intact organ, have the advantage of retaining their native structure within the biological membrane, while being analyzable on a biochip, according to the methods of the invention.

The invention also results from the observation that it is possible to immobilize organelles in a stable and specific manner on biochips for biosensors by optical reflection and to obtain an extremely sensitive quantification of these immobilized organelles, using a method of detecting mass variation or molecular conformation by optical reflection.

Within the meaning of the invention, the term "organelle" means in the following text, unless otherwise specified, an intracellular structure of a eukaryotic cell, delimited by a membrane.

Organelles are generally prepared by lysis of cells containing them.

Preferably, for the implementation of the process of the invention, these are unpurified organelles. By “intact organelle” is meant an organelle whose structure has not been altered by the preparation process. In particular, the membrane protein content was not changed.

Preferably, these are organelles of a size less than 360 nm.

The organelles can be in the native form or, where appropriate, reconstituted. The term “reconstituted organelle” means any proteolipid structure containing any one or more of the specific constituents of these organelles, and in particular a membrane protein of an organelle. The reconstituted organelles are prepared by physicochemical methods, including biochemical methods, or from cells expressing them, naturally or not, originating from sonication. of a cell expressing on its surface a recombinant protein of a native organelle, in particular a membrane protein of a native organelle.

By “device for detecting variation in mass or molecular conformation by optical reflection”, unless specifically stated, is meant within the meaning of the invention any device making it possible to detect variations in mass or molecular conformation at the surface of a biochip appropriate, based on optical reflection methods, internal or external, and in particular ellipsometry or spectroscopy of evanescent waves.

A first aspect of the invention therefore relates to a method of identifying or assaying organelles in a liquid sample, said method being characterized in that it comprises: a) coupling, to the surface of a biochip usable in an optical detection device, for example of detection of variation in mass or of molecular conformation by optical reflection, of a ligand capable of specifically binding to a membrane constituent of the organelles to be identified or to be measured, b) bringing into contact of the liquid sample with the surface of the biochip containing the coupled ligand, for example by injecting the sample into a device allowing this contacting, c) detecting a variation of an optical signal, and in particular a variation in mass or molecular conformation at the surface of the biochip by optical reflection, said detection allowing the identification and / or the determination of the organelles bound to the surface d e the biochip.

Within the meaning of the invention, the term "identification" means that the method at least makes it possible to determine whether the sample contains the organelles sought or not. The term “assay” means that the method at least makes it possible to determine whether the sample contains more or less organelles than a control sample, hereinafter indicated by the term “relative quantity organelles ”or“ relative organelle rate ”, and where appropriate, to determine the mass of organelles immobilized on the biochip and its concentration in a sample.

The first step of the method consists in preparing a biochip allowing the fixation of the organelles to be detected or measured.

The biochip chosen will naturally be adapted to the optical detection device chosen. In particular, it will be a biochip adapted to a device chosen to detect a variation in mass or molecular conformation by optical reflection. In a particular embodiment, the detection of mass variation or molecular conformation by optical reflection is based on spectroscopy of evanescent waves, in particular on surface plasmon resonance.

In this case, the biochip preferably comprises a solid metallic support, for example gold or silver. Examples of biochips for optical biosensors by surface plasmon resonance are described in patents EP044922, US 5,242,828 and US 5,436,161. Such biochips are marketed by BIACORE AB.

A person skilled in the art will select an appropriate ligand according to the organelle to be identified, the ligand having to have sufficient affinity with a specific membrane component of the organelle in order to immobilize

The organelle on the surface of the biochip when the ligand is coupled to the biochip. The ligand can be any type of molecule, and in particular a polypeptide, a polysaccharide, a polynucleotide or a lipid, or a natural or synthetic physiological or pharmacological ligand.

In a first embodiment, the ligand is an antibody or antibody fragment directed against an antigen expressed on the surface of the organelle to be identified. Step (a) of the method involves coupling the chosen ligand to the biochip. The term “coupling” means within the meaning of the invention any method aiming to allow a physical association between the ligand and the solid support constituted by the biochip. It is preferable, however, that the interaction be sufficiently stable so that the ligand does not dissociate during the implementation of the method. Preferably, the interaction is stable enough to allow repeated use of the biochip without loss of detection sensitivity.

The coupling can be direct or indirect. By "direct coupling" is meant a covalent bond between the ligand and the biochip support. Through

"Indirect coupling", a first ligand is covalently linked to the chip, a second ligand reversibly to this first ligand. According to an alternative procedure, the second ligand can be preincubated with the preparation containing the organelles before they are brought into contact with the first ligand.

As an example of indirect coupling, the first ligand, covalently attached to the biochip is an anti-Fc or anti-IgG mouse antibody which specifically binds to a second antibody capable of recognizing the organelle to be identified in the sample. . The first ligand is advantageously not very sensitive to pH shock, so as to regenerate the biochip after use by acid shock while allowing successive and reproducible immobilizations of the second ligand. An example of anti-Fc antibody not very sensitive to pH shock is sold in particular by the company BIACORE AB under the name of anti-mouse Fcγ Ref. BR-1000-50.

Preferably, the indirect coupling will be sufficiently stable so as to perform organelle binding measurements over a 24 hour period.

In a particular embodiment aimed at detecting synaptic vesicles or related vesicles expressing neuroendocrine markers, there may be mentioned as examples of ligands capable of being coupled to the biochip, the antibodies or fragments of antibodies specifically recognizing the antigens expressed on the surface of these organelles. A non-exhaustive list of antibodies recognizing antigens of synaptic vesicles includes for example the anti: -VAMP, -synaptophysin, - synaptotagmine, -SV2, the vesicular transporter of glutamate.

Advantageously, in order to detect synaptic vesicles or any organelle expressing VAMP or synaptophysin, anti-synaptophysin or anti-VAMP antibodies or one of their functional fragments will be used. These antibodies have already been described in the state of the art.

When one of these antibodies is used as a ligand, it has been advantageously found by the inventors that the biochip could also be regenerated, after use, by means of a concentrated detergent solution, for example octylglucoside. Preferably, octylglucoside is used in a 100 mM solution. This allows in particular to completely regenerate the synaptophysin / anti-synaptophysin interaction while solubilizing the membrane of the organelles. At least 40 regeneration / immobilization cycles are then possible on the same antibody coupled to the biochip, preferably from 10 to 100 regeneration / immobilization cycles. The alternative use of regeneration procedures by variation of pH and by octylglucoside allows a theoretical capacity of several hundred analyzes.

