Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54306—Solid-phase reaction mechanisms
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54326—Magnetic particles

Definitions

• the present invention relates to controls that can be used to secure the results of multiplex analysis methods comprising one or more steps.
• a multiplex analysis method makes it possible to detect, simultaneously, the possible presence of several analytes within the same sample.
• Multiplex analysis has several advantages, such as saving time by allowing multiple analytes to be analyzed at the same time, less consumption of reagents and consumables, and also less sample needed for analyte detection.
• a classically used positive control consists of verifying the detection of a known compound used in a known quantity and which corresponds to an analyte whose possible presence is sought in a given sample.
• this type of control makes it possible to validate only the overall implementation of the analysis method and does not make it possible to validate each step of the method.
• WO2004 / 046685 uses controls to check the quality of reagents used in immunohistochemistry tests.
• a control slide comprising a series of dilutions of different control compounds is used. Positive staining of the control slide at a control compound should be observed only if a specific antibody of said control compound or a reporter or substrate of said control compound is used during the immunolabeling process. This method of verifying the quality of the reagents requires the use of a large number of control compounds on the control slide.
• the present invention is based on the demonstration by the inventors of control means making it possible to guarantee the reliability of the results obtained, by verifying that each step of a multiplex analysis method has proceeded correctly (and not by checking only one overall implementation of the process).
• the inventors provide controls that make it possible to validate the deposition of the sample itself and more generally the different steps of the process.
• the use of the controls according to the invention also makes it possible to identify potential faults existing during the implementation of the multiplex analysis method, and to modify, for example, the corresponding step or steps to improve the implementation of the analysis method.
• the present invention also has the advantage of being simple to implement, requiring only a limited number of controls, by not adding additional steps to the analysis method and not requiring the use additional equipment (for example, not requiring the use of a spectrophotometer). Indeed, the steps of the analysis method are carried out in a single location (for example, the tube or well in which the sample is placed) and the controls, for example in the form of spots or beads, are treated in same time as the spots or beads used for the detection of analytes.
• the first type of control according to the invention is the control of the deposit of a sample which makes it possible to check the deposit of the sample.
• the check of the sample deposit also makes it possible to verify the deposition of one or more analyte detection ligands.
• Deposit of the sample or an additive or “addition of the sample or additive” means the placing in the presence of the sample or at least one additive with compounds of interest fixed on a solid support.
• At least one is meant in this application one or more, “several” meaning in particular two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen. , sixteen or more than sixteen.
• a detection ligand By “deposition of a detection ligand, a reporter or a substrate” or “addition of a detection ligand, a protractor or a substrate”, it is meant to bring into the presence of a detection ligand, a reporter or a substrate with compounds of interest fixed on a solid support and any compounds attached to said compounds of interest.
• the second type of control is the control of the deposition of an analyte detection ligand which makes it possible to verify the deposition of one or more detection ligands (for example a mixture of detection ligands) of analytes to be detected. in the sample. This control also validates the incubation and washing steps.
• the third type of control is the control of the deposit of a protractor which makes it possible to verify that the step (s) of revelation proceeded correctly.
• the control of the deposition of a reporter is useful in case of indirect labeling of the detection ligands.
• controls according to the invention are particularly suitable for the implementation of micro-array multiplex analysis methods, for example on a solid support of the microplate type, or in a liquid chip, for example on a solid support of the type balls.
• 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 a tissue sample, such as tissue obtained by biopsy , a cell or a set of cells, a plant extract, or combinations thereof.
• a biological fluid such as a blood sample, derived from blood (such as plasma or serum), urine, cerebrospinal fluid, saliva, or a tissue sample, such as tissue obtained by biopsy , a cell or a set of cells, a plant extract, or combinations thereof.
• 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.
• the sample Before being analyzed, the sample may undergo one or more preliminary treatment steps, such as dilution, centrifugation, heat and / or chemical treatment, cell lysis (for example by one or more chaotropic agents, one or more reducing agents 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, which is in itself a test known to those skilled in the art.
• the sample may also be a mixture of at least two samples that may be of the same kind or of a different nature.
• 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 and / or blood-derived samples.
• An analyte to be detected in the sample may be any type of compound, natural or synthetic, that it is desired to detect and / or quantify in a sample.
• An analyte may for example be a protein, a peptide, a glycoprotein, a carbohydrate, a lipid, a cell, an organelle, a virus or a nucleic acid.
• the cell may be an animal cell, a plant cell, a bacterial cell, a metazoan cell, a yeast cell, a fungus cell, or a protozoan.
• 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.
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• analogue thereof such as phosphorothioates or thioesters
• An analyte or at least one of the analytes or the analytes is (are) 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.
• the analyte (s) are not nucleic acids.
• the analyte (s) is, for example, selected from the group consisting of an antigen, an antibody, an antibody fragment, a hapten, a hormone, a hormone receptor and an enzyme.
• antigen is meant here a natural, recombinant or synthetic molecule recognized by antibodies or cells of the immune system and capable of generating an immune response when presented under conditions appropriate to the immune system of a host. It can be a molecule, in particular a polypeptide, comprising or consisting of at least one epitope that can be linear or conformational.
• linear epitope denotes a polypeptide, in particular a peptide, comprising or consisting generally of 3 to 15, more generally 5 to 15 amino acids, and preferably at least 6, 8, 10 or 12 amino acids, capable of bind to a molecule of antibodies directed against said antigen.
• formational epitope denotes 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 this antibody is directed. antibodies, but which, because of the folding of the polypeptide chain, are found close to each other in space, and can thus form a motif that can be recognized by an antibody.
• An antigen is, for example, a protein (in particular a native or recombinant protein), a peptide (for example a synthetic peptide), a glycoprotein, a carbohydrate or a lipid, said peptide possibly being associated or not with a carrier molecule, for example BSA (bovine serum albumin).
• carrier molecule (also called “carrier molecule”) is meant especially in the present application a protein carrier molecule or carbohydrate.
• 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) or a co-polymer. 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 (particularly a monoclonal antibody or a polyclonal antibody), for example an immunoglobulin.
• An example of a carrier molecule is BSA.
• the carrier molecule is not an antibody.
• hapten is meant here 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 or at least one of the analytes is preferably a compound for diagnosing a condition of a subject, pathological or otherwise, or of diagnose the risks of developing a condition, pathological or not.
