CONTROL MARKER FOR IMPLEMENTING SPOT ANALYSIS METHODS

专利摘要:
The present invention relates to the use of a control marker for the implementation of spot analysis methods, in particular in the context of multiplex analyzes. The present invention thus relates to solid supports containing said control marker, their method of preparation and their use in analytical methods. The present invention makes it possible to check the presence, the location and / or the integrity of the spots at the end of the analysis process, and thus to secure the results obtained, by guaranteeing that the result rendered results from a present spot. , integrates and localizes.
公开号:FR3019901A1
申请号:FR1453169
申请日:2014-04-09
公开日:2015-10-16
发明作者:Agnes Roseline Claude Pouzet；Vincent Doury；Laurent Emmanuel Fournier；Christophe Rene Roger Vedrine
申请人:Bio Rad Innovations SAS；
IPC主号:

专利说明:

[0001] FIELD OF THE INVENTION The present invention relates to the use of a control marker for the implementation of spot analysis methods, in particular in the field of framework of multiplex analyzes. The present invention thus relates to solid supports containing said control marker, their method of preparation and their use in analytical methods. The present invention makes it possible to check the presence, the location and / or the integrity of the spots at the end of the analysis process, and thus to secure the results obtained by guaranteeing that the result rendered results from a present spot, integrated and localized. STATE OF THE ART A multiplex analysis method makes it possible to detect, simultaneously, the possible presence of several analytes within the same sample. A multiplex analysis method is typically carried out by means of a solid support comprising spots, for example a microplate comprising spots in each well, the spots being each intended to detect an analyte or to serve as a control. It is clear to those skilled in the art that a risk related to spot technology is the absence of deposition, the elimination or the degradation of one or more spots during the process for preparing the solid support, in particular of a microplate, or during the implementation of the analysis method by means of said solid support. The device for depositing the samples or the reagents can, in fact, accidentally come into contact with one or more spots, thus altering their surface, for example by forming a streak in one or more spots, or tearing off partially or totally one or several spots. For example, in the article by Bastarache et al. (Accuracy and Reproducibility of a Multiplex Immunoassay platform a validation study'Immunol Methods, Mar 31, 2011, 367 (1-2) 33-39) are presented defects which were observed at the end of the test on the luminescence signal , such as spot irregularities, the presence of comets which causes the contamination of a spot by a neighboring spot and the absence of expected signal at the theoretical location of the spot. However, when a negative result is given at the end of an analysis process, this result must result from the absence of the analyte to be detected in the sample, and not from an absence or a degradation of the detection spot of the corresponding analyte. Securing analysis methods is essential, especially for their use in diagnosis in humans, for example to verify the absence of viral or bacterial contamination of a blood sample for transfusion. The manufacture of a solid support for a spot analysis consists in depositing, on the surface of the solid support, solutions comprising a capture ligand of the analyte to be detected, so as to form spots. A quality control of the solid support is then carried out after the manufacture of the solid support, in order to keep only the solid supports having intact and well-formed spots. Thus, document US2006 / 0063197 describes the use of a fluorophore in the deposition solution intended to form the spots of a microarray. The fluorophore is used to control the quality of each spot at the end of the microarray preparation process. Moreover, only a weak residual fluorescence signal is detected at the spots before the addition of the substrate, during the implementation of an ELISA test. WO2012 / 142397 discusses flow issues in microfluidic devices containing microarrays and describes a microarray assembly comprising a grating chamber with an input for the sample at a first end, a microarray and an output of the microarray. sample at a second end connected to a waste chamber, the cross sectional area at the first end of the grating chamber being wider than that at the second end. This document also describes a method for controlling the manufacturing quality of a microarray, by measuring the fluorescence emitted by an internal quality control fluorophore at the spots of a network and by coding the information relating to each spot of the network on the network. a bar code, a memory device or radio-identification, this coded information being associated with the microarray. Moreover, when implementing a multiplex analysis method, the signal corresponding to the analyte to be detected can be detected at a theoretical reading gate. This theoretical reading grid is generally defined at the end of the multiplex analysis method, from the signal detected at a spot serving as a positive control. However, given the reading scales, any offset in the positioning of the spots during the manufacture of the solid support and / or in the positioning of the solid support in the signal detection device with respect to the theoretical reading gate causes a shift. in the actual positioning of the spots with respect to the theoretical reading grid and therefore has an influence on the detection sensitivity of the analytes. There is therefore still a need for solutions to secure the results obtained at the end of a spot analysis process, in particular by making it possible to check the presence, the location and / or the integrity of the spots at the end. of the analysis method and / or by making it possible to optimize the detection of the analytes, for example by improving the detection sensitivity of the analytes. DETAILED DESCRIPTION The present invention is based on the detection of control markers which can be deposited, for the formation of spots on a solid support, together with capture ligands specific for the analytes to be detected in a sample, these control markers. being able to produce a signal at the end of an analysis process carried out by means of said solid support, without interfering or to a negligible extent with the detection of the analytes. The control markers according to the invention thus make it possible to check the presence, the location and / or the integrity of the spots at the end of an analysis method. These control markers are referred to here as "resistant control markers". The terms "method of analysis" and "method of detecting at least one analyte" are here synonymous. The terms "produce", "product" or "production" apply to any type of signal, and in particular to any type of electromagnetic radiation, whether it be a radioactive signal, a light emission or absorbance. When the detected signal is a fluorescence or luminescence signal, the "produced signal" is in particular an "emitted signal", and by "producing a signal" and "producing a signal" is then understood to mean "emitting a signal" and "sending a signal" respectively. By "detection of at least one analyte" (or "detect at least one analyte") is meant, in the present application, detection of the presence (or detect the presence) of said analyte (s) and / or quantification said analyte (s) (or quantify said analyte (s)). For the purposes of the invention, the term "end of an analysis method" means after the spots have been placed in the presence of the sample to be analyzed, of a ligand or of detection ligands of one or more analyte, where appropriate at least one (first) reporter of a detection marker coupled to an analyte detection ligand and, if appropriate, at least one (second) reporter of a marker of detection coupled to said first reporter. Surprisingly, the inventors have thus demonstrated resistant control markers which can be fixed on a solid support, which do not interfere with the analysis method itself and which remain perfectly detectable (in particular because they remain at least partially fixed on the solid support) after the implementation of the various steps of the analysis method. When the signal produced by the resistant control marker at the end of the analysis process makes it possible to delimit a spot meeting the quality criteria of a spot, in particular presence, location and / or integrity, the signal corresponding to an analyte can then be detected at this spot. In particular, the resistant control markers according to the invention can be used in an analysis method in which the presence of at least one analyte is detected by a signal emitted in chemiluminescence, preferably via the reaction of a peroxidase enzyme. with a luminol substrate and / or a luminol analogue, for example isoluminol, and / or a derivative thereof.
[0002] The resistant control markers according to the invention have the advantage of allowing the control of the spots of a solid support at the end of an analysis process, and not only at the end of the process for preparing said solid support. Thus, the present invention makes it possible to guarantee the results rendered to the user. In particular, the present invention makes it possible to guarantee that a negative result results from the absence of the analyte in a sample and not, for example, from an absence of spot or from an offset in the reading of the spot. In other words, the present invention makes it possible to eliminate false negative results (also called "false negatives") which would be linked to a defect of a spot (ie a spot that does not comply with the quality criteria) and / or of reading a spot at the end of the analysis method and which are not detectable in the conventionally used multiplex analysis methods, in which the reading gate is adjusted theoretically, for example on the signal emitted by a positive control spot. The present invention also ensures that a positive result is a result of the presence of the analyte in a sample. For example, it may happen to detect a signal corresponding to the marker of an analyte at the theoretical location of a spot (as defined by a theoretical reading grid, for example adjusted to the signal emitted by a control spot), while there is no spot at this location; a false positive result (also called "false positive") would then be rendered during the implementation of a conventional analysis method; by using a resistant control marker according to the invention in the spots of the solid support, no signal corresponding to said resistant control marker will be detected at this theoretical location of a spot and the analysis method will lead to an absence of result, despite the detection of a signal corresponding to the marker of the analyte at that location. Another advantage of the present invention resides in the fact that the resistant control markers make it possible to define the reading gate at the end of the analysis method. However, there may be differences between a theoretical reading grid defined according to the physical parameters of the solid support and that defined at the end of an analysis method using said solid support. These differences may for example result from a shift between the expected theoretical spot grid and the grid actually obtained at the end of the analysis process (for one or more spots); this offset is thus corrected by the device described in the present application. It is thus more reliable to define the reading grid of a solid support at the end of the analysis method, preferably with the same detection device as that used to detect the signal produced by the detection marker from minus one ligand for detecting an analyte and preferably concomitantly. Thus, the resistant control markers according to the invention also make it possible to secure the results of a spot analysis method, by improving the sensitivity of the detection of analytes by defining a reading grid for the detection of analytes from the location of the spots detected at the end of the analysis process. Finally, the preparation of a solid support whose spots comprise a resistant control marker according to the invention is simple to implement and does not add any additional step, the resistant control marker being, for example, simply mixed with the compound of interest, such as a capture ligand, in the solution to be deposited to form the spot. The present invention particularly relates to a solid support for a secure detection of at least one analyte in a sample, at least one spot for the detection of an analyte comprising at least one resistant control marker and at least one ligand capturing an analyte, a process for preparing such a solid support and a method for the secure detection of at least one analyte in at least one sample using said solid support.
[0003] By "at least one" is meant, in the present application, one or more, "several" meaning in particular two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen. , fifteen or more than fifteen. Similarly, by "at least x" is meant, in the present application, x or more of x, and in particular x + 1, x + 2, x + 3, x + 4, etc., "x" being an integer greater than or equal to 2, for example 2 or 3. A particularly preferred method of analysis according to the invention is a multiplex analysis method. The present invention also relates to a method of selecting a resistant control marker, and the use of at least one resistant control marker in at least one spot for detecting an analyte for securing a detection method of at least one analyte in a sample.
[0004] The present invention also provides a device suitable for improved detection of at least one analyte in a sample. Sample The sample to be analyzed is preferably a biological sample. The biological sample may be a biological fluid, such as a blood sample, derived from blood (such as plasma or serum), urine, cerebrospinal fluid, saliva or combinations thereof, or a tissue sample, such as a tissue obtained by biopsy, a cell, a set of cells, a plant extract, or their combinations.
[0005] A blood derivative refers to any product, in particular fluid, obtained from a blood sample. The sample to be analyzed may also be a culture medium and / or a culture supernatant. Before being analyzed, the sample may undergo one or more preliminary treatment steps, such as dilution, centrifugation, heat treatment, cell lysis, solubilization, denaturation (for example by one or more chaotropic agents, one or more agents). reducers and / or by heating), extraction, PCR (Polymerase Chain Reaction) reaction, addition of unlabeled detection ligand or combinations thereof. The addition of an unlabeled detection ligand is particularly useful for the implementation of a neutralization test. The sample may also be a mixture of at least two samples that may be of the same kind or of a different nature. By way of example of a mixture of samples of a different nature, mention may be made of a mixture of blood and serum, a mixture of blood and plasma, a mixture of serum and plasma, or a mixture of blood, serum and plasma. A preferred sample according to the invention is a sample or a mixture of blood samples and / or derivative (s) of the blood (in particular plasma and / or serum). Analyte An analyte to be detected in the sample can be any type of compound, natural or synthetic, that one wishes to detect and / or quantify in a sample. An analyte may for example be a protein, a peptide, a glycoprotein, a carbohydrate, a cell, an organelle, a virus or a nucleic acid. The cell may be an animal cell, a plant cell, a bacterial cell, a protozoan, a metazoan cell, a yeast cell or a mushroom cell.
[0006] A nucleic acid denotes a polymer of nucleotides connected by phosphodiester bonds, such as a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA) or an analogue thereof, such as phosphorothioates or thioesters, in single-stranded form or double strand.
[0007] The analyte or at least one of the analytes is thus for example selected from the group consisting of an antigen, an antibody, an antibody fragment, a hapten, a hormone, a hormone receptor, an enzyme or a nucleic acid. . By "antigen" is meant for the purposes of the present application a natural, recombinant or synthetic molecule recognized by antibodies or cells of the immune system and capable, when presented under conditions appropriate to the immune system of a host to induce an immune response. An antigen may be a molecule, particularly a polypeptide, comprising or consisting of at least one epitope that may be linear or conformational. The term "linear epitope" refers to a polypeptide, in particular a peptide, comprising or consisting generally of 3 to 15 amino acids, more generally 5 to 15 amino acids, preferably at least 6, 8, 10 or 12 amino acids, capable of to bind to an antibody molecule directed against said antigen. The term "conformational epitope" refers to a three-dimensional structure recognized by an antibody and determined by the juxtaposition of several amino acids in space, which may be non-contiguous in the peptide sequence of the protein (or polypeptide) against which it is directed. this antibody, but which, because of the folding of the polypeptide chain, are found close to each other in space, and can thus form a pattern that can be recognized by an antibody. An antigen within the meaning of the invention is, for example, a protein (in particular a native protein or a recombinant protein), a peptide (for example a synthetic peptide), a glycoprotein, a carbohydrate or a lipid; said peptide may or may not be associated with a carrier molecule. By "carrier molecule" (also called carrier molecule) is meant in particular a carrier protein or carbohydrate molecule, in particular a carrier protein. A carrier molecule may be a natural or non-natural polypeptide (in particular a protein or a peptide) (for example a recombinant protein or a synthetic peptide), a functionalized polymer (of the dextran, polysaccharide or poly-lysine type), a mixed polymer (in particular a co-polymer of different amino acids, for example a lysine-tyrosine co-polymer). A carrier molecule may be an antibody (in particular a monoclonal antibody or a polyclonal antibody), for example an immunoglobulin (also called Ig).
[0008] An example of a molecule or support protein is BSA (bovine serum albumin). By "hapten" is meant in the present application a molecule of low molecular weight capable of being recognized by the immune system, but which is immunogenic only when coupled to a carrier molecule. An analyte is preferably a compound for diagnosing a condition in a subject, pathological or otherwise, or diagnosing the risks of developing a condition, pathological or not. An example of a non-pathological condition is a pregnancy.
[0009] The subject may be a man, a non-human animal or a plant. The non-human animal is preferably a mammal, such as a cat, dog, monkey, rabbit, mouse or rat. The term "man" is used broadly and includes a man or woman of any age, such as an infant, a child, a teenager, an adult or an elderly person.
[0010] In a preferred embodiment, at least one analyte is selected from an antigen or an antibody. When the analyte or an analyte is an antigen, it is preferably an antigen for diagnosing an infection, for example an infection caused by a virus, bacteria, fungus, protozoan or metazoan.
[0011] When the analyte or an analyte is an antibody, it is preferably an antibody for diagnosing an infection, for example an infection caused by a virus, bacteria, fungus, protozoan or metazoan. In another preferred embodiment, at least one analyte is a nucleic acid.