Different methods of coupling molecules, and in particular of coupling polypeptides to solid supports of the biochip type, such as silanes, polymer films, alkanethiols and hydrogels, are well known to those skilled in the art. Advantageously, the biochip contains a biocompatible porous matrix, for example a hydrogel (formed of polysaccharide such as dextran). Such a hydrogel may suitably contain hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy, or vinyl groups for covalent attachment of the ligand. In the particular application of optical reflection methods, the change in composition on the surface of a biochip, by adsorption, desorption, fixation of an analyte or release of an analyte, is associated with a change in the angle of reflection of a light reaching the surface of the biochip. Step (b) therefore consists in bringing the liquid sample into contact with the surface of the biochip containing the coupled ligand, for example by injecting the liquid sample to be analyzed into a device allowing this contacting.

The device chosen will naturally be adapted to the chosen optical detection method, for example to a method of detecting mass variation or molecular conformation by optical reflection. For the implementation of step b), a preferred example of a device is a device allowing the flow of a flow of the liquid sample to the surface of the biochip, for example by means of microfluidic circuits. Such devices are described in particular in US patent 5,313,264.

Commercially available devices are suitable devices allowing mass variation detection by surface plasmon resonance and using a microfluidic circuit for bringing the sample into contact with the biochip.

Organelles likely to be present in the sample specifically bind to ligands coupled to the biochip. The quantity of immobilized organelles is thus determined according to the method of the invention by detection of variation in mass or of molecular conformation by optical reflection, naturally using the appropriate controls to deduce from the detected interaction, the non-specific interactions (noise background).

The liquid sample can be any type of sample prepared either from cells capable of containing organelles, or from a biological medium in which they have been released, or by any process consisting in generating proteolipid structures containing proteins synaptic vesicles, in particular proteins of the membrane of synaptic vesicles, for example by induction of expression of a protein of the membrane of synaptic vesicles in non-neuroendocrine cells. In the first case, the preparation generally consists of a lysis of the cells so as to obtain a release of the organelles. In order to obtain better stability of the organelles, preferably, the organelles released into the solution are not purified after lysis of the cells. Advantageously, the solution containing the organelles for the implementation of the methods of the invention is a very dilute solution, in particular in comparison with the organelle solutions conventionally prepared in the prior art. For example, the solution containing the organelles comprises between 1 ng and 1 μg of total proteins, preferably between 10 ng and 200 ng of total proteins, for example between 30 ng and 80 ng. More particularly, as in the second or third case, it may be a sample prepared from cells of a biological organism, and in particular a sample of tissues or biological fluids such as blood, cerebrospinal fluid, urine or saliva. The sample can be taken from an animal (in particular a mammal and preferably a man) or a plant. The sample can in particular be carried out on a healthy individual or a patient suffering from a pathology. The pathology can in particular be cancer, a neurodegenerative pathology, an infectious pathology and in particular a viral, bacterial or parasitic pathology. In a sample prepared from cells of a biological organism and in particular from a tissue sample, or from a culture of cells, the ligand coupled to the biochip is chosen so as to be specific for the membrane of the organelles to be detected. in particular to avoid interactions, for example, with constituents of the plasma membrane of cells. The liquid sample can also be prepared from a cell culture. The analyzed sample can be prepared for storage, for example, by freezing or freeze-drying. In a particular embodiment, the organelles contained in the sample are vesicles of exocytosis. The term "exocytosis vesicles" means any organelle containing constituents intended to be secreted outside the cell, or comprising membrane constituents intended to be incorporated into the plasma membrane. These organelles include in particular synaptic vesicles, secretory granules (or vesicles with dense hearts) or even SLMVs.

In a preferred embodiment, the liquid sample is prepared from neural tissues for the identification and / or determination of synaptic vesicles.

After implementing the method by SPR detection, it appears that the phenomenon of capture of synaptic vesicles on the biochip is irreversible (less than 5% dissociation in 24 hours for example on an anti-synaptophysin antibody). This very strong affinity makes it possible to quantify synaptic vesicles over a very wide concentration range, which is particularly important for methods using protein biochips. A calibration line can be established to correlate the concentration of one of the components of the synaptic vesicle membrane (or the concentration of synaptic vesicles) with the signal measured on the biosensor.

The proteins in the membrane of synaptic vesicles are well-studied synaptic markers. In certain neurodegenerative diseases of the Alzheimer type (Terry et al., 1991; Sze et al., 2000), amyotrophic lateral sclerosis (Ikemoto, Acta neuro, 2002), degeneration begins with the presynaptic compartment where the vast majority are located. synaptic vesicles. In Alzheimer's disease, a good correlation has been observed between the loss of functions cognitive and decreased synaptophysin (Mucke et al., 2000, US2002040032). In epileptic rat models, a selective decrease in the number of synaptic vesicles associated with a neuronal population has been shown (Hirsh et al., 1999). Conversely, synaptogenesis studies show that the presynaptic elements are put in place before the post-synaptic complexes.

The present method can therefore be used for the diagnosis and / or prognosis of a patient suffering from neurodegenerative diseases, in particular Alzheimer's disease. It relates in particular to the dosage of synaptophysin or other proteins of synaptic vesicles contained in the sampling of tissues or biological fluids from patients suffering from neurodegenerative diseases (see in particular US2002040032).

On the other hand, it is well known that molecules like neurotrophins play a major role in survival and neuronal differentiation in many systems. It has been shown, for example, on hippocampal neurons in culture, that these molecules significantly increase the number of synaptic vesicles (Yamada et al., 2002, Collin et al., 2001). The search for new molecules having an effect comparable to neurotrophin is a major path for the discovery of new treatments against neurodegenerative diseases (see for example

US20020210837). The sensitivity and the speed of implementation of the method according to the invention can be used to demonstrate a loss (neurodegenerative disease, infection with neurotropic viruses, effect associated with the presence of synapto-toxic molecules, see patent US6 , 444,201 ...) or synaptic gain (study on neuronal regeneration, synaptogenesis, screening of anti-neurodegenerative agents ...) in cell culture systems (in particular neuron / target cell co-cultures), slices of neurons kept alive or in animals. The method of the invention is also useful in all research aimed at demonstrate synaptic changes in particular regions of the nervous system (Sze et al., 2000).

The method can more particularly be used for the screening of molecules capable of modulating the growth or the stability of cells containing synaptic vesicles, in particular neurotrophic or anti-neurodegenerative molecules. It can be used for the study of active molecules on axon formation, synaptogenesis or the maintenance of synapses (US2002644433). It can also be used to detect the presence of synaptic vesicles within an embryonic stem cell culture as well as to determine their phenotype, for example by means of anti-vesicular neurotransmitter transporter antibodies.

In another embodiment of the method according to the invention, the liquid sample is extracted from cells isolated from an individual for the purpose of identifying or assaying vesicles containing neuroendocrine markers.

The detection of neuroendocrine markers, among which is synaptophysin, by conventional histology and immunohistochemistry methods occupies an essential place in the positive and differential diagnosis of tumors. The present method advantageously makes it possible to detect and quantify in a sample containing cells originating from tumors, the neuroendocrine markers present in the synaptic vesicles, such as for example synaptophysin or a VAMP, for example VAMP2 or their isoforms. In particular, the method lends itself to the detection of synaptophysin from sections of lung tumors, for example frozen sections with a thickness of 40 μm. Also, the present invention more preferably relates to any use of the method according to the invention, for the positive and differential diagnosis of tumors.