• An example of a non-pathological condition is a pregnancy.
• the subject may be a human, a non-human animal or a plant.
• the non-human animal is preferably a mammal, such as a cat, dog, monkey, rabbit, mouse, rat.
• 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.
• analyte or at least one analyte is an antigen
• it is preferably an antigen for diagnosing an infection, for example an infection caused by a virus, bacteria, fungus or parasite.
• analyte or at least one analyte is an antibody
• it is preferably an antibody for diagnosing an infection, for example an infection caused by a virus, bacteria, fungus or parasite.
• it may be one or more antigen (s) and / or one or more antibodies specific for:
• HIV Human Immunodeficiency Virus
• HIV-1 or HIV-2 HIV-1 or HIV-2
• HBV Hepatitis B Virus
• HCV Hepatitis C Virus
• HPV Hepatitis C Virus
• ⁇ human papillomavirus ⁇ human papillomavirus
• HTLV human T-lymphotropic virus
• 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
• 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
• Chagas disease in particular Trypanosoma cruzi
• a bacterium such as a bacterium capable of causing syphilis (in particular Treponema pallidum) or Lyme disease (in particular a bacterium of the genus Borrelia), in a human or a non-human animal.
• syphilis in particular Treponema pallidum
• Lyme disease in particular a bacterium of the genus Borrelia
• 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, bacteria, fungus or parasite, a marker of resistance to treatment, for example antiviral treatment, antibiotic treatment or treatment against cancer.
• 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, bacteria, fungus or parasite such as hepatitis
• a marker of the course of an infection caused by a virus, bacteria, fungus or parasite such as hepatitis
• a marker of the course of an infection caused by a virus, bacteria, fungus or parasite such as hepatitis
• analytes as described herein can be detected simultaneously in a sample during a multiplex analysis process. This can make it possible to diagnose, in the same sample, 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 resistance marker to treatment in a subject.
• the analytes detected during a multiplex analysis method may be of the same kind (for example, only antibodies or only antigens) or of a different nature (for example, at least one antigen and at least one antibody).
• the analyte (s) to be detected are not labeled with a detection marker.
• the analyte (s) to be detected are selected from an antibody and / or an antigen.
• control compound is meant here a compound naturally present in the sample to be analyzed, preferably in a detectable concentration in all samples of the same kind.
• the compound naturally present in the sample to be analyzed may be a compound initially present in the sample or a derivative of a compound initially present in the sample.
• a derivative of a compound initially present in the sample can be obtained at the end of one or more steps of treatment of the sample prior to the analysis method. This or these steps are in particular as defined above, for example heat and / or chemical treatment, cell lysis, extraction, PCR (Polymerase Chain Reaction) reaction, addition of an unlabeled detection ligand or combinations thereof.
• a derivative of a compound present initially in the sample is, for example, a PCR product and / or a compound modified by a heat and / or chemical treatment, a cell lysis, the addition of a non-detection ligand. marked or their combinations.
• a compound naturally present in the sample to be analyzed is a compound initially present in the sample.
• the compound is therefore present before any prior step of processing the sample.
• a naturally occurring compound in the sample to be analyzed does not include a compound present in the sample at the time of analysis but which has been added or is derived from a compound added to the sample during one or more pre-processing steps of the sample.
• control compound is a compound naturally present in at least one of said samples, preferably in each sample of the mixture.
• sample of the same kind means a sample of the same origin taken in substantially the same way from different subjects or from the same subject at different time intervals and, where appropriate, having undergone the same treatment step or steps.
• a “detectable concentration” is a concentration for detecting the presence of a control compound in a test, including a sandwich-type immunoassay in which a control compound binds to a capture antibody bound to a solid support and is detected by a labeled detection antibody.
• the concentration of a control compound varies little in samples of the same kind.
• the concentration of a control compound varies little in samples of the same kind
• concentration here means a concentration that varies from less than 60%, preferably less than 40%, more preferably less than 20% from one sample to another, the concentration being given in ⁇ g / mL.
• a control compound is present in the sample in a concentration of the same order of magnitude as the concentration of the analyte (s) to be detected, when these analytes are present.
• a control compound is present in the sample in a concentration of the same order of magnitude as the concentration of each of the analytes to be detected.
• the concentration of a control compound is of the same order of magnitude as the concentration of an analyte to be assayed.
• the concentration of the control compound is at most 5 decades less than or greater than that of said analyte to be assayed, when it is present in the sample, preferably at most 4 decades, more preferably at most 3 decades.
• the concentration of a control compound is at most 100 ⁇ 9 ⁇ _, preferably at most 10 ⁇ 9 ⁇ _, more preferably at most 1 ⁇ 9 ⁇ _, for a concentration of an analyte to be assayed which would be 1 ng / ml.
• the concentration of the control compound is of the same order of magnitude as the concentration of an analyte, when the difference between the concentration of the analyte in a sample to be analyzed which contains this analyte, and that of the control compound is at more than 5 decades, preferably at most 4 decades, more preferably at most 3 decades.
• a control compound preferably has a conserved structure within the human or animal population considered for analysis.
• a control compound does not exhibit polymorphism within the population of interest.
• the control compound is preferably not a marker of a disease.
• a control compound is preferably a detectable compound with an antibody.
• control compound is preferably not denatured by the possible pre-treatment step (s) of the sample, so as to be detectable by an antibody.
• the control compound can be a molecule, or a complex of at least two molecules, identical or different.
• a control compound when the sample to be analyzed is a sample of blood or plasma, in particular human blood or human plasma, a control compound may be the transferrin soluble receptor complexed to transferrin, a hormone, by a steroid, a coagulation factor, for example a coagulation factor chosen from factor VIII, IX, X, XI, XII or XIII.
• a control compound is not the factor
• a control compound is not a type G immunoglobulin (IgG), in particular a human IgG, and / or is not an immunoglobulin type M (IgM), in particular a human IgM or, more generally, is not an immunoglobulin, in particular a human immunoglobulin.
• IgG immunoglobulin
• IgM immunoglobulin type M
• the transferrin soluble receptor complexed to transferrin is a complex comprising two soluble transferrin receptor molecules and two transferrin molecules.