[0012] When the analyte or an analyte is a nucleic acid, it is preferably a nucleic acid to diagnose an infection, for example an infection caused by a virus, a bacterium, a fungus, a protozoan or a metazoan . Typically, it may be one or more antigen (s) and / or one or more antibodies and / or one or more specific nucleic acid (s) of: a virus, such as HIV (Human Immunodeficiency Virus), particularly HIV-1 or HIV-2, HBV (Hepatitis B Virus), HCV (Hepatitis C Virus), HPV (human papillomavirus), HTLV (T-Virus) -lymphotropic human), in particular HTLV-I or HTLV-II, a parasite, such as a parasite capable of causing Toxoplasmosis (in particular Toxoplasma gondii), Malaria (in particular a parasite of the genus Plasmodium, for example Plasmodium falciparum). , Plasmodium vivax, Plasmodium ovale, Plasmodium malariae or Plasmodium knowlesi) or Chagas disease (in particular Trypanosoma cruzi) in a human or a non-human animal), or a bacterium such as a bacterium capable of causing syphilis (in particular Treponema pallidum) or Lyme disease (especially a bacterium of the genus Borrelia) in a man or n non-human animal. By "parasite" is meant here a metazoan or protozoan parasitizing an organism and causing parasitosis. A parasite within the meaning of the invention is therefore neither a virus, nor a bacterium, nor a fungus. The analyte or at least one of the analytes may also be a marker of a disease, such as a marker of cardiovascular disease or a marker of diabetes, a marker of the course of a disease, such as hepatitis, a marker of the course of an infection caused by a virus, a bacterium, a fungus or a parasite, a marker of resistance to a treatment, for example an antiviral treatment, an antibiotic treatment or a treatment against cancer . Several (eg, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more than fifteen) analytes as described herein can be detected simultaneously in a same sample or in the same sample mixture during a multiplex analysis method. This can make it possible to diagnose, in the same sample or in the same mixture of samples, one or more infection (s) or disease (s), the evolution of an infection or disease, a condition (pathological or not), a risk of developing a condition (pathological or not) or a marker of resistance to treatment in a subject. The analytes detected during a multiplex analysis method may be of the same nature (for example only antibodies, only antigens or only nucleic acids) or of different nature (for example, at least one antigen and at least one antibody).
[0013] Capture Ligand A capture ligand is a specific molecule of a compound of interest. A capture ligand is preferably specific for an analyte to be detected in a sample. It is usually an antibody, an antigen, a peptide, a carbohydrate, a lipid or a nucleic acid.
[0014] A capture ligand is attached to the surface of a solid support at a spot. In a preferred embodiment, the capture ligand or one of the capture ligands is an antibody, an antigen or a nucleic acid. When the capture ligand or one of the capture ligands is an antibody, it is for example a monoclonal antibody or a polyclonal antibody. Detection Ligand A detection ligand is a specific molecule of a compound of interest. It allows the detection of a compound of interest attached to a capture ligand.
[0015] An analyte detection ligand is specific for an analyte to be detected in a sample. It allows in particular the detection of an analyte attached to a capture ligand. A detection ligand can be an antibody, an antigen, a peptide, a carbohydrate, a lipid or a nucleic acid. It is preferably an antibody or an antigen. In another embodiment, the one or more detection ligands is a nucleic acid. When the detection ligand or one of the detection ligands is an antibody, it is for example a monoclonal antibody or a polyclonal antibody. The detection ligand or one of the detection ligands is preferably a labeled detection ligand, i.e., a detection ligand to which a detection marker is attached (which may be, for example, biotin or peroxidase ). The detection marker is attached (i.e., coupled) to the detection ligand covalently or non-covalently, preferably covalently. The detection ligand or one of the detection ligands may be coupled to a direct or indirect detection marker. When the detection ligand is not labeled, its detection can be carried out using a labeled antibody specific for said detection ligand. A detection ligand may be identical to the capture ligand or to one of the capture ligands used, with the exception of the possible presence of a detection marker, and / or to bind to the analyte of which it is specific at the level of the same zone as that bound by the capture ligand or one of the capture ligands, in particular when the analyte of which it is specific is in the form of a complex having at least two identical binding zones. Thus, when one of the analytes to be detected is an antibody, it is possible to use, as capture ligand and detection ligand of said analyte, the same antigen specific for said antibody or two different antigens but comprising at least one common area (at least one common epitope) allowing recognition by the bivalent antibody.
[0016] A capture ligand and a detection ligand may also be specific for distinct areas at an analyte level. In a particular embodiment, a detection ligand and a capture ligand (for example antibodies) specific for the same analyte (for example an antigen) do not bind at the same location on said analyte. For example, the detection ligand may bind to an area of said analyte of which it is specific which is remote from the binding region of the capture ligand, for example so as to avoid competition of the capture ligand and the detection ligand. with respect to the compound of which they are specific, because of a steric hindrance.
[0017] Detection Ligand Detection Marker A detection marker coupled to the detection ligand may be a direct marker or an indirect marker. A direct marker is a marker whose signal can be detected directly, that is to say without requiring the prior addition of a protractor. A direct marker is, for example, selected from the group consisting of a radioisotope, a fluorophore, a heavy element of the periodic table such as a lanthanide, a luminescent compound, a transition metal such as ruthenium, a chromogen and colored nanoparticles, fluorescent or luminescent.
[0018] A "luminescent" compound may be an electroluminescent compound, a thermoluminescent compound or a chemiluminescent compound. In a preferred embodiment, the luminescent compound is a chemiluminescent compound. An example of a luminescent compound (more specifically a thermoluminescent compound) that can be used as a direct marker consists of silica nanoparticles comprising (for example doped or doped) doped molecules. a dioxetane compound, in particular the 1,2-dioxetane compound, or a derivative of a dioxetane compound, for example a 1,2-dioxetane derivative. An indirect marker is a marker whose signal detection previously requires the addition of a reporter (also called first reporter) and, if said reporter is itself coupled to an indirect detection marker, the addition of a second reporter of the indirect detection marker coupled to said first reporter. A reporter is a substrate of an indirect marker or a molecule specifically binding to an indirect marker, said molecule being itself a direct or indirect marker or itself being coupled to a direct or indirect marker.
[0019] An indirect marker is, for example, selected from the group consisting of an enzyme, a ligand of a ligand-receptor pair, a receptor of a ligand-receptor pair, a hapten, an antigen and an antibody. A ligand or receptor of a ligand-receptor pair is, for example, biotin, a biotin analogue, avidin, streptravidin, neutravidin or digoxigenin. A preferred indirect marker according to the invention is an enzyme, preferably an enzyme producing a luminescent compound by reaction with a substrate. Detection of the signal of an indirect marker first requires the addition of a reporter of said indirect marker. An enzyme reporter is, for example, a substrate of said enzyme. A reporter for biotin is, for example, avidin, streptavidin or neutravidin, preferably coupled to a direct label or an indirect label, such as an enzyme or a catalyst.
[0020] A biotin reporter is preferably coupled to a direct or indirect detection marker, such as an enzyme or a catalyst. An example of an enzyme is peroxidase, for example horseradish peroxidase (HRP or POD), alkaline phosphatase or luciferase. A preferred biotin reporter according to the invention is streptavidin coupled to a peroxidase, preferably horseradish peroxidase. The detection of the signal of biotin then requires the addition of streptavidin coupled with peroxidase, and then the addition of the substrate of peroxidase. In a preferred embodiment, the detection ligand or one of the detection ligands is coupled to an indirect detection marker selected from a peroxidase enzyme or a biotin. When the analyte is a nucleic acid, the detection marker or one of the detection markers can be detected in fluorescence, for example using direct or indirect labeling with a fluorophore, in luminescence, preferably in chemiluminescence, for example using direct labeling with a luminescent compound or indirect labeling with an enzyme, especially a peroxidase, producing a luminescent compound by reaction with a substrate. When the analyte is a protein compound, the detection label or one of the detection markers is preferably detected by luminescence, for example using direct labeling with a luminescent compound, in particular a chemiluminescent compound or still indirectly labeling with an enzyme producing a luminescent compound by reaction with a substrate. In a particular embodiment of the invention, the detection marker or one of the detection markers used has for substrate luminol, isoluminol, an acridine, coelenterazine, dioxetane or peroxyoxalic compound, or a derivative thereof, and particularly a compound described in the publication Dodeigne C. et al (2000), Talanta 51, 415-439, "Chemiluminescence as diagnostic tool. A review ». In a particular embodiment, the detection marker or one of the detection markers used has for substrate luminol, a luminol analogue, for example isoluminol, or a derivative thereof. By "luminol" is meant 3-aminophthalhydrazide, also called 5-amino-2,3-dihydro-phthalazine-1,4-dione. The gross formula of luminol is C8H7N302. By way of example, it is possible to use the ELISTAR ETA C Ultra ELISA substrate (Cyanagen, Italy) described in the examples.
[0021] By "isoluminol" is meant 4-aminophthalhydrazide. A derivative of the luminol or luminol analogue is preferably a molecule obtained respectively from the luminol or luminol analogue, by any possible modification (s) (for example chemical and / or enzymatic). A derivative of luminol or a luminol analogue is, for example, a substrate of a peroxidase enzyme, the reaction of said peroxidase enzyme with said luminol derivative or luminol analogue for the production of a chemiluminescent compound. A derivative of isoluminol may be, for example, aminoethylisoluminol (or AEI), aminoethylethylisoluminol (or AEEI), aminobutylisoluminol (or ABI), aminobutylethylisoluminol (or ABEI), aminopentylethylisoluminol (or APEI), aminohexylisoluminol (or AHI), aminohexylethylisoluminol (or AHEI), aminooctylmethylisoluminol (or AOMI) or aminooctylethylisoluminol (or AOEI), as described in the publication Dodeigne C. et al (2000), Talanta 51, 415-439, "Chemiluminescence as diagnostic tool. A review ».
[0022] The present invention is based on the use of one or more resistant control markers (for example a mixture of resistant control markers) making it possible to control the quality of the spot or spots present on the surface of a solid support, in particular the presence, location and / or integrity of the or spots present on the surface of a solid support.
[0023] A control marker is a compound that can be detected, for example by producing a signal. The signal produced may for example be a radioactive signal or a light signal. It may also be a control marker (e.g., a colored control marker) that is detected by light absorption.
[0024] A light signal corresponds to the emission of light, in particular in a given wavelength range. For example, a control marker may be a marker that emits fluorescence, phosphorescence or luminescence light, in particular electroluminescence, thermoluminescence or chemiluminescence.
[0025] When the one or more control markers is a marker emitting luminescent light, it emits preferably chemiluminescent light. As the name suggests, a resistant control marker according to the invention is resistant. By "resistant control marker" is meant here a marker which is detected a signal at the end of an analysis method. A resistant control marker is in particular a marker which remains, in whole or at least partially, fixed at a spot on the surface of a solid support, during the process for preparing the solid support and also during an analysis method employing said support, and which is capable of producing a detectable signal at the end of the analysis method, that is to say including in the presence of a detection marker of a ligand of detection of an analyte and / or one or more reporters in the case of one or more indirect detection markers. The terms "detectable", "signal detectable", "capable of producing a detectable signal" or "producing a detectable signal" at the end of the analysis process mean, particularly in the case where the Resistant control is a fluorophore, that the "signal detected" (the signal produced by the resistant control marker and measured on the imaging sensor at the corresponding pixel area) minus (that is, at which one subtract) the average signal of the surrounding pixels is at least three standard deviations above the signal level of the surrounding pixels. A control marker according to the invention is thus resistant to the steps of washing and incubation with the various reagents used during a method of analysis, such as the detection ligand or ligands, the reporter (s), the or the substrates, the sample, one or more diluents.
[0026] In particular, a resistant control marker according to the invention is resistant to the use of the detection marker (s) of an analyte detection ligand, where appropriate to the (first) reporter of the detection marker (s) of the analyte. an analyte detection ligand and optionally the (second) reporter of the indirect marker coupled to said first reporter. In a preferred embodiment, a resistant control marker according to the invention is resistant to the use of one or more enzymes, in particular alkaline phosphatase and / or peroxidase, and to the substrate (s) of said one or more enzymes. A preferred control marker according to the invention is resistant to the use of a substrate selected from the group consisting of luminol, isoluminol or a derivative thereof. By "resistant to the use of a compound" or "resistant to a compound" is meant here that the control marker is resistant to the use of said compound during a method of analysis. In particular, the control marker is resistant to its contact with said compound. Surprisingly, the inventors have thus demonstrated the resistant nature of control markers that have simply been deposited on the surface of a solid support, mixed with a compound of interest, for example a capture ligand, in order to form spots. Thus, in a particular embodiment of the invention, a resistant control marker according to the invention is not covalently attached to the surface of the solid support and / or is not covalently coupled to a capture ligand. Finally, in a preferred embodiment of the invention, a resistant control marker interferes little or, preferably, does not interfere with the signal produced by an analyte detection ligand detection marker used in a method of analysis. By the expression "signal produced by the detection marker of an analyte detection ligand" is meant the signal produced by the detection ligand detection marker of an analyte itself or corresponding to the analyte marker. detection of the detection ligand. Indeed, for example in the case of an enzyme or other type of indirect labeling, the signal is not produced by the detection ligand detection marker of an analyte itself, but by the reporter or a compound produced by the reaction of a reporter, for example an enzyme, with its substrate; in this case, this signal corresponds to the detection marker of the detection ligand of an analyte. The phrase "a resistant control marker that does not interfere with the signal produced by the detection tag of an analyte detection ligand" means that the functionality of the analyte method for the user is not affected by the presence of the resistant marker, in particular that the decrease in sensitivity is less than 40%, preferably less than 35%, more preferably less than 30%, more preferably less than 25%. According to a particular embodiment, this expression further means that there is no degradation of the specificity.
[0027] The expression "there is no degradation of the specificity" means that the increase in the threshold value is less than or equal to 25%. The threshold value is the average of the signals obtained for negative samples at the end of the analysis process plus 12 times the standard deviation of the signals of these samples.
[0028] A strong control marker can be neutral, positively charged or negatively charged. By "positively or negatively charged molecule" is meant, in the present application, a molecule which has a global charge respectively positive or negative, in particular which comprises globally one, two, three, four or more than four charge (s) respectively positive (s) or negative (s). A resistant control marker may comprise or be coupled to a carrier molecule, for example a protein such as BSA. When the resistant control marker "comprises" a carrier molecule, generally it comprises or consists of a control marker (for example a fluorophore) and a carrier molecule (for example BSA), said control marker being coupled to said carrier molecule . This coupling may make it possible to make a control marker that is not resistant in the absence of coupling resistant. The coupling between a control marker or a resistant control marker and a carrier molecule results from a covalent or non-covalent bond between the resistant control marker and the carrier molecule, preferably a covalent bond. A carrier molecule covalently or non-covalently coupled to the control label or the resistant control label is, for example, selected from the group consisting of BSA, immunoglobulin (also called Ig), dextran, poly -lysine and a mixed co-polymer, in particular a co-polymer of different amino acids (co-polymer lysine-tyrosine for example). A resistant control marker according to the invention may be for example a marker selected at the end of the method of selecting a resistant control marker as defined below.
[0029] Resistant Control Fluorophore A preferred resistant control marker according to the invention is a fluorophore. It is, in this case, called resistant control fluorophore. A fluorophore, also called a fluorochrome or fluorescent molecule, is a substance, in particular a chemical substance or protein (or polypeptide), capable of emitting fluorescence light after excitation with a light energy. A fluorophore generally comprises a plurality of conjugated aromatic rings and / or planar and cyclic molecules which have one or more u bonds.
[0030] A fluorophore may be a fluorescent protein, for example B-Phycoerythrin. A resistant control marker according to the invention, in particular a resistant control fluorophore, is preferably characterized in that its excitation spectrum does not cover the emission spectrum of a detection ligand of a detection ligand. of an analyte or corresponding to a detection marker of an analyte detection ligand (used in an analysis method) and in that its emission spectrum does not overlap or partially overlap the emission spectrum a detection marker of an analyte detection ligand or corresponding to a detection marker of an analyte detection ligand (used in an assay method). The "emission spectrum or excitation spectrum corresponding to a detection ligand detection marker" is, for example, the emission spectrum or the excitation spectrum of a direct marker or a reporter an indirect detection marker coupled to the detection ligand, a reporter of an indirect detection marker coupled to a reporter (in particular a marker for indirect detection of a detection ligand), or a compound produced by the reaction of an enzyme reporter with its substrate. In the case of a resistant fluorophore according to the invention, the excitation spectrum may correspond to the absorption spectrum.
[0031] A preferred resistant control marker according to the invention, in particular a preferred resistant control fluorophore, is a fluorophore characterized in that its excitation spectrum does not cover the emission spectrum of luminol, of one of its analogues, in particular isoluminol, and / or one of their derivatives, and in that its emission spectrum does not overlap or partially cover the emission spectrum of luminol, of one of its analogues, in particular of the isoluminol, and / or a derivative thereof.