More generally, the method according to the invention can be used to detect any variation in the relative quantity of organelles in a biological sample correlated to a treatment with a physiological or pharmacological agent or a genetic manipulation of the biological organism from which the analyzed sample comes.

In addition, the method according to the invention can be applied for the screening of molecules capable of modifying the quantity of a membrane constituent of an organelle or of modifying the capacity of a membrane constituent to bind with its natural ligand.

In particular, the method according to the invention can be used for the detection of toxins in a sample likely to contain them. For the purposes of the invention, the term “toxin” is intended to mean any molecule capable of affecting cell functions and / or viability and directed against a membrane target of an organelle, resulting in a modification of the quantity of the targeted membrane constituent or of its interaction with its natural ligand. Preferably, the method of the invention applies to the detection of neurotoxins whose targets are the membrane constituents of synaptic vesicles. Botulinum and tetanus toxins are the subject of major challenges in the medical sector (treatment of severe neurotoxic disorders which they cause, increasing use in local treatments for dystonia), the food industry (detection / research of the presence of toxins botulinum), the biotechnology sectors (tetanus vaccine is one of the main products in this sector) and the military, insofar as these neurotόxins are likely to be used in a biological weapon. These neurotoxins target the proteins of the SNAREs family (syntaxins, VAMPs, and for example SNAP23 or SNAP25), in particular syntaxin 1, VAMP2 and

SNAP25 present in the membrane of synaptic vesicles. In a preferred embodiment, the method according to the invention is applied to the detection of a botulinum toxin or tetanus toxin in a sample capable of containing it. Preferably, for the detection of neurotoxins, anti-VAMP, anti-SNAP25 or anti-syntaxin antibodies are coupled to the biochip. A sample likely to contain a neurotoxin is brought into contact with a preparation containing organelles comprising targets of botulinum and tetanus toxins on their surface, in particular synaptic vesicles, and the variation, or disappearance, of SNARE proteins, is analyzed. by bringing this treated sample into contact with these antibodies coupled to a biochip. The experiment can also be carried out using a supernatant of a lyophilized cell extract in the homogenization buffer and rehydrated.

There is currently no simple method for measuring the activity of botulinum and tetanus toxins in cell culture systems. The present method makes it possible to quickly and simply obtain a quantification of the effect of these toxins on cell cultures, in particular hippocampal neurons in culture or grain cells of the cerebellum. The method can also be used to determine the presence of these toxins in a medium supposed to contain them, to determine the safety of batches of vaccines, to screen for molecules potentially antagonistic to these toxins or to detect a titer of neutralizing antibodies in the serum from patients treated with these toxins or from immunized animals.

Biochips containing a determined quantity of organelles immobilized on their surface can, because of their remarkable stability, be themselves used as starting materials for the implementation of a method for detecting a bond between an analyte and a membrane constituent of an organelle by methods of detecting mass variation or molecular conformation by optical reflection.

A second object of the invention therefore relates to a method of preparing a biochip usable for the detection of a bond between a analyte and a membrane constituent of an organelle, said method comprising: a) coupling, on the surface of a biochip usable in an optical detection device, for example for detection of variation in mass or of molecular conformation by optical reflection, a ligand capable of specifically binding to a membrane constituent of an organelle, b) bringing a liquid sample containing organelles into contact with the surface of the biochip containing the coupled ligand, for example by injection of the liquid sample in a device allowing this contacting, for obtaining a biochip containing organelles immobilized on its surface, c) if necessary, freezing the biochip obtained in step b) and keeping it cold , followed by thawing, or lyophilization followed by rehydration, for use.

Steps (a) and (b) are implemented similarly to steps

(a) and (b) of the method of identification and determination of organelles described above. Preferably, the biochip and the device chosen for the implementation of steps (a) and (b) are suitable for use of the biochips in an optical detection device, in particular in a mass variation detection device or molecular conformation by optical reflection based on spectroscopy of evanescent waves, in particular on surface plasmon resonance.

Advantageously, the device used in step b) allows, after injection of the sample into it, the passage of a flow of the liquid sample to the surface of the biochip, for example by means of microfluidic circuits.

Examples of such devices have been described previously.

In a preferred embodiment, the organelles contained in the sample and then immobilized on the biochip are native organelles the membrane of which has remained intact after the extraction of the cells containing them, in particular retaining the same proteolipid content as the intracellular organelle before extraction.

In a particular embodiment, the organelles contained in the sample are secretion vesicles.

In a preferred embodiment, the organelles contained in the sample are synaptic vesicles. As described above, the ligand coupled to the surface of the biochip is then chosen, for example, from the group consisting of anti-synaptophysin antibodies and anti-VAMP antibodies.

The invention naturally relates to a biochip usable for the detection of a bond between an analyte and a membrane constituent of an organelle, characterized in that it is capable of being obtained by the preparation method defined above.

A biochip according to the invention, which can be used for the detection of a bond between an analyte and a membrane constituent of an organelle, is characterized in that it comprises: a) a suitable support for detecting a variation in optical signal, by example to detect a variation in mass or molecular conformation on its surface by optical reflection, b) ligands coupled to said support, said ligands being capable of specifically binding to the membrane constituent of an organelle, c) of organelles immobilized on the surface of the biochip by specific binding with said ligands.

In a particular embodiment, the support suitable for measuring a change in mass or molecular conformation by optical reflection at the surface of the biochip is chosen from a support of gold or silver. The the biochip can also comprise a layer of dextran allowing the coupling of the ligands on its surface. Preferred examples of biochips for surface plasmon resonance biosensors have been previously described.

In a preferred embodiment, the organelles immobilized on the surface of the biochips according to the invention are synaptic vesicles.

In a particular embodiment, the synaptic vesicles are immobilized on the surface of the biochip by specific binding with ligands chosen from the group consisting of anti-synaptophysin antibodies and anti-VAMP antibodies.

A third object of the invention relates to a method for detecting a bond between an analyte and a membrane constituent of an organelle, comprising a) obtaining a biochip usable in an optical detection device, for example of detection of variation in mass or molecular conformation by optical reflection and comprising organelles immobilized on its surface, b) bringing the analyte into contact with the surface of the biochip containing immobilized organelles, for example by injection of a liquid sample containing the analyte in a device allowing the analyte to come into contact with the surface of the biochip containing immobilized organelles, c) measuring a variation in optical signal, for example a variation in mass or of molecular conformation on the surface of the biochip- by optical reflection, said measurement allowing the detection of the quantity of analytes bound to the organelles immobilized on the surface of the biochip.