• the transferrin soluble receptor complexed to transferrin is generally present in a concentration of 0.8 ⁇ g / mL to 4 ⁇ g / mL in test samples from a human subject, regardless of the subject's condition. .
• control compound can vary, for example depending on the nature of the sample and / or the condition of the subject from which the sample is derived, it may be advantageous to use at least two different control compounds .
• An additive is a compound or a set of compounds that is (are) not present in the sample, that is, that is (are) not initially present in the sample or which is (are) not derived from a compound initially present in the sample.
• An additive may in particular be an antigen or a hapten, said antigen or said hapten possibly being coupled to a support molecule or not.
• a compound or a compound which is not present in the sample to be analyzed is for example selected from the group consisting of digoxigenin, a plant hormone, an alkaloid , a plant steroid, a nucleic acid and a pesticide.
• the additive is not a nucleic acid.
• Digoxigenin is a steroid extracted from certain plants.
• a pesticide is for example an insecticide, such as an organophosphorus or an organochlorine, a herbicide, such as a triazine or phenyl urea, or combinations thereof.
• a preferred additive according to the invention is digoxigenin coupled to a carrier molecule, an auxin coupled to a support molecule, a triazine coupled to a support molecule, said support molecule being, for example, BSA.
• a preferred additive according to the invention is digoxigenin coupled to BSA.
• the additive does not interfere with the detection of analytes during the implementation of a multiplex analysis method.
• the additive does not interfere with the analytes to be detected, the capture ligand or ligands used, the detection ligand or ligands used, the compound or compounds controls, if necessary the reporter (s), if appropriate the or substrates, and signal detection.
• the additive does not comprise or consist of biotin or a biotin analogue and / or avidin or an avidin analogue (In particular streptavidin or neutravidin), said biotin, avidin or an analogue thereof being grafted or not onto a carrier molecule.
• a capture ligand is a compound attached to a solid support, particularly at a spot or at the surface of a ball.
• a capture ligand is preferably an antibody or antigen that is attached to the solid support, particularly at a spot or at the surface of a ball.
• a capture ligand may be specific for an analyte to be detected in the sample, a control compound or an additive.
• a capture ligand may be an antibody, an antigen, a peptide, a carbohydrate, a lipid or a nucleic acid.
• the capture ligand is not a nucleic acid.
• the capture ligand is selected from the group consisting of an antibody, an antigen, a peptide, a carbohydrate and a lipid.
• a capture ligand is preferably an antibody or an antigen.
• a capture ligand is an antibody, it is for example a monoclonal antibody or a polyclonal antibody.
• a detection ligand is intended to reveal the presence of a compound of which it is specific.
• a detection ligand can be an antibody, an antigen, a peptide, a carbohydrate, a lipid or a nucleic acid.
• the detection ligand is not a nucleic acid.
• the detection ligand is selected from the group consisting of an antibody, an antigen, a peptide, a carbohydrate and a lipid.
• a detection ligand is preferably an antibody or an antigen.
• a detection ligand is an antibody, it is for example a monoclonal antibody or a polyclonal antibody.
• a detection ligand is preferably a labeled detection ligand, i.e. a ligand to which is attached a detection label (which may be for example biotin or a peroxidase), covalently or otherwise.
• a detection ligand is not labeled, its detection can be achieved by using a labeled antibody specific for said detection ligand.
• a detection ligand may be specific for an analyte to be detected in the sample, a control compound or an additive.
• 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 compound of which it is specific at the level of the same area as that bound by the capture ligand or one of the capture ligands. In this case, if said capture ligand and said detection ligand are antibodies, then it is a "homologous sandwich".
• a capture ligand and the detection ligand or one of the detection ligands may be specific for distinct zones at the level of the compound of which they are specific, so as to avoid a competition of the capture ligand and the detection ligand with respect to of the compound of which they are specific, because of a steric hindrance.
• said detection ligand and said capture ligand are antibodies, then it is a "heterologous sandwich".
• a detection ligand and a capture ligand specific for the same compound do not bind at the same location on said compound. More preferably, said detection ligand binds to an area of said compound which is remote from the binding area with said capture ligand.
• a detection ligand is identical to a capture ligand, except for the possible presence of a detection marker, and / or binds to the compound of which it is specific at the level of the same area as that bound by said capture ligand, when the compound of which it is specific is in the form of a complex having at least two identical binding areas.
• a detection marker 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 fluorochrome and 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.
• a “luminescent compound” refers in particular in the present application to an electroluminescent, thermoluminescent or (preferably) 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 compound 1, 2-dioxetane, or a derivative of a dioxetane compound, for example a 1,2-dioxetane derivative.
• An indirect marker is a marker whose signal detection requires the prior addition of a reporter, and optionally the addition of a marker substrate coupled to said reporter.
• a reporter is a substrate of the indirect marker or a molecule specifically binding to the indirect marker, said molecule being itself a direct or indirect marker or itself being coupled to a direct or indirect marker.
• An indirect marker may be for example an enzyme (in particular an enzyme producing a luminescent compound from a substrate), biotin, avidin, streptravidin, neutravidin, a hapten, an antigen or an antibody.
• an enzyme in particular an enzyme producing a luminescent compound from a substrate
• biotin avidin, streptravidin, neutravidin, a hapten, an antigen or an antibody.
• a reporter of an enzyme is for example the substrate of said enzyme.
• a reporter of a luminescent compound is for example an enzyme or a catalyst.
• 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.
• an enzyme is peroxidase, for example horseradish peroxidase
• a preferred biotin reporter according to the invention is streptavidin coupled to a peroxidase, preferably horseradish peroxidase.
• the detection marker or one of the detection markers used is or is substrate luminol (3-aminophthalhydrazide, also called 5-amino-2,3-dihydro-phthalazine-1, 4-dione, of formula C 8 H 7 N 3 O 2 ), isoluminol (also called 4-aminophthalhydrazide), an acridine compound, coelenterazine, dioxetane or peroxyoxalic, or a derivative thereof, and in particular a compound described in Pub. Publication / Gen C. et al (2000), Talanta 51, 415-439, Chemiluminescence as diagnostic tool. A review notes
• the detection marker or one of the detection markers used has for substrate luminol, isoluminol or one of their derivatives.
• a derivative of luminol or isoluminol is preferably a molecule obtained from luminol or isoluminol respectively, by any possible modification (s) (for example chemical and / or enzymatic) .