[0032] "Emission spectrum of luminol, an analogue thereof, in particular isoluminol, or a derivative thereof" means the emission spectrum of the chemiluminescent compound resulting from the reaction of luminol, a its analogues, in particular isoluminol, or a derivative thereof with a peroxidase enzyme.
[0033] Preferably, a resistant control marker according to the invention, in particular a resistant control fluorophore, is characterized in that its emission spectrum does not cover the emission spectrum of luminol, isoluminol or a derivative thereof. . When the emission spectrum of a resistant control marker according to the invention, in particular of a resistant control fluorophore, partially overlaps the emission spectrum of the detection marker of one or more detection ligands or corresponding to the detection marker of one or more detection ligands, it is advantageous to use a filter making it possible to eliminate the wavelengths emitted by said detection marker in order to retain only the specific signal emitted by the fluorophore and / or a filter for eliminating the wavelengths emitted by said fluorophore, to retain only the specific signal emitted by said detection marker. In a preferred embodiment, there is no resonance energy transfer between: the one or more resistant control markers according to the invention, in particular the resistant control fluorophore (s), and the one or more markers of detecting a detection ligand and / or the one or more compounds producing a signal corresponding to the detection ligand detection marker (s) used in the analysis method, in particular between the one or more markers of the detection ligand resistant control according to the invention (in particular the resistant control fluorophore or fluorophores) and a luminescent compound obtained by reaction of luminol, of one of its analogues, for example isoluminol, and / or of one of their derivatives with a peroxidase enzyme. In one advantageous embodiment, the one or more resistant control markers according to the invention, in particular the resistant control fluorophore (s), have excitation wavelength ranges and wavelength ranges. emission which are strictly greater than the emission wavelength ranges of luminol, of one of its analogues, for example isoluminol, and / or of one of their derivatives, or strictly less than the length ranges of emission wave of luminol, an analogue thereof, for example isoluminol, and / or a derivative thereof.
[0034] By way of example, when luminol is used as a substrate, a resistant control marker according to the invention, in particular a preferred resistant control fluorophore according to the invention, emits no light at around 425 nm in particular from 375 nm to 550 nm, preferably from 375 nm to 580 nm, more preferably from 350 nm to 580 nm. In other words, when luminol is used as a substrate, a preferred resistant control fluorophore according to the invention emits light outside wavelengths from 375 nm to 550 nm, from 375 nm to 580 nm. nm or from 350 nm to 580 nm. It can for example emit light only at wavelengths lower (or lower or equal) at 375 nm, 370 nm, 360 nm or 350 nm, or only at higher wavelengths (or greater or equal) at 550 nm, 560 nm, 570 nm, 580 nm, 590 nm or 600 nm.
[0035] The expression "from a value X to a value Y" means that the terminals X and Y are included. In a particular embodiment, surprisingly, the basic pH of the reaction medium containing luminol, an analogue of luminol and / or one of their derivatives, the presence of peroxide, one or more agents promoting electron transfer and an acylation catalyst in the reaction medium do not cause a spectral shift causing a problem in the detection of the signal emitted by the resistant control marker according to the invention and in particular by the fluorophore of resistant control according to the invention. According to a particular embodiment of the invention, a resistant control marker according to the invention, in particular a resistant control fluorophore according to the invention, has an absorption window which does not include the wavelength range. corresponding to the signal emitted by the detection ligand detection marker and in particular which does not include the luminol emission wavelength range. For example, a preferred resistant control fluorophore according to the invention does not absorb wavelengths ranging from 375 to 530 nm, from 375 to 550 nm, or from 350 to 580 nm (inclusive). Thus, one can for example use a fluorophore that absorbs only wavelengths lower (or lower or equal) at 375, 370, 360 or 350 nm, or a fluorophore that absorbs only higher wavelengths (or higher or equal) at 530, 540, 550, 560, 570, 580, 590 or 600 nm.
[0036] According to a particular embodiment, a resistant control marker can be a neutral fluorophore, a positively charged fluorophore or a negatively charged fluorophore or a fluorescent protein, said fluorophore being optionally coupled to a carrier molecule, for example a protein such as BSA. . According to a particular embodiment, a resistant control marker comprises or consists of at least one fluorophore and at least one carrier molecule (for example BSA), said at least one fluorophore being coupled to said at least one carrier molecule, and said at least one fluorophore which may be, for example, a neutral fluorophore, positively charged or negatively charged, or a fluorescent protein. A carrier molecule is in particular as defined above in the "analyte" section. A resistant control fluorophore is, for example, selected from the group consisting of a coumarin, a rhodamine, a carbopyronine, an oxazine, a benzopyrylium derivative, their derivatives, and a phycoerythrin. A resistant control fluorophore is for example chosen from the compounds described in application W000 / 64986 A1 and / or marketed by Atto-Tec. By way of example, mention may be made of Atto 633 carbopyronine marketed by Atto-Tec and its derivatives, in particular an amine derivative, as well as benzopyrylium derivatives such as those marketed by Dyomics, in particular Dye 634. when coupled to a carrier molecule, for example BSA. A preferred carbopyronine according to the invention is a molecule comprising the following basic structure: + N A carbopyronine which can be used as a resistant control fluorophore, such as the amine derivative of Atto 633, has for example the following characteristics: maximum absorption wave = 629 nm, molar absorption coefficient at the maximum absorption wavelength = 1.3 x 105 M-1 cm-1, maximum emission wavelength = 657 nm and quantum yield = 64 `Vo. A benzopyrylium derivative that can be used as a resistant control fluorophore, for example, has the following characteristics: maximum absorption wavelength (in ethanol): 635 nm, maximum emission wavelength (in ethanol) ): 658 nm, and molar absorption coefficient at the maximum absorption wavelength: 200,000 M-1cm-1.
[0037] It is for example the fluorophore called Dye 634 (in form coupled to a carrier molecule, for example BSA) of formula: 3Na + Enzyme as a resistant control marker Another example of a resistant control marker according to the invention is an enzyme producing for example a luminescent compound by reaction with a substrate of said enzyme. Preferably, the luminescent compound is a chemiluminescent compound. An enzyme that can be used as a resistant control marker according to the invention is, for example, an alkaline phosphatase or a luciferase.
[0038] An alkaline phosphatase substrate is, for example, the Lumi-Phos 530 or Lumi-Phos substrate sold by Lumigen. A luciferase substrate is, for example, the luciferin or coelenterazine substrate. When the resistant control marker is an enzyme producing a luminescent compound, the detected signal corresponding to the resistant control marker is the signal emitted by the luminescent compound produced by reaction of said enzyme with a substrate of said enzyme.
[0039] Signal detection The signal produced by the detection ligand detection marker (s) or produced by the resistant control marker (s) is detected directly or indirectly. Depending on the type of label used, the signal may for example be detected in fluorescence or in luminescence, in particular in chemiluminescence. The signal emitted by a fluorophore type marker can be read directly in fluorescence. An enzyme type marker requires the addition of a substrate for the production of a detectable product, for example the addition of a substrate for the production of light. A strong, electroluminescent light-emitting control marker requires the addition of a Ruthenium complex and the addition of tripropylamine (TPA), as well as the application of an electric current to produce light. As indicated above, an indirect marker of biotin type requires the addition of a reporter, preferably a reporter coupled to a direct or indirect detection marker. If the reporter is coupled to an enzyme-like indirect marker, for example peroxidase, it is necessary to add in a subsequent step the substrate of this enzyme, for example luminol or a luminol analogue, such as isoluminol or a derivative of luminol or a luminol analogue. The signal may advantageously be detected by means of a device according to the invention, as defined below. In a preferred embodiment, the signal emitted by the resistant control label (s) is detected in fluorescence and the signal emitted by the detection ligand detection marker (s) is emitted in luminescence, for example in chemiluminescence. Generally, the chemiluminescence reaction is carried out by means of a kit comprising at least two solutions. The first solution comprises the peroxidase substrate, for example luminol, isoluminol and / or a luminol or isoluminol derivative, and an electron mediator; the second solution comprises an oxidant. By way of example, it is possible to use the "Immun-star western C" kit (Bio-Rad, United States), "ELISTAR ETA C Ultra ELISA" (Cyanagen, Italy), "Supersignal West Pico" ( Thermo Scientific, USA), "Chemiluminescent Sensitive Plus HRP" (Surmodics, USA). Solid support for improved detection of at least one analyte in a sample The support (s) used for carrying out the analysis method according to the invention are solid supports.
[0040] According to a particular embodiment, a solid support is obtained by the method for preparing a solid support according to the invention. A solid support may be in any material suitable for carrying out the analysis method. A solid support is for example a support based on a polymer or a mixture of polymers.
[0041] A suitable solid support is, for example, a carrier of polystyrene, polypropylene, poly (meth) acrylate, polybutadiene or combinations thereof. Another example of a suitable solid support is a membrane, for example a nitrocellulose membrane, PVDF (polyvinylidene fluoride), nylon or combinations thereof.
[0042] Yet another example of a suitable solid support is an inorganic support, for example a glass slide and / or a metal support. A preferred solid support is polystyrene or polypropylene. A solid support comprises at least one compartment (also called zone of analysis), preferably at least two compartments. According to a particular embodiment of the invention, a solid support comprises a single compartment. Said single compartment may be a compartment comprising one or more walls. Alternatively, said single compartment may be free of walls and then assimilate to the solid support itself. The bottom of the compartment can then consist of the upper face of the solid support. An example of such a solid support comprising a single compartment with or without one or more walls is a blade or a membrane. According to a particular embodiment of the invention, the solid support, which may be for example a microplate or a membrane, comprises at least two compartments. When the solid support comprises at least two compartments, they are isolated from each other, so that they do not communicate with each other, that is to say, so that the different compositions or solutions used for the analysis can not not move from one compartment to another during the analysis. Thus, a solution added in one compartment does not go into the other compartments. For example, the compartment or compartments comprise or consist of a bottom and one or more walls, the said wall or walls isolating the compartment or compartments from each other so that they do not communicate with each other. Typically, at least one (for example one or two) compartment of the solid support is used per sample to be analyzed.
[0043] In a particular embodiment of the invention where the solid support (for example a slide or a membrane) comprises a single compartment, typically at least one (for example one or two) solid support is used per sample to be analyzed. . The solid support is for example a microplate. In this case, an example of a compartment is a well. A microplate is typically a 96-well or 384-well microplate. The present invention thus relates to a solid support for the detection of at least one analyte in at least one sample, characterized in that said solid support comprises at least one compartment comprising at least one spot, said spot comprising at least one marker resistant control and at least one capture ligand. A resistant control marker is especially as defined above, in particular in the paragraphs "resistant control marker", "resistant control fluorophore" and "enzyme as resistant control marker" or as obtained by the selection method defined below in the "method of selecting a resistant control marker". In particular, said one or more resistant control markers are markers that remain at least partially fixed at said spot on the surface of the solid support during the implementation of a method for detecting at least one analyte, so as to produce a detectable signal at the conclusion of said detection method.
[0044] A compartment of the solid support for analyzing a sample comprises at least one spot, for example two spots, three spots, four spots or five spots, or at least six spots, preferably six spots, seven spots, eight spots, more preferably at least one spot. minus nine spots, for example nine spots, ten spots, eleven spots, twelve spots, thirteen spots, fourteen spots, fifteen spots, sixteen spots or more than sixteen spots.
[0045] "Spot" here means an area on the surface of a compartment of a solid support comprising at least one resistant control marker and at least one compound of interest, for example a capture ligand. The resistant control label (s) and the compound (s) of interest are thus fixed at the same time to said zone on the surface of the compartment by non-covalent physicochemical interactions ( in particular of the weak bonds type, for example, ionic, van der Waals, hydrogen and / or hydrophobic) and / or by covalent bonds.
[0046] A spot may comprise, in addition to the compound (s) of interest, at least one polymer, in particular at least one polymer comprising hydrophilic groups, for example at least one hydrogel. In a particular embodiment, all the spots of a compartment, preferably all the spots of a solid support, comprise a single resistant control marker or a unique mixture of resistant control markers that can be used at the same concentration. in all spots or at different concentrations. Alternatively, different (at least two) resistant control markers and / or different (at least two) mixtures of resistant control markers can be used in the spots of the same compartment of a solid support. A spot corresponds to a well-defined zone, for example ranging from 0.0078 mm 2 to 5.309 mm 2, preferably from 0.196 mm 2 to 3.142 mm 2, more preferably from 0.503 mm 2 to 2.011 mm 2. A spot may be discoidal, cylindrical or hemispherical or approximately discoidal, cylindrical or hemispherical, for example oval, particularly when the solid support is a microplate or a blade. Alternatively, a spot may be square or rectangular (especially a band), for example when the solid support is a membrane, or any other form.
[0047] The spots are obtained by techniques well known to those skilled in the art, such as those disclosed in US Pat. No. 7,470,547 B2, US Pat. No. 6,576,295 B2, US Pat. No. 5,916,524 and US Pat. spot is obtained by depositing at least one drop of a solution containing a determined quantity of at least one resistant control marker and at least one compound of interest at a specific location on the surface of the support compartment solid. When a spot comprises at least one polymer (for example at least one hydrogel), said spot can be obtained by depositing at least one drop of a solution containing a determined quantity of at least one resistant control marker and at least one compound of interest at a specific location on the surface of the compartment on which said polymer has been deposited beforehand. The surface of a compartment is also called "solid phase". A compound of interest is generally a capture ligand, particularly a capture ligand of an analyte. One or more spots of a compartment may serve as a control spot and thus comprise a capture ligand which is not intended to detect an analyte of the sample, or comprise another compound of interest.
[0048] The present invention particularly relates to a solid support for detection of at least one analyte in a sample, characterized in that said solid support comprises at least two compartments comprising at least one spot, said spot comprising at least one control marker resistant and at least one capture ligand.
[0049] In a particular embodiment, some or all of the compartments of a solid support have the same spot composition. In another particular embodiment, a part or all of a solid support or compartments of a solid support comprises or consists of several (for example two) distinct groups (or types) of spots or compartments, each of distinct groups having a different spot composition (by the number of spots and / or by the resistant control label (s) and / or the capture ligand (s) and / or the compound (s) of interest present in the spots of each group). The subject of the present invention is also a process for preparing a solid support for the detection of at least one analyte in at least one sample comprising the following steps: ) depositing, on the surface of at least one compartment, preferably at least two compartments, a solid support, a mixture comprising at least one resistant control marker and at least one capture ligand, to obtain a spot, b ) repeating step a) n-1 time, n being an integer greater than or equal to 1, to obtain n spots intended for the detection of an analyte on the surface of said compartment or compartments, c) optionally saturating the surface of said or said compartments, and d) optionally drying the surface of said compartment or compartments. The solid support, the resistant control marker (s) and the capture ligand (s) are in particular as defined above.
[0050] In step a), a mixture comprising at least one resistant control marker and at least one capture ligand is deposited on the surface of at least one compartment of a solid support, preferably at least two compartments of a solid support, to get a spot. In step a), said one or more resistant control markers are markers which remain at least partially fixed at said spot on the surface of the solid support during the implementation of a detection method of at least an analyte, so as to produce a detectable signal upon completion of said detection method. A resistant control marker is in particular as defined in the present application.
[0051] Said resistant control marker (s) are preferably fluorophores, for example a mixture of fluorophores. The fluorophore (s) may be fluorescent chemical molecules or fluorescent proteins. Said capture ligand or ligands are preferably selected from the group consisting of an antibody, an antigen, a nucleic acid and combinations thereof.
[0052] The mixture is preferably a solution. A resistant control marker is present in the mixture at a concentration which does not interfere with the binding of the capture ligand (s) to the compartment surface and which allows for detection of the signal produced by said control resistant resistor. the end of the analysis process.