Advantageously, the biochip obtained in step (a) is a biochip according to the invention as defined above. The biochip is produced by the immobilization of a sufficient quantity of organelles necessary for the subsequent analysis sought. Since the quantity of organelles can be quantified by the implementation of the first method according to the invention described above, the level of immobilization of organelles can be controlled precisely according to the type of application desired.

According to a preferred embodiment, the measurement of a change in mass or molecular conformation by optical reflection is based on spectroscopy of evanescent waves, in particular on surface plasmon resonance. Preferably, the device used in step b) allows a flow of the liquid sample to pass over the surface of the biochip, for example by means of microfluidic circuits. Examples of such devices have been given above.

The method makes it possible in particular to measure the affinity of any molecule on a membrane constituent of the immobilized organelle. It makes it possible to quantify any molecule present in a sample which specifically binds to a membrane constituent of an organelle. It also makes it possible to determine the presence or absence of a specific membrane constituent of an organelle by detection of a bond between said membrane constituent and a known ligand thereof.

Any type of analyte can be studied. It may in particular be a molecule or a supramolecular complex. By way of examples, the analyte can be a protein, DNA, a lipid, a sugar, and in particular an antibody or antibody fragment, an enzyme, a hormone, a neuromediator, a receptor, a substrate for enzyme, lectin, toxin or pharmacological agent. The analyte can also be included in a biological sample. More particularly, it may be a sample from a tissue or biological fluid sample such as blood, cerebrospinal fluid, urine or saliva. The sample can be taken from an animal (in particular a mammal and preferably a man) or a plant. The sample can in particular be carried out on a healthy individual or a patient suffering from a pathology. The pathology can in particular be cancer, a neurodegenerative pathology, an infectious pathology and in particular a viral, bacterial or parasitic pathology. The analyte can in particular be antibodies directed against membrane constituents present on the surface of synaptic vesicles. They may in particular be anti-phospholipid antibodies. These antibodies can be generated during an autoimmune reaction.

The analyte can also be included in a culture of eukaryotic or prokaryotic cells, bacteria, fungi or yeasts.

The analyte can also be included in an agrifood product, and in particular seeds, fruits, cereals or cooked foods.

Preferably, the analyte can be complexed with high molecular weight complexes so as to increase the mass of the injected molecule and thus increase the sensitivity of the measurement. For example, the analyte can be integrated into artificial liposomes.

In particular, it is possible to repeat steps (b) and (c) with different concentrations of the same analyte making it possible to obtain the binding affinities of different analytes on the same organelle.

The method of the invention as defined above can therefore be applied to the screening of molecules capable of specifically binding to a membrane constituent of an organelle, in particular agonists or antagonists of ligands of membrane receptors.

By way of examples, a sample containing various candidate molecules capable of specifically binding to a membrane constituent will be injected followed by a ligand known to bind specifically to said membrane constituent. Partial inhibition of ligand binding will significant of the presence of a new ligand among the candidate molecules injected.

In addition, the present method also allows the purification of new ligands specifically immobilized on an organelle and their subsequent analysis, for example by mass spectrometry.

The method targets in particular the screening of new ligands of neuroendocrine markers for the molecular diagnosis of these markers. In a preferred embodiment, the analytes tested are antibodies or antibody fragments directed against a membrane constituent of an organelle, preferably of a secretion vesicle and in particular of synaptic vesicles. In particular, these are monoclonal antibodies.

Another use of the method described above relates to the screening of molecules capable of modifying the quantity of a membrane constituent of an organelle or of modifying the capacity of a membrane constituent to bind with its natural ligand. The molecules sought may, for example, be compounds capable of modifying the post-translational structure of the membrane constituents, such as kinases or phosphatases. By way of example, the method makes it possible to compare the binding affinity of a natural ligand with a membrane constituent of the organelle immobilized on the biochip and the affinity of the same ligand injected with or after the injection of a molecule capable of modifying the interaction of said ligand with the membrane constituent of the organelle. Thus, in a particular embodiment, the method comprises, prior to the injection of a ligand of a membrane constituent (the analyte), the injection of a compound capable of modifying the interaction of said ligand with the membrane constituent- of

I'organelle.

The repetition of the binding measurements of different analytes can be applied to the characterization of the organelles contained in a sample, said analytes being chosen from known ligands of constituents. organelle membranes. By “characterization” is meant the determination of the presence on the same organelle of different known membrane constituents and, where appropriate, the evaluation of the relative proportion of each constituent analyzed. These differential profiles are established for example in the context of the development of personalized medicine, in particular for the specific treatment of cancers and neurodegenerative diseases. The invention makes it possible in particular to establish such a characterization, from lyophilized organelles, and in particular from lyophilized synaptic vesicles.

This characterization can make it possible to identify, if necessary, pathological conditions, induced or natural genetic modifications or pharmacological treatments. Examples of modifications of the membrane constituent content of synaptic vesicles are described for example by Shimohama et al., 1998.

By way of example of analytes which can be used for the characterization of organelles, in particular synaptic vesicles, use may be made of:

- specific antibodies directed against each membrane constituent of the organelle, or even antibodies directed against different epitopes of the same membrane constituent in order to identify changes in conformation of said constituent resulting for example from alterations linked to a pathology,

- specific lipid ligands (for example, annexin V),

- all types of natural, recombinant or synthetic ligands, and in particular calmodulin, complexin or alpha-SNAP for the characterization of synaptic vesicles.

A preferred application of the present method relates to the detection of toxins in a sample likely to contain them. Preferably, the method of the invention applies to the detection of neurotoxins whose targets are the membrane constituents of synaptic vesicles. In a preferred embodiment, the method according to the invention is applied to the detection of a botulinum toxin or a tetanus toxin. These neurotoxins target the SNARE proteins (syntaxin 1, VAMP2 and SNAP25) present in the membrane of synaptic vesicles. The effect of these neurotoxins on synaptic vesicles can be analyzed by the detection according to the method of the invention of the binding of an anti-syntaxin 1, anti-VAMP2 or anti-SNAP25 antibody on immobilized synaptic vesicles brought into contact or not with a sample likely to contain said neurotoxins.

Also targeted in the present invention are the uses of biochips containing the organelles immobilized on their surface, as described above,

- for the screening of molecules capable of modifying the quantity of a membrane constituent of an organelle or of modifying the capacity of a membrane constituent to bind with its natural ligand,

- for the screening of molecules capable of specifically binding to a membrane constituent of an organelle,

- for the characterization of the organelles contained in a sample, or

- for the detection of toxins in a sample likely to contain them, preferably botulinum or tetanus toxin.