• a derivative of luminol or isoluminol is in particular a substrate for a peroxidase enzyme, the reaction of said peroxidase enzyme with said luminol derivative or isoluminol 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 Wales Revelation
• the step or steps of revealing corresponds (ent) to the detection of the signal produced by the detection marker or markers.
• the "product signal” is in particular an "emitted signal”.
• the step or steps of revealing depends on the type of marker used.
• the signal produced or emitted by a direct fluorochrome type marker can be read directly in fluorescence.
• An indirect marker of enzyme type, luminescent compound type or biotin type requires the addition of a reporter.
• an indirect marker of biotin type requires the addition of a reporter, preferably a reporter coupled to a marker.
• a reporter is coupled to an indirect marker, for example an enzyme
• an indirect marker for example an enzyme
• a reporter is coupled to peroxidase, it is necessary to add in a subsequent step a substrate of this enzyme, such as luminol.
• a signal is detected in chemiluminescence, said signal being produced by a chemiluminescent compound produced by the reaction of a peroxidase enzyme with its substrate, for example luminol, isoluminol and / or a luminol derivative. or isoluminol.
• a peroxidase enzyme with its substrate
• luminol for example luminol, isoluminol and / or a luminol derivative. or isoluminol.
• This reaction of a peroxidase enzyme with its The substrate generally also requires the presence of an oxidant and, where appropriate, an electron mediator.
• 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 derivative of luminol or isoluminol, and an electron mediator;
• the second solution comprises an oxidant.
• 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).
• the support or supports used for the implementation of the analysis method according to the invention are solid supports.
• 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.
• a suitable solid support according to the invention is, for example, a support of polystyrene, polypropylene, poly (meth) acrylate, polybutadiene or combinations thereof.
• suitable solid support is, for example, an inorganic support, such as glass, and / or a metal support.
• a support may be in the form of a plate, a microplate, a blade, beads, a membrane.
• a suitable solid support is a membrane, for example a nitrocellulose membrane, PVDF (polyvinylidene fluoride), nylon or combinations thereof.
• PVDF polyvinylidene fluoride
• a preferred solid support is polystyrene or polypropylene.
• the multiplex analysis method may be carried out using a single solid support, for example a solid support comprising at least one compartment, said compartment comprising at least two spots, or on a set of solid supports for example a set of balls.
• the controls according to the invention are transposable to use on a single solid support or on a set of solid supports.
• the controls and the means for detecting the analytes are in the form of spots and in the second case, the controls and the means for detecting the analytes are in the form of beads.
• the beads may be in solution or suspension or fixed on another solid support, for example a plate, a microplate, a slide, or a membrane , and in particular fixed to the bottom of one or more wells of a solid support (for example a microplate).
• a sample deposition check is called a spot to check the deposit of a sample or ball to check the deposit of a sample;
• a control of the deposition of one or more analyte detection ligand (s) is called a spot to control the deposition of an analyte or bead detection ligand to control the deposition of a detection ligand of an analyte an analyte;
• a deposit check of a reporter is called a spot to control the deposit of a protractor or ball to control the deposition of a protractor.
• the one or more solid supports are suitable for carrying out a multiplex analysis in the form of a sandwich type immunoassay.
• a solid support according to the invention also has the advantage of being able to detect analytes independently of the matrix of the sample.
• the detection of analytes present in the blood can be implemented by means of a solid support according to the invention from a blood sample or a blood derivative, such as plasma or serum, or a mixture of blood samples and / or blood derivatives.
• Solid support suitable for secure multiplex analysis on spots can be implemented by means of a solid support according to the invention from a blood sample or a blood derivative, such as plasma or serum, or a mixture of blood samples and / or blood derivatives.
• the subject of the present invention is particularly a solid support suitable for multiplex analysis of at least one sample, comprising at least one compartment, said compartment comprising at least one control spot and at least two detection spots of an analyte, characterized in that that said control spot is selected from the group consisting of a spot to control the deposition of a sample, a spot to control the deposition of an analyte detection ligand and a spot to control the deposition of a reporter .
• a solid support comprises at least one compartment (also called zone of analysis), preferably at least two compartments.
• a solid support comprises a single compartment.
• Said single compartment may be a compartment comprising one or more walls.
• said single compartment may be devoid 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.
• a solid support for example a slide or a membrane
• a single compartment typically at least one (for example one or two) solid support is used per sample to be analyzed.
• a 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 move from one compartment to another during the analysis.
• an added solution in one compartment does not go into the other compartments.
• 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.
• a compartment of the solid support is used per sample to be analyzed.
• An example of a compartment is a well.
• a solid support is for example a microplate.
• the microplate is typically a 96-well or 384-well microplate.
• a compartment of the support used to analyze a sample comprises at least three spots, for example three spots, four spots or five spots, or at least six spots, preferably six spots, seven spots, eight spots, more preferably at least 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.
• spot is meant here an area of a compartment of a solid support comprising at least one compound of interest linked to the surface of said compartment, in particular by non-covalent physicochemical interactions (in particular of the weak link type type). for example, ionic, van der Waals, hydrogen and / or hydrophobic) and / or by covalent bonds.
• 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.
• at least is meant in this application one or more, “several” meaning in particular two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, sixteen or more than sixteen.
• a spot corresponds to a well-defined zone, generally of spherical or oval shape and of small size, 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 2.01 1 mm 2 .
• a spot may be discoidal, cylindrical or approximately discoidal or cylindrical, for example oval, in particular when a solid support is a microplate or a blade.
• a spot may be square or rectangular (this may be in particular a band), for example when a solid support is a membrane, or any other form.
• spots are obtained by techniques well known to those skilled in the art, such as those disclosed in patents US 7,470,547 B2, US 6,576,295 B2, US 5,916,524 A and US 5 743960 A.
• a spot is obtained by depositing at least one drop of a solution containing a determined amount of said compound (s) of interest at a specific location on the surface of the compartment.
• 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 said compound (s) of interest at a specific location on the surface of the compartment on which said polymer has been deposited beforehand.
• a spot can also be obtained by in situ synthesis of said compound (s) of interest at a specific location on the surface of the compartment.
• Said compound (s) of interest are in this case qualified probe. It may be a nucleic acid or a peptide (see for example US 5,143,854).