[0053] By way of example, the mixture comprises 0.1 to 100 μg / ml of a capture ligand and 0.01 to 100 μg / ml of a control label, preferably in a buffer solution, for example a solution of TBS (Tris Saline Buffer). The concentration of a resistant control marker in the mixture may for example be determined by the method comprising the following steps: depositing at the surface of a solid support at least two solutions, each solution comprising said resistant control marker and a ligand capture, the concentration of the resistant control marker being increasing from one solution to another and the concentration of the lowest resistant control marker being greater than or equal to a predefined minimum concentration, possibly saturate the surface of the solid support, c that is to say, to put the surface of the solid support in the presence of an agent making it possible to avoid non-specific bonds to the solid support, optionally to dry the surface of the solid support, to put in the presence of spots a sample comprising an analyte whose capture ligand is specific, put in the presence of spots a detection ligand specific for said analyte e, said detection ligand being coupled to a direct or indirect detection marker, when said detection ligand is coupled to an indirect detection marker, adding a reporter (also called first reporter) of said indirect detection marker and when said (first ) reporter is coupled to an indirect detection marker, add a second reporter of the indirect detection marker coupled to said (first) reporter of the indirect detection ligand detection marker, detect a signal produced by the resistant control marker, detect a signal produced by the detection ligand detection marker, and selecting a resistant control marker concentration which makes it possible to detect both the signal produced by the resistant control marker and the signal produced by the detection ligand detection marker.
[0054] The concentration of the resistant control marker preferably makes it possible to detect both the signal produced by the resistant control marker and the signal produced by the detection ligand detection marker, without significant loss of sensitivity compared to a spot. not including the resistant control marker, i.e., the functionality of the assay method for the user is not affected by the presence of the resistant marker, in particular the decrease in sensitivity is less than 40%, preferably less than 35%, more preferably less than 30%, more preferably less than 25%. According to a particular embodiment, the concentration of the resistant control marker further makes it possible to detect the signal produced by the resistant control marker and the signal produced by the detection ligand detection marker without degradation of the specificity. The expression "there is no degradation of the specificity" means that the increase in the threshold value is less than or equal to 25%.
[0055] The threshold value is the average of the signals obtained for negative samples at the end of the analysis process plus 12 times the standard deviation of the signals of these samples. When a detection ligand is coupled to an indirect detection marker, a reporter of said indirect detection marker is added. When said reporter (called first reporter) is itself coupled to an indirect detection marker, for example an enzyme, a second reporter, for example a substrate, indirect detection marker coupled to said first reporter is added.
[0056] The minimum concentration of predefined resistant control marker can, for example, be determined by the following method: depositing at the surface of a solid support at least two solutions, each solution comprising a resistant control marker and not including a ligand of capture, the concentration of the resistant control marker being increasing from one solution to another, to form at least two spots, possibly saturate the surface of the solid support, that is to say put the surface of the solid support in the presence an agent making it possible to avoid non-specific bonds to the solid support, possibly drying the surface of the solid support, possibly performing at least one of the following steps: (i) carrying out one or more washing steps, (ii) setting the the spots in the presence of a detection ligand, (iii) put the spot or spots in the presence of a reporter, (iv) put the spot or spots in the presence of a substrate, detect er the signal produced by the resistant control marker, and select the minimum concentration of resistant control marker that allows the detection of a signal. Deposition of the mixture (or solution) on the surface of the solid support or a compartment of the solid support can be performed manually, but preferably in an automated manner by a suitable device. Thus, as indicated above, a resistant control marker binds to the solid phase of the analysis support at the same time as the capture ligand (s) present in the mixture (or the solution).
[0057] In a particular embodiment of the invention, each spot of a compartment of the solid support intended for the detection of at least one analyte comprises at least one different capture ligand, preferably intended to detect an analyte per spot. However, several spots of a compartment may comprise at least one capture ligand.
[0058] In a particular embodiment of the invention, the same spot may comprise several different capture ligands (for example several antibodies and / or antigens), which are generally specific for the same pathology, infection or disease to be detected (in particular the same virus, the same bacterium, the same fungus or the same parasite), the evolution of an infection or disease, a condition (pathological or not) of a subject, at a risk of developing a condition (pathological or not) or a marker of resistance to a treatment.
[0059] Several spots or all the spots of a compartment may comprise capture ligands intended for the detection of the same analyte; they are, for example, different capture ligands specific for the same analyte or even the same capture ligand present at different concentrations in the spots.
[0060] In a particular embodiment, the resistant control marker or the mixture of resistant control markers is the same in all spots, and it may be added or not at the same concentration in each spot. Alternatively, different (at least two) resistant control markers or different mixtures of at least two resistant control markers may be used in different spots of a compartment. A compartment may also include one or more spots without a strong control marker. However, preferably, all the spots of said compartment comprise a resistant control marker. A compartment may also comprise one or more spots without a capture ligand, but preferably comprising another compound of interest. In a particular embodiment of the invention, all the compartments of the support have the same spot composition. In another particular embodiment of the invention, some or all of the compartments of a solid support comprise or consist of several (for example two) distinct groups of compartments, each of the distinct groups having a different spot composition. Steps a) and b) are performed in part or in all compartments of the solid support, preferably in all compartments of the solid support. The mixture deposited on the surface of at least one compartment of the solid support is incubated for example for a few seconds to a few hours, for example at a temperature of between 4 ° C. and 40 ° C. The method comprises a possible step c) of saturation of the surface of said compartment (s) of the solid support, that is to say of putting the surface of the solid support in the presence of an agent making it possible to avoid non-specific bonds. to solid support. The purpose of the saturation step is notably to prevent the non-specific binding of compounds during the implementation of the analysis method. The agent which makes it possible to avoid non-specific bonds to the solid support is, for example, a saturation solution well known to those skilled in the art.
[0061] In the optional step d), the surface of the compartment or compartments is dried.
[0062] The drying is, for example, carried out at 56 ° C. or 37 ° C. at room temperature. In a particular embodiment, step a), step b) and / or step c) are followed by one or more washing steps.
[0063] The present invention also relates to a method for preparing a solid support as defined above, comprising a subsequent step e) of controlling the quality of the spots. If the control of the quality of a spot is positive, said spot can be used as part of an analysis process. If the control of the quality of a spot is negative, said spot can not be used as part of an analysis method, possibly as well as the compartment containing said spot.
[0064] The present invention also relates to a kit (or kit) characterized in that it comprises or consists of at least one solid support according to the invention or obtained by the preparation process according to the invention and, where appropriate, less a composition or solution to be used to implement an analysis method according to the invention and / or a user manual.
[0065] Device for Improved Detection (or Device for Dual Detection) Another object of the present invention is to provide a device for the detection of at least one analyte in a sample, said device comprising: - means for detecting a first signal and a second signal produced at a solid support, and - means for defining a reading gate from the location of said first signal and for reading said second signal at said reading gate. According to a particular embodiment, this reading corresponds to a quantization of the second signal at the level of the regions of interest. The detection of the first signal can be done before, after or simultaneously with the detection of the second signal. The solid support is in particular as defined above. The first signal may be a fluorescence or luminescence signal, for example in chemiluminescence.
[0066] The second signal may be a signal in fluorescence or luminescence, for example in chemiluminescence. In a preferred embodiment, the first signal is a fluorescence signal and the second signal is a luminescence signal, preferably chemiluminescence (or vice versa). The means for detecting a first signal and a second signal produced at a solid support are called optical bench. An optical bench may for example comprise or consist of: a lighting system, a telecentric lens, a filter wheel, said telecentric lens being preferably coupled on its output lens to said filter wheel, and a camera . The lighting system is preferably a controllable lighting system.
[0067] The lighting system makes it possible to illuminate the solid support with more or less intensity, for example to reveal the fluorescence or the geometry of the solid support used. The telecentric lens is preferably large. In particular, the telecentric lens covers the entire solid support.
[0068] The telecentric lens makes it possible to image the entire surface to be measured without distorting the image, to eliminate the parallax error inherent in the standard lenses and to guarantee a homogeneity of the signal over the entire area viewed. The telecentric lens is preferably coupled on its output lens to a filter wheel.
[0069] The filter wheel makes it possible to present different filters between the telecentric lens and the camera (also called image taking camera). In a preferred embodiment, it is thus possible to select the wavelengths reaching the camera sensor in order to be able, in one case, to separate the excitation signals from the lights from those emitted by at least one resistant control marker. (in particular at least one fluorophore) by the presence of a filter and, in the other case, to collect the maximum of the signal produced by at least one detection marker of an analyte detection ligand by the presence a neutral window does not filter signal but ensuring the maintenance of the optical properties of the whole. The camera makes it possible to take an image with varying exposure times.
[0070] The optical bench thus defined makes it possible to take a succession of images of the same medium, with lighting parameters, filtering and / or taking different images, such as: an image in the visible (also called positioning image) for example by using illumination of the illumination system, but no specific filter, - a fluorescence image (also called a detection image) using lighting of the illumination system and a filter specific for the desired fluorescence an image without illumination and without a specific filter (also called an analysis image), for example to reveal a signal in chemiluminescence, means for defining a reading gate from the location of said first signal and for reading the second signal at said reading gate may comprise or consist of an imaging system. In an advantageous embodiment, the optical bench is therefore associated with an imaging system that makes it possible to implement an analysis method comprising several steps that notably guarantee the accuracy and robustness of the analysis performed, said steps comprising: 1. search and positioning of the solid support and the compartment or compartments of the solid support, in particular thanks to the positioning image; this treatment aims to reduce any mechanical positioning error of the solid support and makes it possible to use a mechanism that does not have a high positioning accuracy; 2. from the position of the compartment or compartments determined in step 1, positioning in each compartment of a theoretical grid representing the theoretical position of each of the spots; this positioning can be done from a reference grid, for example described in the analysis system, said reference grid indicating the coordinates of each of the spots with respect to a reference point of the compartment; the reference point of the compartment is, for example, the center of the compartment; 3. search, using the detection image made with the illumination and the fluorescence filter of all fluorescent events existing in the compartment or compartments of the solid support, and possibly the rejection of certain events to the using a selection of events by their size and shape so as to eliminate those that are totally outside the expected specifications; 4. comparison between: o the position of the events previously detected and considered as valid, o the theoretical position of the expected spots as defined in step 2; 5. for each of the expected spots, association with the closest detected event and being in intersection with the theoretical spot surface so as to form "theoretical spot" / "fluorescent event" pairs making it possible to detect the missing spots. For each theoretical spot, a distinct and unique fluorescent event must exist; 6. characterization of each of the spots detected by criteria of diameter, shape, distance from the theoretical position to ensure the integrity of the spots detected; 7. for each of the detected spots, definition of the corresponding region of interest, called reading grid, in particular defined by the surface and the coordinates of the spot detected; and 8. reading the second signal on the reading gate defined in step 7, in particular quantizing the second signal on the analysis image at this region of interest, that is to say from the reading grid. Method of analysis with spot quality control The analysis method according to the invention allows the detection of at least one analyte in at least one sample. The present invention particularly relates to a method for detecting at least one analyte in at least one sample comprising the following steps: a) placing a sample to be analyzed in the presence of the spot or spots of a compartment of a solid support, said spot or at least one of said spots comprising at least one resistant control marker and at least one capture ligand of an analyte, b) placing at least one spot of said compartment at least one analyte detection ligand, said analyte detecting ligand being coupled to a direct or indirect detection marker, c) when at least one analyte detecting ligand is coupled to an indirect detection marker, bringing said one or more spots of said analyte compartment a reporter of said indirect detection marker, d) when the reporter used in step c) is coupled to an indirect detection marker, put in the presence of spot or spots of said compartment a r supplying the indirect detection marker coupled to said reporter used in step c), e) detecting a signal produced by at least one resistant control marker in said compartment, f) defining a reading gate from the location of the detected signal in step e), g) optionally, detecting a signal produced by at least one detection marker of an analyte detection ligand, and h) optionally, reading the signal detected in step g) on the reading gate defined in step f), said one or more resistant control markers being markers which remain at least partially fixed at the spot on the surface of the solid support during the implementation of said detection method of at least one analyte, so as to produce a detectable signal in step e).
[0071] Steps a) to d) are always performed before steps e) to h). Step g) can be performed before or after or simultaneously with step e). In a particular embodiment, step g) is performed before or after step e). Step f) is always performed after step e). Step f) is always performed after step e) and before step h), and can be performed before, after or simultaneously in step g). Step h) is always performed later in steps e) to g). Steps a) to f) and, where appropriate, step g) and / or step h) are in particular carried out for each compartment of a solid support comprising at least one spot comprising at least one resistant control marker and at least one capture ligand of an analyte, wherein a sample is analyzed. Steps g) and / or h) may not be performed if the reading gate defined in step f) indicates no zone for reading the signal to be detected in step g). If step h) is performed, step g) is also performed. The method may advantageously comprise one or more washing steps, for example between each or some of the steps a) to d).
[0072] The washing of each compartment intended for the analysis of a sample comprises at least one cycle, preferably 3 to 6 cycles of distribution and suction of a volume (for example 400 μL) of a solution of washing (for example a solution of 0.01M Tris NaCl buffer, pH 7.4, supplemented with 0.1% Tween 20).
[0073] According to a particular embodiment of the invention, no operation and in particular no pipetting, dispensing, stirring, suctioning or washing step is performed between steps e) and g), whatever the order in which the steps e), f) and g) are performed. The expression "putting a compound X in the presence of the spots of a compartment" means that the compound X is added to a compartment comprising said spots, said compartment being intended to analyze a sample. When at least two compounds are to bring into contact with one or more spots of a compartment during the same step and / or when at least two of the steps a) to d) are carried out simultaneously, said compounds can be in the presence of said spot or spots separately, that is to say provided in the form of separate compositions; alternatively, said compounds or some of said compounds may be brought into contact with one or more spots of a compartment in the form of one or more mixtures. The detection method is in particular implemented using a solid support as defined above or obtained by the preparation method as defined above. The one or more resistant control markers, the spot (s), or the capture ligand (s) of an analyte are in particular as defined above. In particular, the one or more resistant control markers are markers which remain at least partially fixed at said spot on the surface of the solid support during the implementation of said method of detecting at least one analyte, so that produce a detectable signal during step e), that is to say when the analysis process has proceeded without deterioration of a spot. The present invention particularly relates to a process as defined above, characterized in that said, said or one of said resistant control markers is (are) a fluorophore (s), for example one or more fluorescent chemical molecules or one or more fluorescent proteins, for example a mixture of fluorophores. The present invention particularly relates to a method as defined above, characterized in that said, said or one of said resistant control markers is a resistant control marker, in particular a fluorophore, whose excitation spectrum does not cover not the emission spectrum of the signal emitted by or corresponding to the detection marker of said one or more analyte detection ligands and whose emission spectrum does not overlap or partially cover the emission spectrum of or corresponding to the detecting said one or more analyte detection ligands. The subject of the present invention is, for example, a process as defined above, characterized in that said, said or one of said resistant control markers is a resistant control marker, in particular a fluorophore, whose excitation spectrum does not does not cover the emission spectrum of the signal emitted by a luminescent compound obtained by reaction of luminol and / or an analogue and / or a derivative of luminol or luminol analogue with a peroxidase enzyme and whose spectrum of emission does not cover or partially overlap the emission spectrum of a luminescent compound obtained by reaction of luminol and / or an analogue and / or a derivative of luminol or luminol analogue with an enzyme peroxidase.