The features of the invention mentioned above, as well as others, will appear more clearly in the light of the examples presented below. DESCRIPTION OF THE FIGURES

Figure 1: Figure 1 illustrates the specific capture of approximately 2000 RU of synaptic vesicles on a biochip by means of anti-synaptophysin antibody 171B5. Sn10: subcellular fraction Sn10 diluted to 1/20 not pre-incubated with the antibody 171 B5 and injected on a biochip to which the antibody 171B5 is coupled

Sn10 in the presence of an excess of anti-synaptophysin antibody: subcellular fraction Sn10 diluted to 1/20 and pre-incubated with the antibody 1.71 B5 injected on a biochip to which the antibody 171 B5 is coupled

Figure 2: Figure 2 illustrates the ability of a 100 mM octylglucoside solution to regenerate synaptic vesicles immobilized on an anti-synaptophysin 171B5 antibody. Each cycle consists of an injection of diluted Sn10 fraction, followed by an injection of 100 mM octylglucoside (40 μl to 5 μl / min).

Figure 3:

Figure 3 illustrates the capture of synaptic vesicles from lysates of cultured hippocampal neurons, seeded at different densities. Lysate of 28,800 cells Lysate of 9,000 cells

Lysate of 4500 cells.

Figure 4: 23 samples from lung tumor samples were analyzed for the presence of neuroendocrine markers.

The horizontal line represents the signal level above which the detection is considered to be positive (4 resonance units value below which the signal is no longer considered reliable). Small cell tumors represented by diamonds. Non-small cell tumors represented by squares. Healthy samples (control) represented by a triangle.

Figure 5: Figure 5 illustrates the demonstration of a variation in the amount of synaptic vesicles in neurons in culture treated with a neurotrophic factor

Cells treated: hippocampal neurons (20,000 cells / cm ² ) treated after 7 days of culture with 37.5 ng / ml of BDNF (Brain-derived neurotrophic factor, R&D Systems) for 7 days. Untreated cells: hippocampal neurons grown under the same untreated conditions.

Figure 6: Figure 6 illustrates the effect of botulinum toxin B (Bont / B) on the immobilization of synaptic vesicles on a biochip.

Sn10 previously incubated with a toxin concentration range for 20 hours is analyzed on a biochip coupled with an anti-VAMP, anti-synaptophysin or anti-synaptogyrin antibody.

The biochip comprises two to four analysis tracks placed in series: a non-specific antibody is captured on the control track (700 pg) and an anti-VAMP antibody on the test track (700 pg). Analysis of Sn10 (after 20 hours of incubation at 37 ° C) allows the determination of synaptic vesicles on the biochip. The presence of BoNT / B toxin abolishes the signal on the anti-VAMP2 antibody. The sensitivity is 3fM, which makes it possible to detect an amount of 0.04 pg of toxin (experiment representative of three independent experiments analyzed in triplicate). Figure 7: Example of characterization of synaptic vesicles from cultured neurons whether or not treated with tetanus toxin.

The culture is treated (treated cells) or untreated (untreated cells) with tetanus toxin (100 ng / ml) for 39 hours. The specific antibodies of the synaptic vesicles are then injected successively:

1: anti-CSP (Cysteine String Protein)

2: proton pump

3: vesicular glutamate anti-transporter

4: anti-SNAP25 5: anti-synapsin

6: anti-SV2A

7: anti-synaptotagmin

8: anti-synaptogyrin

9: anti-synaptophysin 10: anti-VAMP Nt polyclonal

11: anti-VAMP 1H7 12: anti-VAMP 60-88 13: anti-VAMP 3D10

Figure 8: Figure 8 illustrates a calibration curve for the assay of synaptophysin.

A dilution range of Sn10 (containing a known concentration of synaptophysin) is injected into an anti-synaptophysin antibody. The samples are injected at a flow rate of 5 μl / min. The binding speed of the synaptic vesicles is constant and proportional to the amount of synaptophysin present in the sample. The inset shows that linearity is observed over a wide range of synaptophysin concentrations. Estimating that there are approximately 20 copies of synaptophysin per synaptic vesicle, a second abscissa axis makes it possible to relate the amplitude of the SPR signal to the concentration of synaptic vesicles present in the medium. EXAMPLES

1. Materials and methods

1.1 Antibodies

The anti-synaptophysin antibody used to capture synaptic vesicles is the 1 1 B5 antibody (Lévêque et al. 2000).

The anti-synaptotagrnin I CL 41.1, anti-synaptogyrin Cl 80.1, anti-synapsin I Cl 46.1 and anti-Rab5 Cl 621.3 and anti-VAMP2: Cl 69.1 monoclonal antibodies are marketed by the company Synaptic Systems.

Polyclonal anti-bassoon antibodies and monoclonal antibody. anti-syntaxinl 3 are sold by the company Stressgen.

The anti-VAMP2 Nter polyclonal antibody (directed against the Nter part of the VAMP, Léveque et al. 2000) and anti-VAMP 60-88 (polyclonal antibody directed against the rat VAMP2 peptide 60-88) were provided by Dr Takahashi's laboratory (Mitsubishi Kasei Institute of Life Science, Tokyo, Japan). Antibodies 1 H7 and 3D10 (monoclonal anti VAMP2 whose epitopes are located in region 19-59 of rat VAMP2), as well as the anti SNAP 25 antibody (BR05, Lévêque et al, 2000) were provided by the Dr Kosaki (Department of Veterinary Science, College of Agriculture, Osaka Prefecture University, Sakai, Osaka, Japan). The anti-syntaxin monoclonal antibodies: 6D2, 9H1, 4D4, 10F5, 6H1 were also provided by the laboratory of Dr Takahashi as well as the anti-synaptotagmin antibodies (1 D12 and anti Nterminal region of synaptotagmin I), anti-CSP ( Lévêque et al. 2000), anti-Rab3A, Munc 18, NSF, sortillin, complexin, GluR2, Neurexin, alpha-beta SNAP and synaptophysin (6H2 directed against a luminal region)

The anti-synapsin IIa monoclonal antibody is sold by the company Translaboratories. The Developmental Studies Hybridoma Bank anti-SV2A antibody was developed by NICHD and stored by the University of Iowa, Department of biological Sciences, lowa City, IA 52242. Non-immune IgGNIS mouse IgGNIS (non-specific IgG) were obtained from Interchim.

1.2 Preparation of synaptic vesicles from rai brain

The fractionation procedure is based on the work of Lévêque et al. 2000.

A fresh rat brain is homogenized with a Teflon homogenizer in 10 ml of a cold sucrose buffer (10 mM HEPES, NaOH, 0.32 M sucrose, 1 mM EDTA, 1 mM Phenylmethylsulfonyl fluoride, pH 7.4). The homogenized sample is centrifuged at 10000xg for 15 minutes. The supernatant (Sn10) (containing approximately 3 mg of protein / ml) is filtered and used immediately or stored at -20 ° C or lyophilized for later use.

1.3 Method for detecting variation in mass or molecular conformation by optical reflection All the experiments described below were carried out on a biosensor of the Biacore type (Biacore X or Biacore 3000). The temperature of the biosensor is controlled at 25 ° C. All the experiments were carried out with a control track placed in series and a test or experimental track. All the results presented correspond to specific measurements (signal from the control track subtracted from the signal from the test track) expressed in resonance units (RU): one resonance unit corresponds to a mass of 1 pg of protein equivalent with technology Biacore.