• the surface of the compartment is also called "solid phase”.
• a compound of interest is generally a capture ligand, a carrier molecule coupled to an indirect marker or an indirect marker.
• the capture ligand, the carrier molecule coupled to an indirect marker and the indirect marker are in particular as defined above.
• each compartment of a solid support comprises the same number of spots.
• each compartment of a solid support may comprise the same number of spots and the same spot composition.
• a support may comprise one or more compartments without spot, or with a number and / or a different spot composition (s).
• Part or all of a support may for example comprise at least two distinct groups (or types) of spots or compartments, each of the distinct groups having a different number and / or spot composition (s).
• a compartment comprises at least one control spot (for example at least one spot for monitoring the deposition of a sample), preferably at least two control spots, and at least two detection spots of an analyte.
• a compartment generally comprises at least one spot per analyte to be detected, each analyte can for example correspond to an infection or a disease to be detected, to the evolution of an infection or disease, to a condition (pathological or not) of a subject, to a risk of developing a condition (pathological or not) or to a marker of resistance to a treatment.
• spots of a compartment may also be intended for the analysis of the same analyte.
• the spot for controlling the deposition of a sample or for controlling the deposition of an analyte detection ligand preferably comprises a capture ligand.
• the capture ligand is in particular as defined above.
• the same spot can comprise several different capture ligands (for example several antibodies and / or antigens), which are generally specific for the same infection or disease to be detected (in particular specific for the same virus, the same bacterium or of the same parasite), or specific of the same evolution of an infection or disease, the same condition (pathological or not) of a subject, the same risk of developing a condition (pathological or not) or the same resistance marker to a treatment.
• capture ligands for example several antibodies and / or antigens
• the subject of the present invention is particularly a solid support suitable for multiplex analysis of at least one sample, comprising at least one compartment, said compartment comprising at least one control spot and at least two detection spots of an analyte, characterized in that said spot control is a spot for controlling the deposition of a sample or a spot to control the deposition of an analyte detection ligand.
• Said compartment may further comprise at least one spot for controlling the deposit of a protractor.
• the spot for controlling the deposition of a sample or for controlling the deposition of an analyte detection ligand comprises a or at least one capture ligand, the capture ligand or ligands not being a nucleic acid.
• the spot for controlling the deposition of a sample or for controlling the deposition of an analyte detection ligand preferably does not comprise nucleic acid.
• the spot for monitoring the deposition of a sample or for monitoring the deposition of an analyte detection ligand comprises one or at least one capture ligand selected from the group consisting of an antibody, an antigen, a peptide, a carbohydrate and a lipid.
• the subject of the present invention is particularly a solid support suitable for multiplex analysis of at least one sample, comprising at least one compartment, said compartment comprising at least two control spots and at least two detection spots of an analyte, characterized in that said control spots are selected from the group consisting of a spot to control the deposition of a sample, a spot to control the deposition of an analyte detection ligand and a spot to control the deposition of a protractor .
• a solid support according to the invention allows a secure multiplex analysis.
• the subject of the present invention is a solid support suitable for multiplex analysis of at least one sample, comprising at least one compartment, said compartment comprising at least two control spots and at least two detection spots.
• an analyte characterized in that said control spots are selected from a spot to control the deposition of a sample and a spot to control the deposition of an analyte detection ligand.
• the compartment or compartments of the solid support may for example comprise at least two spots for controlling the deposition of a sample or at least two spots for controlling the deposition of an analyte detection ligand.
• the solid support compartment or compartments comprise at least one spot for monitoring the deposition of a sample and at least one spot for controlling the deposition of an analyte detection ligand.
• the compartment or compartments comprise at least one spot for monitoring the deposition of a protractor.
• the present invention also relates to a solid support suitable for multiplex analysis of at least one sample, comprising at least one compartment, said compartment comprising at least one spot for controlling the deposition of a sample, at least one spot for controlling the deposition of an analyte detection ligand, at least a spot for controlling the deposition of a reporter, and at least two detection spots of an analyte.
• the compartment or compartments of the solid support may therefore comprise several spots for controlling the deposition of a reporter, for example two or three spots to control the deposition of a reporter, for analyzes involving at least two different indirect markers.
• each spot to control the deposition of a reporter is specific to a marker.
• the multiplexed analysis method of a sample is made with a set of beads.
• the present invention thus also relates to a set of beads suitable for multiplex analysis of a sample, comprising at least one control ball and at least two analyte detection beads, characterized in that the control ball is selected from the group consisting of a ball to control the deposition of a sample, a ball to control the deposition of an analyte detection ligand and a ball to control the deposition of a protractor.
• At least one ball X is meant, in the present application, at least one ball X or at least one type (or group) of beads X, “ball X” may mean “ball control”, “ball to control depositing a sample ",” ball to control the deposition of an analyte detection ligand “or” ball to control the deposition of a protractor ".
• a “type of ball” within the meaning of the invention comprises or consists of several balls (for example 10, 50, 100, 200, 300 or 500 balls), which are identical to one another or to one another. less in several balls on the surface of which are fixed the same or the compounds of interest.
• At least two balls X is meant, in the present application, respectively, at least two balls X or at least two types (or “groups”) distinct balls X
• ball X may mean “Ball control”, “ball of detection of an analyte”, “ball to control the deposit of a sample”, “ball to control the deposit of an analyte detection ligand” or “ball to control the tabling of a rapporteur ".
• Each ball is also covered with at least one compound of interest linked to the surface of the ball, also called “solid phase”.
• a set of beads used to analyze a sample comprises at least three beads (or types of beads), for example three, four or five beads (or types of beads), or at least six beads (or types of beads), preferably six, seven, eight balls (or types of beads), more preferably at least nine balls (or types of balls), for example nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or sixteen balls (or types of balls), or more than sixteen balls (or types of marbles).
• ball By “ball”, “particle”, “microbead” or “microparticle” is meant in the present application any particle, preferably of spherical or approximately spherical shape, of size ranging from 0.3 ⁇ to 100 ⁇ in diameter , preferably from 0.5 ⁇ to 40 ⁇ . Such particles are manufactured for example by Luminex, Merck and Dynal.