[0074] The subject of the present invention is particularly a process as defined above, characterized in that: the signal produced by at least one detection marker of an analyte detection ligand is the light emitted by a chemiluminescent compound obtained by reacting a peroxidase with luminol, a luminol analogue and / or a derivative of luminol or a luminol analogue, and the signal produced by the one or more resistant control markers is a light emitted outside the luminol lengths; waves ranging from 375 nm to 550 nm, from 375 to 580 nm, or from 350 to 580 nm (the terminals being included). Preferably, the one or more resistant control markers have an excitation spectrum and, preferably, an emission spectrum outside wavelengths ranging from 375 nm to 550 nm, from 375 to 580 nm, or from 350 at 580 nm (limits included). The method of analysis is, for example, an immunotest and in particular an enzyme immunoassay, such as an Enzyme-Linked ImmunoSorbent Assay (ELISA). The analysis method can also be used for the detection of nucleic acids. In step a), a sample to be analyzed is placed in the presence of the spot or spots of a compartment of the solid support. When several samples are analyzed and the solid support comprises several compartments, step a) comprises bringing a sample into contact with the spot or spots of at least one compartment of a solid support, said solid support comprising at least as many compartments comprising at least one spot comprising at least one resistant control marker and at least one capture ligand of an analyte that the number of samples to be analyzed or more solid supports are used.
[0075] When several samples are analyzed and the solid support comprises a single compartment, step a) comprises adding a sample to the compartment of a solid support (or, when said compartment is assimilated to the solid support itself). same, adding a sample to said solid support), and at least as many solid supports comprising a compartment comprising at least one spot comprising at least one resistant control marker and at least one capture ligand of at least one analyte as the number of samples to be analyzed. When multiple samples are analyzed, a different compartment is used for each sample. It is also possible to use several compartments of one or more solid supports to analyze the same sample, in particular if said compartments have a different spot composition. Steps a) and b) can be performed simultaneously, step a) before step b) or step b) before step a). When step b) is performed before step a), the method does not include a washing step between these two steps a) and b).
[0076] When present, steps c) and d) are preferably carried out after steps a) and b) and there is preferably at least one washing step between steps a) and b) and steps c) and d). When present, steps c) and d) can be performed simultaneously or step c) before step d). When step d) is carried out before step c), the method does not include a washing step between these two steps d) and c). When present, step d) is preferably carried out after step c) and there is at least one washing step between steps c) and d). In step b), at least one analyte detection ligand is brought into contact with the spot or spots of said compartment. Step b) is in particular carried out in each compartment intended for the analysis of a sample. The detection ligand or ligands of an analyte are in particular as defined above.
[0077] Preferably, step b) comprises adding at least one detection ligand of each analyte to be detected. When different detection ligands of an analyte are used, they may be added simultaneously or successively, some or all of the ligands may be provided as separate compositions or one or more mixtures. Part of the detection ligands of an analyte may be added simultaneously to step a), followed preferably by a washing step before adding the remainder of the detection ligands of an analyte. When detection ligands of an analyte are added successively in step b), each addition of at least one analyte detection ligand may be followed by a compartment washing step for analysis. of a sample.
[0078] The carrying out of steps c) and d) depends on the detection ligand detection marker and, if appropriate, the detection marker detection marker of the detection ligand detection marker. Thus, step c) is performed when at least one analyte detection ligand is coupled to an indirect detection marker.
[0079] Preferably, the detection ligands of an analyte are coupled to the same detection marker. If at least two analyte detection ligands are coupled to different indirect detection markers, step c) is performed for each indirect detection marker, in order to detect the signal of each marker. Step d) is carried out when at least one reporter (also called first reporter) used in step c) is coupled to an indirect detection marker. The reporter used in step d) is called the second reporter. When step d) is carried out, step c) is therefore also performed In step e), a signal produced by at least one resistant control marker is detected in said compartment.
[0080] If at least two resistant control markers are used, step e) preferably comprises detecting a signal produced by each of said resistant control markers. The detection of the signal produced by at least one resistant control marker makes it possible, in particular, to localize each spot comprising at least one resistant control marker in each compartment of the solid support. The signal produced by at least one resistant control marker is preferably a fluorescence emitted signal. In step f), a read pattern is defined from the location of the signal detected in step e).
[0081] The reading grid indicates precisely in which zone (s) of the compartment the second signal must be read (or "analyzed" or "taken into account" or "interpreted"). In particular, a second signal detected in step g) outside the one or one of the zones defined in the reading grid is not taken into account in the reading (or "the analysis" or "the interpretation ") of the second signal in step h). It is particularly advantageous to delimit said zones precisely according to the contour of each spot. The step f) of defining the reading gate therefore comprises, in particular, checking the quality of the spots, starting from the signal detected in step e) which corresponds to at least one resistant control marker. By "spot quality" is meant the presence, location and / or integrity of the spot or spots. Spot integrity includes spot size and shape. A control of the quality of a spot is particularly positive if the spot is present in the compartment at a position conforming to that expected, if it has a well-defined outline, if its shape conforms to the acceptability criteria, for example if it is a discoidal shape, approximately discoidal, for example oval or with a circularity greater than 80%, and if it does not intersect another spot of the compartment. "Circularity greater than 80%" means that the detected spot has a circularity sufficient to ensure that it has not been damaged by all the treatments carried out during the analysis process.
[0082] For example, a control of the quality of a spot is positive if a signal is detected in step e), this signal has a well-defined contour, a discoidal shape, approximately discoidal, for example oval or with a higher circularity at 80%, and if it does not intersect the signal produced by another spot of the compartment. If no signal is detected in step e) at a spot or if the spot does not respond to the quality check for at least one of the reasons indicated above, the second signal possibly detected at step g) at said spot is not taken into consideration when reading the second signal in step h), as well as possibly the second signal detected at each spot of the compartment corresponding to said spot.
[0083] In steps e) and g), the detection of a signal preferably comprises the acquisition of a signal. The use of one or more resistant control marker (s) according to the invention thus makes it possible to perform a reading of the second signal at the precise locations where the spots are located, making it possible to improve the sensitivity of the analysis, while securing the results achieved, in particular by eliminating false positive or false negative results related to a spot defect.
[0084] The subject of the present invention is particularly the process as defined above, characterized in that the signal produced by at least one detection marker for an analyte detection ligand is a luminescent signal, in particular a chemiluminescent signal. for example light emitted by reaction of luminol and / or an analogue and / or a luminol derivative or luminol analogue with a peroxidase enzyme. The present invention more particularly relates to a method as defined above, characterized in that the signal produced by at least one resistant control marker is detected in fluorescence and the signal produced by at least one detection marker of a Detection ligand of an analyte is detected in luminescence, especially in chemiluminescence. In a preferred embodiment, at least steps e) to h) are implemented by means of the same device, for example a device as described in the section "device for improved detection". In another preferred embodiment, all the steps of the method are implemented by means of the same device, for example a device as described in the section "device for improved detection".
[0085] The present invention also relates to a method of selecting a resistant control marker comprising the following steps: a) depositing a marker to be tested on the surface of a solid support, for forming at least one spot, b) optionally saturating the surface of the solid support, c) optionally drying the surface of the solid support, d) performing at least one of the following steps: (i) performing one or more washing steps, (ii) put the spot or spots in the presence of a detection ligand, (iii) put the spot or spots in the presence of a protractor, (iv) put the spot or spots in the presence of a substrate, e) select a marker which produces a signal at the end of step d). The marker to be tested is a compound that produces (eg emits) a detectable signal. For example, the marker to be tested is a fluorophore or a luminescent compound.
[0086] The solid support is in particular as defined above. Step a) comprises or consists of depositing a marker to be tested on the surface of a solid support, to form at least one spot. The deposition of the marker can be done manually or automatically by means of a suitable device. The marker to be tested is generally deposited in the form of a solution comprising said marker. Step a) may comprise the deposition of the same marker to be tested in several spots, at increasing concentrations.
[0087] The solution deposited in step a) may or may not comprise a capture ligand. The method comprises a possible step b) of saturation of the surface of the solid support. The purpose of the saturation step is notably to prevent the non-specific binding of compounds during the implementation of the analysis method. In step c), the surface of the solid support is optionally dried.
[0088] Generally, step a) and / or step b) are followed by one or more steps a ') and / or b') of washing. Step d) comprises or consists in carrying out at least one of the following steps, preferably at least two of the following steps, more preferably at least three of the following steps: (i) carrying out one or more washing steps, (ii) carrying out the spot or spots in the presence of at least one detection ligand, (iii) placing the spot or spots in the presence of at least one reporter, and / or (iv) putting the spot or spots in the presence of at least one substrate. In a preferred embodiment, step d) comprises at least step (i), step (ii), step (iii) and step (iv). Steps (i) to (iv) can be performed in any desired order.
[0089] However, in a preferred embodiment, step d) comprises the following steps and in the following order: step (ii), step (i), step (iii), step (i), step (iv) and step (i). Preferably, the one or one of the reporters of step (iii) is the reporter of a detection marker coupled to at least one detection ligand of step (ii) and / or the one or one of the substrates of the step (iv) is a reporter of a detection marker coupled to a reporter.
[0090] For example, the or one of the detection ligands of step (ii) is coupled to a biotin-type detection marker, the one or one of the reporter of step (iii) is a streptavidin-type reporter coupled to an enzyme , for example a peroxidase, and / or the one or one of the substrates of step (iv) is a substrate of said enzyme, for example luminol, a luminol analogue, and / or a derivative of luminol or the like luminol. The selection method comprises a step e) of selecting a marker that produces a signal at the end of step d). Preferably, step e) further comprises selecting a marker which does not interfere or little with the detection of the signal produced by a detection ligand detection marker. The selection process according to the invention may also comprise: optionally in a first step: the steps a) to c) above, in which the spot or spots formed in step a) do not comprise a capture ligand , o optionally a first preselection step comprising the selection of a marker which produces a detectable signal at the end of step c), o optionally steps d) to e) on the same support, preferably said steps n with said selected marker in the first preselection step, followed by a second preselection step comprising selecting a marker that produces a detectable signal at the end of step d), in a second step, steps a) to e) above, wherein the spot or spots formed in step a) comprise a capture ligand, steps d) to e) being preferably carried out only with a marker selected at ladit e first preselection step and / or selected at said second preselection step.
[0091] The present invention also relates to the use of at least one resistant control marker in at least one spot intended to detect an analyte, to secure a method for detecting at least one analyte in a sample or at least one sample.
[0092] By "securing a method for detecting at least one analyte in a sample or at least one sample", the aim here is to guarantee the reliability of the results obtained after said detection method, in particular by avoiding the presence of "false negative "or" false positives ".
[0093] A "false negative" is a negative result reflecting the absence of one or more analytes to be detected in a sample, while said one or more analytes were present in the sample and should have been detected. A "false positive" is a positive result reflecting the presence of one or more analytes to be detected in a sample, while the said analyte (s) were absent in the sample. Securing the method for detecting at least one analyte in a sample is obtained in particular by controlling, at the end of the detection process, the quality of the spot (s) intended for the detection of an analyte and / or by improving the sensitivity of the detection of analytes, through the use of at least one resistant control marker.
[0094] The present invention thus relates to the use of at least one resistant control marker in at least one spot for detecting an analyte for securing a method for detecting at least one analyte in a sample or at least one sample, characterized in that it comprises: controlling the quality of said spot, after said spot has been placed in the presence of the sample and at least one detection ligand of an analyte to be detected, and / or reading the signal produced by at least one detection marker of an analyte detection ligand to be detected on a reading gate defined from the location of the signal produced by said one or more resistant control markers. The present invention particularly relates to the use of at least one resistant control marker in at least one spot for detecting an analyte for securing a method for detecting at least one analyte in a sample or at least one sample, characterized in that it comprises: controlling the quality of said spot, after said spot has been placed in the presence of the sample, of at least one detection ligand of an analyte to be detected coupled to a detection marker indirectly, a reporter (also called first reporter) of said indirect detection marker, optionally a second reporter of an indirect detection marker coupled to said first reporter, and / or - reading the signal produced by at least one detection marker of an analyte detection ligand to be detected on a reading gate defined from the location of the signal produced by said one or more control markers r ésistants.
[0095] The present invention particularly relates to the use as defined above, characterized in that the control of the quality of the spot includes or consists of controlling the presence, location and / or integrity of the spot. The reading gate indicates in which zone (s) of the compartment the signal produced by the one or more detection markers of an analyte detection ligand must be read. Other characteristics and advantages of the invention will emerge more clearly from the examples which follow, given by way of illustration and not limitation.
[0096] FIG. 1: Use of Different Fluorophores and Resulting Images at the Different Steps of the Protocol Described in Example 1. FIG. 2: Acquisition of the 12-well fluorescence signal comprising 9 spots at the end of an analysis process.
[0097] Figure 3: Fluorescence image: Comparison of the theoretical reading grid (continuous white circles) with respect to the actual position of the fluorescence spots (dashed white circles) on the basis of the signal produced by the resistant control marker. Figure 4: Chemiluminescence image: Improvement of the accuracy of the analysis method by defining the reading grid on the real position of the spots detected thanks to the resistant control marker (dashed lines), versus the theoretical position grid (continuous lines ). Figure 5: Chemiluminescence image: Improvement of the accuracy of the analysis process by checking the integrity of the detected spots before validating the result rendering.
[0098] Figure 6: Diagram of an optical bench comprising a lighting system, a telecentric lens, a filter wheel, said telecentric lens being coupled on its output lens to the filter wheel and a camera. Examples Example 1: Example of a method Selection of a Resistant Control Marker Material and Method Within each well of a polystyrene microplate (Greiner, Germany) well drops are deposited 500 nl drops of a fluorophore solution in the buffer used. typically for spotting antigens or antibodies. The following fluorophores are used in this example: Atto 633-amine (supplier: ATTO-TEC, Germany), Dye 634-carboxylic acid (supplier: Dyomics, Germany), APC (Allophycocyanine) (supplier: Febico, Taiwan), B- Phycoerythrin (Febico, Taiwan). The bottom of each well of these microplates has molecule adsorption capabilities known to those skilled in the art. The surface of each well thus obtained is saturated with a saturation solution in itself known to those skilled in the art; the plates are then dried; after a step of rehydrating these plates, a substrate solution containing luminol (ELISTAR ETA C Ultra ELISA (Cyanagen, Italy) (see Example 2) is then added at a rate of 50 μl / well.The fluorescence images are different steps of the protocol described above (i.e. after the drop deposition, saturation, drying, rehydration, and after the addition of the luminol-containing substrate solution), using the Chemidoc ™ MP System (Bio-Rad) having the appropriate filters for fluorophores Results Figure 1 describes the use of different fluorophores and the resulting images at different stages of the protocol described above.