The characterization experiments were carried out on a microchip (CMS sensor chip. The immobilization protocol is the same as that used for the CMS biochip. After immobilization of the synaptic vesicles, the surface of the biochip is saturated with 50 μl of a IgGNIS antibodies (50μg / ml) at 15 μl / min before contacting specific antibodies. Example 1: Control of the specificity of the immobilization of synaptic vesicles on biochips

Figure 1 illustrates the specific capture of synaptic vesicles immobilized on biochips. A Sn10 fraction containing synaptic vesicles is diluted in a TBS buffer to a protein concentration of 0.15 mg / ml, and preincubated or not with an excess of anti-synaptophysin antibodies (100 μg / ml). The samples are then injected (50 μl to 5 μl / min) on a biochip on which 0.5 ng of anti-synaptophysin antibody have been immobilized beforehand in the test track and 0.5 ng of non-immune antibody in the control track .

FIG. 1 illustrates the specific capture of approximately 2 ng of protein equivalent of synaptic vesicles on a biochip by means of anti-synaptophysin antibodies 171B5. The analysis of the immobilization rate of each preparation makes it possible to measure an inhibition of 88% of the immobilization of synaptic vesicles when the Sn10 fraction has been pre-incubated with the antibody 171B5. The signal generated by the immobilization of the synaptic vesicles is specific with regard to the following criteria: • the passage of the biological sample on a “control” track on which a non-specific antibody is coupled generates a signal which represents only 5 to 10 % of the signal collected on the "experimental" track.

Immunological and morphological analyzes demonstrate that this non-specific signal measured on the control track does not correspond to proteins of the synatic vesicles, • the preincubation of the Sn10 fraction with an excess of the specific antibody largely inhibits the binding of vesicles to the As an experimental track, the use of antibodies directed against specific markers of other organelles does not generate any specific signal whereas a majority of antibodies specific to synaptic vesicles generates a positive signal as shown in Table 1 below. Table 1: Immunological characterization on biochip of the material immobilized with anti-synaptophysin mAb (171B5) coupled with an anfiFcγ antibody.

- signal <0 RU

+ signal 0-10 RU ** ivi: monoclonal

++ signal 10-50 RU P: polyclonal

+++ signal> 50 RU * Products marketed In addition, analysis by electron microscopy makes it possible to observe spherical vesicles with a diameter close to 50 nm immunomarked with an anti-VAMP antibody (results not shown).

Example 2: Regeneration of the biochip

Several consecutive immobilizations of small quantity can be carried out on the same biochip. Nevertheless, the inventors have developed methods for regenerating biochips making it possible to implement the methods according to the invention on the same biochip several hundred times.

The inventors have selected an anti-synaptophysin monoclonal antibody having a dissociation ^rapid kinetics, atypical compared to most anti-synaptic vesicles antibody. Prolonged injection of a concentrated detergent solution (such as 100 mM octylglucoside) completely regenerates the synaptophysin / antisynaptophysin interaction while dissolving the vesicle membrane.

After immobilization of a non-specific mouse antibody (1, 4 ng) on the control track, and of an anti-synaptophysin antibody on the experimental track (1, 4 ng), 6 μl of the Sn10 fraction (0.5 mg / ml) are injected. Each cycle consists of an injection of the Sn10 sample, followed by an injection of 100 mM octylglucoside (40 μl to 5 μl / min). As shown in Figure 8, the decrease in capture is limited (23% loss over 40 cycles) and linear over time, which allows it to be taken into account in an injection / regeneration program.

Forty regeneration / immobilization cycles are therefore possible with octylglucoside on the same antibody coupled to the biochip.

The inventors also immobilized a monoclonal antibody against synaptic vesicles) with an anti-Fc antibody. While retaining the immobilization properties described above, this procedure also has the advantage of orienting the anti-synaptic vesicle antibody in a functional manner. By selecting an anti-Fc antibody little sensitive to pH shock, the biochip containing the immobilized organelles can be regenerated several tens of times at an acidic pH.

The alternating use of these two protocols makes it possible to carry out several hundreds of assays on a single biochip.

Example 3: Detection of synaptic vesicles from different amounts of neurons in culture and analysis of the sensitivity of the method according to the invention

Primary cultures of hippocampal neurons are prepared from 18-day rat embryos according to the method described in Garrido et al., 2001. Four coverslips per culture dish are seeded with different densities of dissociated cells. On day D14, the cells are washed three times with PBS buffer. The cells are then lysed for 3 minutes with 500 μl (per culture dish) of lysis buffer (5 mM HEPES, 1 mM EDTA, 1 mM PMSF, pH 7.4) and resuspended by scraping. The lysed cells are passed 9 times through a 27G needle and centrifuged for 15 minutes at 12,000 x g at 4 ° C. The cell supernatants are injected (100 μl to 1 μl / min) on the biochip functionalized with an anti-synaptophysin antibody (171 B5).

The sensitivity of the organelle detection method according to the invention is thus illustrated by the following experiment: by diluting a neuron culture lysate to 20,000 cells / cm2 and by extrapolation, it has indeed been determined that it was possible to detect the presence of vesicles from an equivalent of two neurons. The method according to the invention, rapid and sensitive, is therefore particularly well suited to detection organelles in biopsies or samples of biological fluids, including blood samples.

EXAMPLE 4 Use of the Organelle Detection Method for the Diagnosis of Small Cell Lung Tumors

21 samples of lung tumors (xenografts) and 2 of healthy lungs were provided by the laboratory of M. F. Poupon (Institut Curie). Each sample is homogenized in a 5 mM HEPES-NaOH buffer, 1 mM EDTA, 1 mM PMSF, pH 7.4 (10 mg of tumor per ml of buffer). The samples are then centrifuged for 30 min at 10,000 x g. The supernatant is diluted 40 times in a 10 mM HEPES-NaOH buffer, 0.4 M NaCl, pH 7.4, containing 1 mM spermine before being injected (40 μl at 5 μl / min). The biochip is coupled with an anti-synaptophysin antibody (171B5). The 23 samples were analyzed. The results are presented in FIG. 4. The 9 samples from small cell tumors (the characteristic of which is to contain neuroendocrine markers) are positive as well as 3 samples of “non-small cell” tumors. However, it is expected that 10 to 30% of non-small cell samples nevertheless also contain neuroendocrine markers (Camaghi C. et al., 2001). The horizontal line represents the signal level above which the detection is considered positive (4 resonance units value below which the signal is no longer considered reliable). The results demonstrate that the method of the invention is suitable for the detection and quantification of neuroendocrine markers, in particular for the positive and differential diagnosis of tumors.