• a ball that can be used in the context of the present invention can also be a particle in the form of a cube or block or approximately in the shape of a cube or block, the length of the sides of which would be, for example, from 0.3 ⁇ to 100. ⁇ , preferably from 0.5 ⁇ to 40 ⁇ .
• a ball according to the invention is preferably composed of one or more inert polymers with respect to the constituents of the biological samples; it is solid and insoluble in the samples to be analyzed.
• inert polymer examples include a polyester, polyether, polyolefin, polyamide, polysaccharide, polyurethane or cellulose.
• One or more functional groups may be incorporated with the at least one inert polymer to allow attachment or coupling of one or more compounds of interest (e.g., proteins, peptides, glycoproteins, lipids, carbohydrates, or nucleic acids).
• compounds of interest e.g., proteins, peptides, glycoproteins, lipids, carbohydrates, or nucleic acids.
• These functional groups can be selected from the group consisting of amine (-NH2) or ammonium (-NH3 + or -NHR) functions, where R represents an aliphatic chain, preferably a chain:
• alcoholic functions (-OH), carboxylic functions (-COOH), isocyanate functions (-NCO), thiol functions (SH) or epoxy functions.
• the monomers most commonly used to introduce carboxylic functions -COOH into polyolefins are acrylic acid or methacrylic acid.
• a ball is indeed coated with one or more compounds of interest, using any suitable method well known to those skilled in the art.
• the binding of the compound or compounds of interest on the surface of a ball can be done by electrostatic attraction, affinity interaction, hydrophobic interaction and / or covalent coupling.
• the compound (s) of interest are attached to the surface of the ball by covalent coupling.
• a bead comprising carboxyl-surface chemical functions
• these chemical functions can be converted into an activated ester form by reaction with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and the N-hrydroxysulfosuccinimide.
• a compound of interest such as an antibody, a protein or a peptide, can then be grafted, by a free amino group present on said compound of interest, onto the activated ester groups of each bead.
• the conversion to an activated ester form and the coupling of the compound of interest on a ball are carried out according to procedures that are well known to those skilled in the art (see, for example, US Pat. No. 7,141,362).
• the beads or a portion of the beads of a bead set are preferably magnetic beads, such as beads from the Luminex xMAP ® technology, to allow recovery of the balls between the individual process steps, in particular the after the washing steps.
• Each ball is marked with a code so as to be able to differentiate it from the other balls of the set of balls, for example by a fluorescence code or a barcode.
• the marking of a ball with a code can be achieved by any appropriate method well known to those skilled in the art, for example as described in documents EP 1 802 710 and EP 1 049 807.
• a compound of interest is generally a capture ligand, a carrier molecule coupled to an indirect marker, or an indirect marker.
• the capture ligand, the carrier molecule coupled to an indirect marker and the indirect marker are in particular as defined above.
• the set of beads according to the invention comprises a ball (or a type of bead) for the desired control and at least one bead (or at least one type of bead) per analyte to be detected.
• a ball or a type of bead
• the ball for controlling the deposition of a sample or for controlling the deposition of an analyte detection ligand preferably comprises a capture ligand.
• the capture ligand is in particular as defined above.
• the same bead (or each bead of a given type of bead) may be covered with several different capture ligands (for example several antibodies and / or antigens), which are generally specific for the same infection to be detected, and in particular of the same virus, the same bacterium or the same parasite.
• capture ligands for example several antibodies and / or antigens
• the set of beads therefore comprises at least one control ball (for example at least one ball to control the deposition of a sample), preferably at least two control beads, and at least two analyte detection beads.
• At least one set of beads is used per sample to be analyzed.
• the present invention particularly relates to a set of beads suitable for multiplex analysis of at least one sample, comprising at least one control ball and at least two analyte detection beads, characterized in that said control ball is a ball to control the deposition of a sample or ball to control the deposition of an analyte detection ligand.
• Said set of beads may further comprise at least one ball for controlling the deposition of a protractor.
• the ball for controlling the deposition of a sample or for controlling the deposition of an analyte detection ligand comprises one or at least one capture ligand, the one or more capture ligands being not a nucleic acid.
• the ball for controlling the deposition of a sample or for controlling the deposition of an analyte detection ligand preferably does not comprise nucleic acid.
• the ball for monitoring the deposition of a sample or for controlling the deposition of an analyte detection ligand comprises one or at least one capture ligand selected from the group consisting of an antibody, an antigen, a peptide, a carbohydrate and a lipid.
• the present invention particularly relates to a set of beads suitable for multiplex analysis of a sample, comprising at least two control beads and at least two analyte detection beads, characterized in that said control beads are selected in the group consisting of a ball to control the deposition of a sample, a ball to control the deposition of an analyte detection ligand and a ball to control the deposition of a protractor.
• the subject of the present invention is a set of beads suitable for multiplex analysis of a sample, comprising at least two control beads and at least two analyte detection beads, characterized in that said control beads are selected from a ball to control the deposition of a sample and a ball to control the deposition of a detection ligand an analyte.
• the set of beads may, for example, comprise at least two beads to control the deposition of a sample or at least two beads to control the deposition of an analyte detection ligand.
• the set of beads comprises at least one ball for monitoring the deposition of a sample and at least one ball for controlling the deposition of an analyte detection ligand.
• the present invention also relates to a set of beads suitable for multiplex analysis of a sample, comprising at least one ball for monitoring the deposition of a sample, at least one ball for controlling the deposition of a detection ligand. an analyte, at least one bead for monitoring the deposition of a reporter and at least two analyte detection beads.
• the set of beads may therefore comprise at least two beads to control the deposition of a reporter, for example two or three beads to control the deposition of a reporter, for analyzes involving at least two different indirect markers.
• each ball to control the deposition of a reporter is preferably marker specific.
• the set of balls according to the invention allows a secure multiplex analysis.
• the control of the deposit of a sample makes it possible to check the placing in the presence of the sample with the spots of a compartment of the solid support or with a set of balls.
• the control of the deposition of a sample comprises at least one capture ligand, in particular an antibody or an antigen, specific for a control compound naturally present in the sample to be analyzed.
• the control compound is in particular as defined above.
• a signal detected in the control of the deposition of a sample makes it possible to validate the step of depositing the sample.
• the deposition of a sample consists in adding the sample to be analyzed into a compartment of the solid support according to the invention comprising at least one control spot and at least two analyte detection spots or to bring the sample in contact with the sample. least one set of balls according to the invention.