[0099] The fluorescence obtained with the 4 fluorophores is perfectly visible after the droplets have been deposited on the plate. This fluorescence persists for Atto 633-amine, for allophycocyanin and for B-Phycoerythrin after washing the plates. It also persists after addition of the substrate solution containing luminol for Atto 633-amine and for B-Phycoerythrin, very weakly for allophycocyanin. On the other hand, no residual fluorescence is visible at the end of the washing step with the Dye 634-carboxylic acid and a fortiori after addition of the substrate solution. Thus, Atto 633-amine and B-Phycoerythrin are resistant control markers according to the present invention. Furthermore, as illustrated hereinafter in Example 2, Dye 634 coupled to BSA is also a resistant control marker according to the invention.
[0100] Example 2: Absence of impact of the presence of a resistant control fluorophore on the detection of an analyte of interest Material and Method (i) Preparation of the microplate Within each well of a polystyrene microplate (Greiner , Germany) are filed in rows using a robot spotteur, drops of 50 nL of a solution containing the capture ligand / s and a selected fluorophore according to the method described in the application. The capture ligand solution can be: - either an antigen or mixture of antigens that can consist of a recombinant protein associated with one or more synthetic peptides as part of an antibody detection test, or an antibody or a mixture of antibodies directed against the desired marker in the case of an antigen detection assay. These capture ligand solutions contain the selected fluorophore, for example the Atto 633-amine (Atto-tec, Germany) or a Dye 634-BSA complex, obtained according to a protocol known to those skilled in the art from Dye 634 (Dyomics, Germany) as ester-NHS (ester-N-Hydroxysuccinimide) coupled to BSA; these fluorophores are added at the appropriate dose, determined for each; for Dye 634-BSA, the indicated dose corresponds to the concentration of the Dye 634-BSA complex. The bottom of each well of these microplates has adsorption capabilities of these different proteins, as known to those skilled in the art. The spots thus obtained are saturated with a saturation solution in itself known to those skilled in the art. The plates are then dried. (ii) Implementation of the analysis method Description of the various elements used during the implementation of the analysis method: Reporter The Streptavidin-POD reporter (S-POD) is streptavidin (Roche, Germany) coupled to peroxidase (Roche Germany) according to the method described by Nakane and Kawaoi [J Histochem Cytochem (1974) Vol. 22, No. 12. pp. 1084-1091] in itself known to those skilled in the art.
[0101] Washing solution 10 mM Tris buffer solution, pH 7.4, containing: 218 mM NaCl, Tween 20 ™ (Sigma brand) 0.1%, Proclin 300 ™ (Supelco brand) 0.002%.
[0102] Revelation Substrate The ELISTAR ETA C Ultra ELISA revealing substrate (Cyanagen, Italy) consists of two solutions: XLSE024L Luminol enhancer solution (A) and XLSE024P Peroxide solution (B).
[0103] Description of the various steps carried out: The test protocol comprises the following steps: Step 1: 1. In each well of a microplate (including the spots) are distributed: - 40 μl of sample: the sample may be for example a serum or control sample - 40 μl of diluent 2. The mixture is incubated for 40 minutes at 37 ° C with shaking. 3. Three successive washes with at least 300 μl of washing solution are carried out. 4. Follow an incubation step in the presence of the detection ligand then washing under the same conditions as point 3. 5. Then a step of incubation of the reporter and washing. 6. Finally, an ultimate revelation step, comprising the addition of 25 μl of each of the revealing substrate solutions B and A. 7. The mixture is incubated for 1 minute at 37 ° C. with stirring. 8. The readings are performed with a Chemidoc ™ reader for fluorescence measurement and a Qview ™ reader for chemiluminescence measurement. The results of the readings are directly processed by an image analysis system and recorded in Relative Units of Luminescence or Relative Light Unit (RLU); Also relative fluorescence intensity or Relative Fluorescence Intensity (RFI). 30 Results Each data corresponds to an average of a triplicate; the fluorescence values correspond to the level of fluorescence signal deduced from the background noise. Results obtained with the Atto 633-amine fluorophore added in the antibody solution specific for the marker of interest to be detected: Table 1: Impact of the presence of the Atto 633-amine fluorophore on the detection of the analyte for different doses of fluorophore. El, E2 and E3 are positive samples, containing the antigen to be detected, at different levels of positivity. Fluorophore Atto 633-amine Sample Composition of the solutions Signal in comparison of the deposited chemiluminesc signal in chemiluminescence RLU compared to the absence of fluorophore El Antibody without fluorophore Antibody + Fluo 0.1μg / ml Antibody + Fluo 0.354 / m1 Antibody + Fluo 1 , 2μg / ml Antibody + Fluo 2.5μg / ml 1489.5 1726 1703 1377 1438 1210 reference 16% 14% -8% -3% -19% Antibody + Fluo 5μg / ml E2 Antibody without fluorophore Antibody + Fluo 0.1μg / ml Antibody + Fluo 0.354 / m1 Antibody + Fluo 1.2μg / ml Antibody + Fluo 2.5μg / ml 4651 5033 4861 3679 4115 3779 reference Antibody + Fluo 5μg / ml 8% 5% -21% -12% -19% E3 Antibody without fluorophore Antibody + Fluo 0.1μg / ml Antibody + Fluo 0.354 / m1 Antibody + Fluo 1.2μg / ml Antibody + Fluo 2.5μg / ml 2188 2117 2264 1650 1479 1226 reference Antibody + Fluo 5μg / ml -3% 3% -25% -32% -44% The results presented in Table 1 show the absence of impact of the presence of the Atto 633-amine fluorophore on the detritus. ction of the analyte for fluorophore doses of 0.354 / ml or less. Calculation of the limit of detection of the analyte shows the absence of impact of the presence of the fluorophore up to a dose of 1.24 / ml. The presence of the capture ligands only slightly modifies the fluorescence (see Table 2), which remains very important and perfectly detectable at the end of the analysis.
[0104] Table 2: Impact of the presence of capture ligands on the fluorescence signal (case of Atto 633-amine fluorophore). El and E3 are positive samples, containing the antigen to be detected, at different levels of positivity. N1 is a negative sample, not containing the antigen to be detected. Atto 633-NH2 Sample Signal Signal Composition in Comparison of Fluorescence Signal to Absence of Labels El Deposit Solutions Fluorescence Chemistry of Interest RLU luminescence -21% 26843 RLU 21158 Fluorophore only 0.14 / m1 54 Antibodies + Fluo 0.1μg / ml 1726 E3 Fluorophore alone 0.14 / m1 29039 56 -31% Antibody + Flua 0.1μg / ml 20021 2117 NI Fluorophore alone 0.14 / m1 24999 57 -29% Antibody + Fluo 0.1μg / ml 17641 49 5 Results obtained with the Dye 634-BSA fluorophore added in the antibody solution specific for the marker of interest to be detected: The results presented in Table 3 show the absence of impact of the presence of the fluorophore Dye 634-BSA on the detection of the analyte for fluorophore doses of 6 μg / ml or less, dose compatible with the detection of fluorescence at the end of analysis and the implementation of the data processing method as described in this application. Table 3: Impact of the presence of the Dye 634-BSA fluorophore on the detection of the analyte for different doses of fluorophore. E1, E2 and E3 are positive samples, containing the antigen to be detected, at different levels of positivity. Fluorophore Dye 634-BSA Sample Composition of the solutions Signal in chemi- Comparative of the chemiluminescence signal with respect to the absence of fluorophore deposited luminescence RLU El Antibody without fluorophore 1856 1716 1782 1562 1379 1297 reference -8% -4% -16% - 26% -30% Antibody + Fluo 3 μg / ml Antibody + Fluo 6 μg / ml Antibody + Fluo 12 μg / ml Antibody + Fluo 25 μg / ml Antibody + Fluo 50 μg / ml E2 Antibody without fluorophore 5576 5471 4908 4133 4231 4629 reference Antibody + Fluo 3 pg / m1 -2% Antibody + Fluo 6μg / ml Antibody + Fluo 12 pg / m1 Antibody + Fluo 25 pg / m1 Antibody + Fluo 50 pg / m1 -12% -26% -24% -17% E3 Antibody without fluorophore 2183 2397 2184 1714 1445 1347 reference Antibody + Fluo 3 pg / m1 10% Antibody + Fluo 6μg / ml Antibody + Fluo 12 pg / m1 Antibody + Fluo 25 pg / m1 Antibody + Fluo 50 pg / m1 0% -21 % -34% -38% Results obtained with the Atto 633-amine fluorophore added in the antigen solution corresponding to the antibodies to be detected: The results presented in Table 4 show the absence of impact of the presence of the Atto 633-amine fluorophore on the detection of the analyte for the fluorophore dose of 0.14 / ml. The presence of the capture ligands (see Table 5) modifies the fluorescence, but the signal remains very important, perfectly detectable at the end of analysis and usable to implement the data processing method as described in the present application.
[0105] Table 4: Impact of the presence of the Atto 633-amine fluorophore on the detection of the analyte for different doses of fluorophore. Samples S1 and S2 are positive samples, containing antibodies reacting selectively and respectively vis-a-vis the two types of antigens used: recombinant protein or synthetic peptide. Fluorophore Atto 633-amine Sample Composition of the solutions Signal in comparison of chemiluminescence luminescence chemiluminescence deposit RLU compared to the absence of fluorophore S2 Antigens without fluorophore Antigens + Fluo 0.1 pg / m1 Antigens + Fluo 0.354 / m1 Antigens + Fluorescence 1.2 μg / m1 Antigens + Fluo 2.5 μg / ml 410 355 351 366 330 247 reference -13% -14% -11% -20% -40% Antigens + Fluo 5 μg / ml Si Antigens without fluorophore Antigens + Fluo 0.1 pg / m1 Antigens + Fluo 0.354 / m1 Antigens + Fluo 1.2 pg / m1 Antigens + Fluo 2.5μg / ml 1638 1547 1215 1261 1253 1194 reference Antigens + Fluo 5μg / ml -6% -26% -23 % -24% -27% Table 5: Impact of the presence of capture ligands on the fluorescence signal (case of Atto 633-amine fluorophore). Samples S1 and S2 are positive samples, containing antibodies reacting selectively and respectively vis-a-vis the two types of antigens used: recombinant protein or synthetic peptide. N2 is a negative sample, containing no antibodies recognizing the antigens used. Fluorophore Atto 633-amine Sample Composition of the signal signal in comparison of the fluorescence signal with respect to the absence of markers deposited solutions fluorescence chemistry of interest RLU luminescence RLU N2 Fluorophore alone 0.14 / m1 Antigens + Fluo 0.1 pg / m1 70881 70 -47% 37781 67 S2 Fluorophore alone 0.14 / m1 Antigens + Fluo 0.1 pg / m1 72852 93 -45% 39877 355 51 Fluorophore alone 0.14 / m1 Antigens + Fluo 0.1 pg / ml m1 73721 117 -47% 39096 1547 Results obtained with the Dye 634-BSA fluorophore added in the antigen solution corresponding to the antibodies to be detected: Table 6: Impact of the presence of the Dye 634-BSA fluorophore on the detection of the analyte for different doses of fluorophore. Sample S1 is a sample containing antibodies reactive with respect to the recombinant protein used. fluorophore Dye 634-BSA Sample Composition of the solutions deposited Signal in chemi- Comparative of the luminescence signal in RLU chemiluminescence compared to the absence of fluorophore Si Antigens without fluorophore Antigens + Fluo 3μg / ml Antigens + Fluo 6μg / ml Antigens + Fluo 12μg / ml Antigens + Fluo 25μg / ml Antigens + Fluo 504 / m1 1066 1218 1003 1277 1291 1247 reference 14% -6% 20% 21% 17% The results presented in table 6 show the absence of impact of the presence of the fluorophore Dye 634-BSA on the detection of the analyte regardless of the dose of fluorophore. The fluorescence signal obtained at the end of the analysis is perfectly detectable and usable for implementing the data processing method as described in the present application. Example 3: Use of a Resistant Control Marker for Securing a Method of Detecting at Least One Analyte in a Sample Material and Method (i) Preparation of the Microplate Within each well of a polystyrene microplate (Greiner, Germany) are filed in rows using a robot spotteur, drops of 50 nL of a solution containing the capture ligand / s and a selected fluorophore according to the method described in the application. The capture ligand solution can be: - either an antigen or mixture of antigens that can consist of a recombinant protein associated with one or more synthetic peptides as part of an antibody detection test, or an antibody or a mixture of antibodies directed against the desired marker in the case of an antigen detection assay. These capture ligand solutions contain the Atto 633-amine selected fluorophore (Atto-tec, Germany) at the appropriate dose of between 0.1 to 0.5 μg / mL. The bottom of each well of these microplates has adsorption capabilities of these different proteins, as known to those skilled in the art. The spots thus obtained are saturated with a saturation solution in itself known to those skilled in the art. The plates are then dried. (ii) Implementation of the analysis method Description of the various elements used during the implementation of the analysis method: I. Reporter The Streptavidin-POD reporter (S-POD) is streptavidin (Roche, Germany) coupled to peroxidase (Roche Germany) by the method described by P. Nakane and A.
[0106] Kawaoi [J Histochem Cytochem (1974) Vol. 22, No. 12. pp. 1084-1091] in itself known to those skilled in the art. He. Thinners a) Diluent step 1 50 mM Tris buffer solution, pH 7.5 containing: 150 mM NaCl, 20 mM EDTA, mouse IgG (Meridian) at 500 μg / mL, 15% cow's milk (100% skimmed) , 10% Sheep Serum, 0.095% NaN3. b) Diluent Conjugates 1 50 mM Tris Buffer Solution, pH 7.5 containing: 150 mM NaCl; 20 mM EDTA, 0.1% Chaps, 10% Glycerol, 0.095% NaN3. c) Conjugate Diluent 2 50 mM Citrate Buffer Solution, pH 6.7 Containing: 150 mM NaCl, 5.6 mM EDTA, 2% Triton, 10% Sheep Serum, Mouse IgG 500 μg / mL, Proclin 300TM (Supelco brand) 0.5%, cow's milk (100% skimmed) 15%, Glycerol 10%. NaN3 at 0.095%. d) Streptavidin-POD reporter diluent 50 mM citrate buffer solution, pH 6.7, containing: 2053 mM NaCl, Tween 20 ™ (Sigma brand) 0.5%, Proclin 300 ™ (Supelco brand) to 0.5%, cow's milk (100% skimmed) to 7%, Glycerol 20%. e) Wash Solution 10 mM Tris buffer solution, pH 7.4, containing: 218 mM NaCl, Tween 20 ™ (Sigma brand) 0.1%, Proclin 300 ™ (Supelco brand) 0.002% . f) Revelation substrate The ELISTAR ETA C Ultra ELISA revealing substrate (Cyanagen, Italy) consists of two solutions: XLSE024L Luminol enhancer solution (A) and XLSE024P Peroxide solution (B). III. Reaction cuvettes Immunological reactions take place in 96-well microplates (Greiner, Germany) made of polystyrene with a maximum volume of 392 μL per well.
[0107] IV. Samples The negative samples (serum or plasma) used come from the French blood establishment of Lille. V. Optical bench The optical bench used consists of the following elements: a lighting system emitting a red light centered on the wavelength of 620 nm and assembled in such a way that it illuminates the lower face of the microplate of homogeneously, a telecentric lens made to imitate the entire surface of the microplate, a filter wheel inserted between the telecentric lens output lens and the camera, a camera having the ability to make images with exposure times between 0.001 seconds and 250 seconds. a carrier frame that supports and positions all the elements including the microplate. The optical bench is designed and assembled so that it takes the images of the lower face of the microplate. The focus of the lens is made in such a way that the inner face of the microplate wells is clear. The filter wheel is capable of presenting two different filters: a filter centered on the wavelength of 680 nm making it possible to let only the signal corresponding to the light emitted by the fluorophore, a filter making it possible to pass all the wavelengths between 400nm and 700nm. Description of the various steps carried out: The test protocol comprises the following steps: Step 1: 1. In each well of a microplate (including the spots) are successively distributed: + 20 μl of diluent step 1 + 20 1. Diluent of conjugates 1 comprising the analyte detection ligands to be assayed in the first step + 40 μl of sample 2. The mixture is incubated for 40 minutes at 37 ° C. with shaking. 3. Three successive washes with at least 400 μl of washing solution are carried out.
[0108] Step 2: 4. In each reaction well are dispensed 50 μl diluent conjugates 2 containing the analyte detection ligands to be assayed in the second step. 5. The mixture is incubated for 15 minutes at 37 ° C with stirring. 6. The washing steps (idem point 3) are carried out. Step 3: 7. 501. 1 L of the S-POD reporter is dispensed into each reaction well. 8. The mixture is incubated for 15 minutes at 37 ° C with stirring. Step 4: 9. 25 μL of revealing solution "B" is dispensed into each reaction well. 10. 25 μl of revealing solution "A" are dispensed into each reaction well. 10. The mixture is incubated for 1 minute at 37 ° C with stirring. 11. The acquisition of the fluorescence signal is carried out for 10 seconds. 12. The luminescence signal is acquired for 180 seconds. Results A) Persistence of the resistant control marker after an analysis procedure. The fluorescent signal of the resistant control marker of each of the 9 spots is clearly identifiable and perfectly measurable on the basis of an analysis method in FIG. 2. B) Importance of redefining the reading grid at the end of the analysis method: comparison of the regions of interest obtained in fluorescence by defining the reading gate with respect to theoretical positions versus defining the reading gate from the signal emitted in fluorescence by the resistant control marker. In Figure 3, the continuous white circles show the theoretical position of the spots, the dashed white circles show the actual position detected. The spots are clearly offset from their expected theoretical position.
[0109] This image demonstrates the relevance of the method that allows to always target the position of the actual spot detected by the resistant control marker, which does not induce error on the reading of the signal emitted by the detection ligand detection marker of the analyte.
[0110] C) Importance of redefining the reading grid at the end of the analysis process: comparison of the regions of interest obtained in chemiluminescence by defining the reading grid with respect to theoretical positions versus defining the reading grid from the signal emitted in fluorescence by the resistant control marker.
[0111] The location of the actual spots (dashed lines) was obtained on the basis of the signals produced by the resistant control marker and applied to the signal acquisition image of the chemiluminescence analyte ligand detection marker. shown here in Figure 4. The theoretical positioning of the spots is indicated in continuous lines and clearly shows an offset with respect to the actual position. The comparison between the median chemilumence values of the pixels below the expected theoretical positions (solid lines) and the actual detected positions (dashed lines) in fluorescence is shown in Table 7.
[0112] Table 7: Signal measured from the analysis of the theoretical presence region of a spot versus signal measured from the analysis of the actual positional region of this same spot Spot 1 Spot 3 Spot 5 spot 9 (top left) (top right) (middle) (bottom right) Position 319 301 58 3387 theoretical Position 689 693 138 5233 real Analysis of the region containing a signal producing spot provides significantly higher results than the region of theoretical presence of this same spot. The quantization of the signal and the accuracy of the results obtained are thus improved based on the actual positioning grid detected by the resistant control marker.
[0113] D) Example of a degraded spot measured after an analysis process and which could have given a false result in the absence of verification of the integrity of the spots.
[0114] The location of the actual spots (dashed lines) was obtained on the basis of the signals produced by the resistant control marker and applied to the signal acquisition image of the chemiluminescence analyte ligand detection marker. presented here (see Figure 5). The theoretical positioning of the spots is indicated in continuous lines On the image (see Figure 5), the spot located in the center of the well is deformed. The discontinuous white line that surrounds it shows that the software has detected the actual shape of the spot, which makes it possible to analyze its integrity before validating the rendering of the result. In this case, the circularity parameter of the actual shape of the spot makes it possible to eliminate this spot and not to make a false analysis value.