Example 5: Detection of a modification in the number of synaptic vesicles originating from neurons treated with a neurotrophic factor

Hippocampal neurons (20,000 cells / cm ² ) are treated after 7 days of culture with 37.5 ng / ml of BDNF (Brain-derived neurotrophic factor, R&D Systems) for 7 days. After hypotonic lysis of the neurons, the lysis supernatants are injected (100 μl to 1 μl / min) onto 0.7 ng of anti-synaptophysin antibody or of non-specific antibodies. As shown in FIG. 5, the treatment with BDNF induces a modification of the detection signal of the synaptic vesicles, from the start of the injection, confirmed by obtaining a higher plateau (% increase: 29% +/- 4.8, n = 3).

EXAMPLE S Detection of Botulinum Toxin B by Loss of Immobilization of Synaptic Vesicles on Biochips

The experiment consists in measuring a loss of binding of synaptic vesicles of rat brain, on an anti-VAMP antibody, following the incubation with botulinum toxin B. The monoclonal anti-VAMP antibody used to capture the vesicles is directed against the Nterminal part of the. VAMP (Cl 69.1). Purified Botulinum toxins B and E were supplied by the laboratory of Dr. Kosaki (Department of Veterinary Science, College of

Agriculture, Osaka Prefecture University, Sakai, Osaka, Japan).

The biochip comprises two to four analysis tracks placed in series: a non-specific antibody is captured on the control track (700 pg) and an anti-VAMP antibody on the test track (700 pg). In certain experiments, anti-synaptophysin (171B5) or anti-synaptogyrin (1D12) antibodies were also placed in series on the following two tracks in order to verify the integrity of the vesicular preparation for prolonged incubation times.

It has been found that it is possible to detect the presence of toxin (1-10 pM), after incubation times of 0.5 - 1.5 hours (80% cleavage at 10 pM 30 min at 37 ° C ) (results not shown). However, better sensitivity was obtained for longer incubation times, for example 12 to 20 hours, as in the example in FIG. 6. Only the epitope of VAMP disappears. There is no decrease in capture of these same vesicles on an anti-synaptophysin or anti-synaptogyrin antibody. The Sn10 fraction (3 mg / ml) was prepared in the presence of 5 mM DTT without other protease inhibitors.

The concentrated toxin (0.6 μM) is incubated 45 min at 37 ° C in a buffer

HEPES-NaOH 10 mM, DTT 10mM, NaCI 0.15M, pH 7.4. The Sn10 fraction is diluted 3000 times in the cleavage buffer: 50 mM HEPES-NaOH pH 7.4, 20 mM ZnCI ₂ , 0.02% BSA, 0.16 mM DTT, pH 7.4 and then incubated in the presence or absence of varying concentrations of toxin. At the end of the incubation, the NaCI concentration is adjusted to 0.4 M and part of the sample is injected onto the biochip.

On a Biacore type device, the sample (50 μl) is injected at a flow rate of 2 μl / min. The race buffer consists of 10 mM HEPES-NaOH, 0.15 M NaCl, pH 7.4.

It was observed that the botulinum toxin B (600 pM) heated to 80 ° C or else not reduced as well as the botulinum toxin E (600 pM), used as a negative control, are inactive in this test (experiments carried out over time short).

The detection limit is of the order of 3 fM of botulinum toxin B (approximately 0.04 pg of toxin), ie 0.45 pg / ml. The presence of DTT and the significant dilution of the Sn10 fraction are two important parameters making it possible to measure an immobilization of synaptic vesicles incubated at 37 ° C. for several hours. The botulinum toxin B activity assay is currently performed very often in vivo by LD50 in mice (US20030143651). It has a higher sensitivity to in vitro tests. The method of the present invention applied to the determination of botulinum toxin as described above proves to be 250 times more sensitive than the test for detecting the activity of botulinum toxin B in vivo. EXAMPLE 7 Characterization of Synaptic Vesicles Coming from Neurons in Culture Treated or Not with Tetanus Toxin

For this experiment, the tetanus toxin was supplied by Dr Boquet (INSERM U452, Nice). The hippocampal neurons are cultured as described in Figure 3. The culture is treated or not with tetanus toxin (100 ng / ml) for 39 hours. After hypotonic lysis, the synaptic vesicles (approximately 2 ng of protein equivalent) are immobilized on an anti-synaptophysin antibody. The biochip is then saturated with a non-mouse specific antibody.

The specific antibodies of the synaptic vesicles are then injected successively (20 μl to 30 μg / ml, 15 μl / min). Each injection is preceded by an injection of non-specific antibodies at the same concentration. The percentages of inhibition of binding of anti-VAMP antibodies by the action of tetanus toxin are 94%, 87%, 87% and 89% respectively as illustrated in FIG. 7.

Example 8 Linearity of the dose-response curve for the detection of synaptophysin and or synaptic vesicles from Sn10.

Different dilutions of Sn10 containing known concentrations of synaptophysin are made in TBS buffer. These dilutions are injected at 5 μl / min onto a biochip on which 0.8 ng of anti-synaptophysin antibody on the test track and 0.8 ng of non-immune antibody on the control track have been immobilized beforehand. The injections are carried out at a slow rate and under conditions which are not saturating for the specific antibody (use of 1 to 2% of the capacity for capturing the antibody). The speed of binding of the synaptic vesicles is then a straight line whose slope is calculated and reported as a function of the concentration of synaptophysin contained in the sample. Based on the estimate that there are approximately 20 copies of synaptophysin per synaptic vesicle (Fernandez Chacon and Sudhof 1999), it is also possible to determine the concentration of synaptic vesicles in the injected sample. A calibration curve is thus obtained for the assay of synaptophysin and / or synaptic vesicles. The detection limit is 50 attomoles (2pg) of synaptophysin (2.5 attomoles of synaptic vesicles), which corresponds to a gain in sensitivity of 60 compared to the assay of this protein in ELISA (Schiaf ef a /, 1996) .

BIBLIOGRAPHICAL REFERENCES

Carnaghi et al., 2001, Ann. Oncol. 12 (Suppl.) 119-123

Collin et al., Eur. J. Neurosci., 2001, 13, 1273-82

Fernandez-Chacon and Sudhof Annu. Rev. Physiol. 1999, 61, 753-776

Garrido et al., 2001, EMBO J., 20, 5950-5961

Hirsh et al., Nat. Neurosci., 1999, 2, 499-500

Ikemoto Acta Neuro 2002, 103, 179-187

Jahn and Sudhof, J. Neurochem., 1993, 61, 12-21

Lévêque et al., J. Neurochem., 2000, 74, 367-74

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Claims

1. Method for identifying or assaying organelles in a liquid sample, characterized in that it comprises: a) the coupling, on the surface of a biochip usable in an optical detection device, of a ligand capable of specifically bind to a membrane constituent of the organelles to be identified or measured, b) bringing the liquid sample into contact with the surface of the biochip containing the coupled ligand, c) detecting a variation in optical signal, said detection allowing the identification and / or the determination of the quantity of organelles bound to the surface of the biochip.