• a control compound then binds to the capture ligand at the sample deposition control.
• a control compound thus fixed can be detected by a specific detection ligand of the control compound.
• a control compound detection ligand is specific for a zone of the control compound remote from the region of the control compound to which the capture ligand used specifically binds, so as to avoid competition of the capture ligand and detection ligand to the control compound due to steric hindrance.
• the detection ligand or one of the detection ligands may be specific for one of the two molecules and the capture ligand or one of the specific capture ligands of the other molecule of the complex.
• the detection ligand or one of the detection ligands and the capture ligand or one of the capture ligands may be specific for the same molecule, and even the same region of this molecule, when the complex comprises at least two of these identical molecules, thus allowing the simultaneous binding of the detection antibody and the capture antibody on the complex.
• a depot control may comprise, as a capture ligand, an antibody specific for either transferrin or soluble transferrin receptor.
• the ligand or one of the capture ligands is an antibody specific for the transferrin soluble receptor, in order to avoid interference with free transferrin that may be present in a blood sample.
• the ligand or one of the deposition control capture ligands is specific for the soluble transferrin receptor and the detection ligand or one of the control or control compound detection ligands is either specific to the transferrin, which is specific for the soluble receptor of transferrin.
• control of the deposition of a sample also makes it possible to verify that one or more analyte detection ligands have been brought into contact with the spots of a compartment of a solid support or with an assembly.
• said analyte detection ligand or ligands are not added in same time as the sample to be analyzed, but for example at a later stage, simultaneously with the addition of the detection ligand (s) of the control compound.
• the control compound detection ligand or ligands it is generally meant that the said analyte detection ligand or ligands are added in the form of a solution comprising the said analyte detection ligand (s). and the detection ligand (s) of the control compound.
• said one or more analyte detection ligands and said one or more control compound detection ligands may be added in the same step but as separate solutions.
• a sample to be analyzed comprises a compound that could interfere with the detection ligand of the control compound
• sample in particular after the step of depositing the sample followed by at least one washing step.
• all or part of the analyte detection ligands can be added at the same time as the detection ligand of the control compound.
• the deposition control then also makes it possible to validate the placing in the presence of these analyte detection ligands with the spots of a compartment of the solid support or with the set of beads.
• a blood sample includes both transferrin-soluble receptor complexed with transferrin and free transferrin, i.e. unrelated to the receptor.
• the ligand or one of the detection ligands is a transferrin-specific antibody and is added at the same time as the sample, it may partially bind to free transferrin and the detection of the control compound would be distorted.
• said detection ligand of the control compound is brought in a subsequent step, that is to say after the step of depositing the sample followed by at least one washing step.
• the sample deposition control or controls do not include a factor XIII-specific antibody (ie neither an antibody specific for the tetrameric form, nor a specific antibody for a factor XIII subunit). ).
• the sample deposition control or controls do not include a factor XIII-specific capture ligand (ie, neither a tetramerically specific capture ligand, nor a specific subunit capture ligand). factor XIII).
• the sample deposition control or controls do not comprise a specific capture ligand (in particular a specific antibody) of an IgG, an IgM or more generally an immunoglobulin (in particular particularly human IgG, human IgM or human immunoglobulin). It may be advantageous to use at least two controls for the deposition of a sample, these controls detecting different control compounds and / or the same control compound but with different sensitivities, for example when the concentration of the control compound can vary, for example example depending on the nature of the sample and / or the condition of the subject from which the sample originated.
• the control of the deposition of an analyte detection ligand makes it possible to verify the placing in the presence of one or more analyte detection ligands deposited simultaneously with the detection ligand of an additive and / or of one or more analyte detection ligands deposited simultaneously with an additive, with the spots of a compartment of a solid support or with a set of beads.
• deposited simultaneously with the additive detection ligand is generally meant that said one or more analyte detection ligands are added in the form of a solution comprising said analyte detection ligand (s) and the one or more detection ligands of an additive.
• said one or more analyte detection ligands and / or additive detection ligand (s) may be added at the same step but as separate solutions.
• “Simultaneously deposited with an additive” generally means that said one or more analyte detection ligands are added in the form of a solution comprising said analyte detection ligand (s) and an additive. Alternatively said one or more analyte detection ligands and / or additive (s) may be added at the same step but as separate solutions.
• the control of the deposition of an analyte detection ligand also makes it possible to verify the incubation and washing steps.
• concentration of the additive placed in the presence of the spots of a compartment of the solid support or of a set of balls is known, a variation of the detected signal makes it possible to identify a deficiency of the process in the steps of the process , and in particular incubation and washing steps. This can in particular make it possible to avoid "false positives" resulting from incubation steps and / or deficient washing steps, that is to say unrealized or poorly performed steps.
• a "false positive” is a positive result reflecting the presence of one or more analytes to be detected in a sample, while said one or more analytes were not present in the sample and thus should not have been detected.
• the control of the deposition of an analyte detection ligand comprises at least one capture ligand specific for an additive that is not present in the sample to be analyzed and that is used during the implementation of the method multiplexed analysis.
• An additive is in particular as defined above.
• a signal detected at the level of the control of the deposition of an analyte detection ligand makes it possible to validate the deposition step of the analyte detection ligand (s) deposited at the same time as the detection ligand of a additive.
• An additive placed in the presence of spots and / or a set of beads binds to the capture ligand at the level of the control of the deposition of an analyte detection ligand.
• An additive thus fixed can be detected by a specific detection ligand of said additive.
• a process control capture ligand may be an antibody specific for digoxigenin.
• a ligand for detecting an additive is also an antibody specific for digoxigenin.
• analyte detection ligand deposition controls may be advantageous to use several different analyte detection ligand deposition controls and therefore as many different additives, for example if the analyte detection ligands are added at several different stages, especially during at least three different steps.
• control compound which binds to a capture ligand of a sample deposition control during the implementation of the analysis method of the invention is detected by a specific detection ligand of said control compound ( immunological sandwich type), and
• an additive which binds to a capture ligand of a control of the deposition of an analyte detection ligand during the implementation of the analysis method is detected by a specific detection ligand of said additive (format immunological sandwich type).