权利要求:
Claims (13)
[0001]
REVENDICATIONS1. A method for detecting at least one analyte in at least one sample comprising the following steps: a) placing a sample to be analyzed in the presence of the spot or spots of a compartment of a solid support, said spot or at least one of said spots comprising at least one resistant control marker and at least one analyte capture ligand, b) bringing into the presence of at least one spot of said compartment at least one analyte detection ligand, said analyte detection ligand being coupled to a direct or indirect detection marker, c) when at least one detection ligand of an analyte is coupled to an indirect detection marker, bringing into the presence of one or more spots of said compartment a reporter of said indirect detection marker , d) when the reporter used in step c) is coupled to an indirect detection marker, bringing into the presence of said spot or spots of said compartment a reporter of the indirect detection detector coupled with audi t reporter used in step c), e) detect at least one signal produced by at least one resistant control marker in said compartment, f) define at least one reading gate from the location of at least one signal detected in step e), g) optionally, detecting a signal produced by at least one detection marker of an analyte detection ligand, and h) optionally, reading the signal detected in step g) on the reading gate defined in step f), said one or more resistant control markers being markers which remain at least partially fixed at the spot on the surface of the solid support during the implementation of said detection method of at least one analyte, so as to produce a detectable signal in step e).
[0002]
2. Method according to claim 1, characterized in that the signal produced by at least one detection marker of an analyte detection ligand is a luminescent signal.
[0003]
3. Method according to claim 1 or 2, characterized in that said, said or one of said resistant control markers is a fluorophore.
[0004]
4. Method according to claim 3, characterized in that said, said or one of said resistant control markers is a fluorophore whose excitation spectrum does not cover the emission spectrum of the signal emitted by or corresponding to the detection marker of said or said analyte detection ligands and whose emission spectrum does not overlap or partially overlaps the emission spectrum of or corresponding to the detection marker of said one or more analyte detection ligands.
[0005]
5. Method according to one of claims 1 to 4, characterized in that: the signal produced by at least one detection marker of an analyte detection ligand is the light emitted by a chemiluminescent compound obtained by reaction of a peroxidase with luminol, a luminol analogue and / or a derivative of luminol or a luminol analogue, and the signal produced by the one or more resistant control markers is a light emitted outside the wavelengths going to from 375 nm to 550 nm.
[0006]
6. A method of selecting a resistant control marker comprising the following steps: a) depositing a test marker on the surface of a solid support, to form at least one spot, b) optionally saturating the surface of the solid support, c) optionally drying the surface of the solid support, d) performing at least one of the following steps: (i) carrying out one or more washing steps, (ii) placing the spot or spots in the presence of at least one detection ligand, (iii) putting the spot or spots in the presence of at least one protractor, (iv) placing the spot or spots in the presence of at least one substrate, e) selecting a marker that produces a signal at the end of the step d).
[0007]
7. Solid support for the detection of at least one analyte in a sample, characterized in that said solid support comprises at least one compartment comprising at least one spot, said spot comprising at least one resistant control marker and at least one ligand of capture, said one or more resistant control markers being markers which remain at least partially fixed at said spot on the surface of the solid support during the implementation of a method for detecting at least one analyte, so producing a detectable signal upon completion of said detection method.
[0008]
8. Support according to claim 7, characterized in that said, said or one of said resistant control marker (s) is (are) a resistant control mark (s) selected (s) by the method according to claim 6 and / or as defined in one of claims 3 to 5.
[0009]
9. A process for preparing a solid support for the detection of at least one analyte in a sample comprising the following steps: a) depositing, on the surface of at least one compartment of a solid support, a mixture comprising at least minus one resistant control marker and at least one capture ligand, to obtain a spot, said one or more resistant control markers being markers which remain at least partially fixed at said spot on the surface of the solid support during setting implementing a method for detecting at least one analyte, so as to produce a detectable signal at the conclusion of said detection method, b) repeating step a) n-1 times, n being a higher integer or equal to 1, to obtain n spots intended for the detection of an analyte on the surface of said compartment or compartments, c) possibly saturating the surface of said compartment or compartments, and d) optionally drying the surface of said or said s compartments.
[0010]
The method according to claim 9, characterized in that said at least one capture ligand is selected from the group consisting of an antibody, an antigen, a nucleic acid and combinations thereof.
[0011]
11. The method of claim 9 or 10, characterized in that said one or more resistant control markers are fluorophores.
[0012]
A device for detecting at least one analyte in a sample, said device comprising: means for detecting a signal produced by at least one resistant control marker and a signal produced by at least one ligand detection marker for detecting an analyte at a solid support, said means comprising a lighting system, a telecentric lens, a filter wheel and a camera, and means for defining a reading gate from the signal location produced by at least one resistant control marker and for reading the signal produced by at least one detection marker of an analyte detection ligand at said reading gate.
[0013]
13. Use of at least one resistant control marker in at least one spot for detecting an analyte for securing a method for detecting at least one analyte in a sample, characterized in that it comprises: the control of the quality of said spot, after said spot has been placed in the presence of the sample and at least one detection ligand of an analyte to be detected, and / or the reading of the signal produced by at least one detection marker of an analyte detection ligand to be detected on a reading gate defined from the location of the signal produced by said one or more resistant control markers.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