2. Method according to claim 1, characterized in that the optical detection method is a method of detecting mass variation or molecular conformation by optical reflection based on spectroscopy of evanescent waves, in particular on surface plasmon resonance.

3. Method according to one of claims 1 or 2, characterized in that the contacting of the liquid sample with the surface of the biochip is obtained by injection of the liquid sample into a device allowing this contacting, for example by means of microfluidic circuits.

4. Method according to any one of claims 1 to 3, characterized in that the liquid sample is extracted from neuronal cells for the identification and / or determination of synaptic vesicles.

5. Method according to claim 4, characterized in that the ligand coupled to the surface of the biochip is chosen from the group consisting of antibodies directed against a constituent of the membrane of the synaptic vesicles.

6. Method according to claim 4, characterized in that the ligand coupled to the surface of the biochip is chosen from the group consisting of anti-synaptophysin antibodies and anti-VAMP antibodies.

7. Method according to claim 5 or 6, characterized in that the biochip is regenerated by means of a concentrated detergent solution, for example octylglucoside.

8. Method according to any one of claims 1 to 3, characterized in that the liquid sample is extracted from isolated cells or from a biological fluid taken from an individual for the identification or determination of vesicles containing neuroendocrine markers.

9. Use of a method according to any one of claims 1 to 8, for the detection of a variation in the relative quantity of organelles in a biological sample correlated to a treatment with a physiological or pharmacological agent or genetic manipulation of the biological organism from which the biological sample analyzed is derived.

10. Use of a method according to any one of claims 1 to 8, for the screening of molecules capable of modifying the quantity of a membrane constituent of an organelle or of modifying the capacity of a membrane constituent to bind with a natural ligand.

11. Use of the method according to claim 4, for the screening of molecules capable of modulating cell growth containing synaptic vesicles, in particular neurotrophic or anti-neurodegenerative molecules.

12. Use of the method according to claim 7 or 8, for the diagnosis and / or prognosis of a patient suffering from neurodegenerative diseases, in particular Alzheimer's disease.

13. Use of the method according to claim 7, for the positive and differential diagnosis of tumors.

14. Method for preparing a biochip usable in an optical detection device, and comprising organelles immobilized on its surface, said method comprising: a) coupling, to the surface of a biochip usable in an optical detection device, a ligand capable of specifically binding to a membrane constituent of an organelle, b) bringing a liquid sample containing organelles into contact with the surface of the biochip containing the coupled ligand, thus making it possible to obtain a biochip containing organelles immobilized on its surface, c) if necessary, freezing the biochip obtained in step b) and keeping it in the cold, followed by thawing, or its lyophilization followed by rehydration, view of its use.

15. Method according to claim 14, characterized in that the optical detection device chosen is a device for detecting mass variation or molecular conformation by optical reflection based on spectroscopy of evanescent waves, in particular on surface plasmon resonance.

16. Method according to claim 14 or 15, characterized in that bringing a liquid sample into contact with the surface of the biochip is obtained by a device allowing the passage of a flow of the liquid sample on the surface of the biochip, for example by means of microfluidic circuits.

17. Method according to any one of claims 14 to 16, characterized in that the organelles contained in the sample are vesicles of exocytosis or secretion.

18. Method according to any one of claims 14 to 17, characterized in that the organelles contained in the sample are synaptic vesicles.

19. Method according to claim 18, characterized in that the ligand coupled to the surface of the biochip is chosen from the group consisting of anti-synaptophysin antibodies and anti-VAMP antibodies.

20. Biochip usable for the detection of a bond between an analyte and a membrane constituent of an organelle, characterized in that it is capable of being obtained by a method according to any one of claims 14 to 19.

21. Biochip usable for the detection of a bond between an analyte and a membrane constituent of an organelle, characterized in that it comprises a) a support suitable for detecting a variation in optical signal, for example a variation in mass or of molecular conformation on its surface by optical reflection, b) ligands coupled to said support, said ligands being capable of specifically binding to a membrane constituent of an organelle, c) organelles immobilized on the surface of the biochip by specific bond with said ligands.

22. Biochip according to claim 21, characterized in that the organelles immobilized on its surface are synaptic vesicles.

23. The biochip according to claim 21, characterized in that the synaptic vesicles are immobilized on the surface of the biochip by specific binding with ligands chosen from the group consisting of anti-synaptophysin antibodies and anti-VAMP2 antibodies.

24. Biochip according to any one of claims 21 to 23, characterized in that the support suitable for detecting a variation in optical signal is a support suitable for measuring a change in mass or molecular conformation on its surface by optical reflection, chosen among a medium of gold or silver.

25. A method of detecting a bond between an analyte and a membrane constituent of an organelle comprising: a) obtaining a biochip usable in an optical detection device, for example detection of variation in mass or conformation molecular by optical reflection and comprising organelles immobilized on its surface, b) bringing the analyte into contact with the surface of the biochip containing immobilized organelles, c) measuring a variation in optical signal, said measurement allowing the detection of the quantity of analytes bound to the organelles immobilized on the surface of the biochip.

26. Method according to claim 25, characterized in that the biochip obtained in step (a) is a biochip according to any one of claims 19 to 24.

27. Method according to any one of claims 25 or 26, characterized in that the measurement of a variation in optical signal is a measurement of variation in mass or of molecular conformation by optical reflection based on spectroscopy of evanescent waves, in particular on surface plasmon resonance.

28. Method according to any one of claims 25 to 27, characterized in that bringing the analyte into contact with the surface of the biochip is obtained by injecting the liquid sample into a device allowing the passage of a flow of the liquid sample on the surface of the biochip, for example by means of microfluidic circuits.

29. Use of a method according to any one of claims 25 to 28 for the screening of molecules capable of binding, specifically to a membrane constituent of an organelle, in particular of agonists or antagonists of ligands of membrane receptors .

30. Use of a method according to any one of claims 25 to 28 for the screening of molecules capable of modifying the quantity of a membrane constituent of an organelle or of modifying the capacity of a membrane constituent to bind with its natural ligand.

31. Use of a method according to any one of claims 25 to 28 for the detection of toxins in a sample capable of containing them.

32. Use according to claim 31, in which the toxin to be detected is a botulinum toxin or the tetanus toxin.

33. Use of a method according to any one of claims 25 to 28 for the characterization of the organelles contained in a sample, said analytes being chosen from known ligands of membrane constituents of organelles.

34. Use of a biochip according to any one of the claims

19 to 24,

- for the screening of molecules capable of modifying the quantity of a membrane constituent of an organelle or of modifying the capacity of a membrane constituent to bind with its natural ligand,

- for the screening of molecules capable of specifically binding to a membrane constituent of an organelle,

- for the characterization of the organelles contained in a sample, or, - for the detection of toxins in a sample likely to contain them, in particular of botulinum or tetanus toxin.