US20060063197A1|2004-09-17|2006-03-23|Anderson Bart R|Quality control and normalization methods for protein microarrays|

---

US20120196767A1|2009-09-21|2012-08-02|Akonni Biosystems, Inc.|Microarray based sample detection system|

---

US5451504A|1991-07-29|1995-09-19|Serex, Inc.|Method and device for detecting the presence of analyte in a sample|

---

CA2325986A1|1998-03-27|1999-10-07|Genentech, Inc.|Antagonists for treatment of cd11/cd18 adhesion receptor mediated disorders|

---

US20020090649A1|1999-12-15|2002-07-11|Tony Chan|High density column and row addressable electrode arrays|

---

JP3913978B2|2000-12-13|2007-05-09|富士フイルム株式会社|Image analysis method and apparatus|

---

WO2007041452A2|2005-09-30|2007-04-12|New York University|Methods for preparation of live body tissue for examination|

---

US7329860B2|2005-11-23|2008-02-12|Illumina, Inc.|Confocal imaging methods and apparatus|

---

US9767342B2|2009-05-22|2017-09-19|Affymetrix, Inc.|Methods and devices for reading microarrays|

---

CA2786713C|2010-01-19|2018-03-06|Sirigen Group Limited|Novel reagents for directed biomarker signal amplification|

---

US8236574B2|2010-03-01|2012-08-07|Quanterix Corporation|Ultra-sensitive detection of molecules or particles using beads or other capture objects|

---

FR3019654B1|2014-04-04|2020-10-30|Bio Rad Innovations|CONTROLS FOR THE IMPLEMENTATION OF MULTIPLEX ANALYSIS METHODS|

---

FR3019899B1|2014-04-09|2017-12-22|Bio-Rad Innovations|USE OF A COLOR TO IMPROVE SIGNAL DETECTION IN A METHOD OF ANALYSIS|

---

FR3019901B1|2014-04-09|2020-10-30|Bio Rad Innovations|CONTROL MARKER FOR THE IMPLEMENTATION OF ANALYSIS METHODS ON SPOTS|

---

FR3019900B1|2014-04-09|2017-12-22|Bio-Rad Innovations|USE OF ABSORBENT PARTICLES FOR IMPROVING SIGNAL DETECTION IN A METHOD OF ANALYSIS|

---

WO2017058827A1|2015-09-29|2017-04-06|Essenlix Corp.|Method of detecting an analyte in a sample|FR3019654B1|2014-04-04|2020-10-30|Bio Rad Innovations|CONTROLS FOR THE IMPLEMENTATION OF MULTIPLEX ANALYSIS METHODS|

---

FR3019901B1|2014-04-09|2020-10-30|Bio Rad Innovations|CONTROL MARKER FOR THE IMPLEMENTATION OF ANALYSIS METHODS ON SPOTS|

---

FR3019899B1|2014-04-09|2017-12-22|Bio-Rad Innovations|USE OF A COLOR TO IMPROVE SIGNAL DETECTION IN A METHOD OF ANALYSIS|

---

FR3019900B1|2014-04-09|2017-12-22|Bio-Rad Innovations|USE OF ABSORBENT PARTICLES FOR IMPROVING SIGNAL DETECTION IN A METHOD OF ANALYSIS|

---

EP3845902A1|2017-06-23|2021-07-07|NanoTemper Technologies GmbH|Methods for measuring inter- and/or intra-molecular interactions|

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优先权:

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申请号 | 申请日 | 专利标题

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FR1453169A|FR3019901B1|2014-04-09|2014-04-09|CONTROL MARKER FOR THE IMPLEMENTATION OF ANALYSIS METHODS ON SPOTS|FR1453169A| FR3019901B1|2014-04-09|2014-04-09|CONTROL MARKER FOR THE IMPLEMENTATION OF ANALYSIS METHODS ON SPOTS|

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RU2016143732A| RU2016143732A3|2014-04-09|2015-04-08|

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SG11201608446RA| SG11201608446RA|2014-04-09|2015-04-08|Control marker for implementing analysis methods on spots|

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US15/302,512| US10139404B2|2014-04-09|2015-04-08|Control marker for implementing analysis methods on spots|

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PCT/EP2015/057639| WO2015155254A1|2014-04-09|2015-04-08|Control marker for implementing analysis methods on spots|

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CA2945040A| CA2945040A1|2014-04-09|2015-04-08|Control marker for implementing analysis methods on spots|

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SG10202109120YA| SG10202109120YA|2014-04-09|2015-04-08|Control marker for implementing analysis methods on spots|

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EP15713924.7A| EP3129782B1|2014-04-09|2015-04-08|Control marker for implementing methods of analysis on spots|

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JP2016561694A| JP7017310B2|2014-04-09|2015-04-08|Control marker for performing analytical methods on spots|

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AU2015243288A| AU2015243288B2|2014-04-09|2015-04-08|Control marker for implementing analysis methods on spots|

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CN201580030404.8A| CN106461652B|2014-04-09|2015-04-08|Control markers for carrying out an analytical method on spots|

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US16/199,509| US10921318B2|2014-04-09|2018-11-26|Control marker for implementing analysis methods on spots|

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US17/146,622| US20210164973A1|2014-04-09|2021-01-12|Control marker for implementing analysis methods on spots|