• sandwich-type immunological formats make it possible to achieve a high level of specificity and / or sensitivity, in comparison with other immunological formats and in particular indirect formats.
• control of the deposition of a protractor makes it possible to verify the placing in the presence of a detection marker reporter with the spots of a compartment of a solid support or with a set of balls.
• Control of the deposition of a reporter is of interest only in case of indirect labeling of at least one analyte detection ligand.
• the control of the deposition of a reporter comprises an indirect marker or a carrier molecule coupled to an indirect marker.
• the indirect marker of the control of the deposition of a reporter is identical to the indirect marker coupled to at least one analyte detection ligand.
• a single and same indirect marker is used for labeling the detection ligand or ligands, and therefore only one control of the deposition of a reporter can be used.
• a signal detected at the level of the control of the deposition of a reporter makes it possible to validate the step of depositing the reporter of the corresponding detection marker.
• the reporter thus binds to the detection marker at the level of the control of the deposition of a reporter and a signal is obtained, if necessary after adding a marker substrate of said reporter, in case of control of the deposition of a positive reporter.
• control of reporter deposition includes biotin or a carrier molecule coupled to biotin.
• the subject of the present invention is thus a multiplex analysis method for detecting at least n analytes in at least one sample, n being an integer greater than or equal to 2, said method comprising at least steps a), c ) and e), at least steps a), c) and f) or at least the following steps a), b), c) and d):
• I detection ligands of p analytes to be detected and, where appropriate, at least one additive, I being preferably greater than or equal to p,
• step f optionally, in particular when said or one of said reporters of step f is coupled to a marker, in particular to an indirect marker (for example an enzyme), putting at least a second reporter of said marker coupled to the reporter of the step f) (for example a substrate, such as luminol, isoluminol or a derivative thereof) in the presence of the spots of said compartment or said set of beads,
• a marker in particular to an indirect marker (for example an enzyme)
• putting at least a second reporter of said marker coupled to the reporter of the step f) for example a substrate, such as luminol, isoluminol or a derivative thereof
• I, ⁇ , I ", m, p and y being integers greater than or equal to 0 and the sum m + p + y being greater than or equal to 1.
• a method for detecting at least n analytes may include detecting a number of different analytes that is less than n when at least two analytes to be detected are identical. For example, at least two spots of a compartment of a solid support or at least two balls (or types of beads) of a set of beads are intended for the detection of the same analyte.
• the number "n” is generally equal to the number of detection spots of an analyte of a compartment of a solid support or the number of beads (or types of beads) for detecting an analyte of a set of beads .
• the sum m + p + y is from 1 to n.
• the sum m + p + y is equal to n, for example when there are an analyte detection spots in a compartment of a solid support or n balls (or types of beads) for detecting an analyte of a set of balls.
• steps a) to e) can be performed simultaneously and / or successively (for example, some of these steps can be performed simultaneously while others are performed successively), at least for two of them in the order a) to e) or in another order, step a) always being the first step performed; when the process comprising step f) and possibly step g), these steps are always carried out subsequently to steps a) to e).
• a solid support comprises at least one compartment, said compartment comprising at least one spot for controlling the deposition of a sample, or a set of beads comprises at least one a ball to control the deposit of a sample.
• a solid support comprises at least one compartment, said compartment comprising at least one spot for controlling the deposition of a protractor, or a set of beads comprises at least a ball to control the deposit of a protractor.
• step d) preferably comprises bringing the spots or the set of beads into contact with at least one ligand for detecting said additive (s)
• a solid support comprises at least one compartment, said compartment comprising at least one spot for controlling the deposition of a detection ligand an analyte, or a set of beads comprises at least one ball for controlling the deposition of an analyte detection ligand.
• step e At least one additive is brought into contact with the spots or the set of beads in step b ) and at least one detection ligand of said additive or additives is brought into contact with the spots or the set of beads in step d).
• the expression "put compound X in the presence of a set of beads” means that compound X is brought into contact with at least one set of beads for analyzing a sample in any suitable container containing said set of beads.
• a suitable container is a microtube, a microplate or a reaction vessel.
• the or the compounds X can be put separately in the presence of the spot or spots of a compartment or the ball or beads or sets of beads for analyzing a sample, that is to say in the form of separate compositions.
• these compounds X or some of these compounds X may be placed in the presence of the spot or spots of a compartment or the ball or beads or sets of beads for analyzing a sample in the form of one or more mixtures.
• the different compounds are placed in the presence of the spots of each compartment or of each set of beads for a certain duration, for example from 1 second to 2 hours, preferably 1 minute to 1 hour, more preferably 5 minutes to 50 minutes, more preferably still 10 minutes to 40 minutes.
• the temperature of an incubation may for example be 4 ° C, a temperature of 19 ° C to 24 ° C, 37 ° C or 40 ° C.
• steps b), d), e), f) and g) are well known to those skilled in the art. They allow for example the formation of antigen-antibody complexes, marker-reporter.
• Step a) consists in providing a solid support as defined above comprising at least one compartment or at least one set of balls as defined above.
• Step a) means in particular that the multiplex analysis method is implemented by means of said solid support or said set of balls, that is to say by using said solid support or said set of balls.
• the multiplex analysis method according to the invention is therefore implemented either by using a solid support with compartment (s), for example of the microplate type, or with a set of beads.
• step a) consists in providing:
• a solid support comprising at least x compartments or several solid supports so as to have at least x compartments with all the solid supports (for example, when a solid support has only one compartment, which can, in a particular embodiment, to assimilate to the solid support itself, to provide at least x solid supports), or at least x sets of balls.
• the multiplex analysis method according to the invention allows for example the detection of n analytes in at least 1 sample, at least two samples, at least 5 samples, at least 10 samples, preferably at least 20 samples, by at least 40 samples, at least 60 samples or at least 80 samples.
• n is an integer greater than or equal to 2, for example 2, 3, 4, 5, preferably greater than or equal to 6, for example 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18 or greater than 18.
• steps are not performed simultaneously, they are usually followed by one or more washing steps.
• a washing step makes it possible to eliminate the compounds that are not bound to the spots or to the balls or to the various compounds bonded to said spots or to said balls.
• a washing step consists of at least one cycle, preferably at least two cycles, more preferably 3 to 6 cycles, of distribution (for example a volume of 400 ⁇ ) and aspiration of a washing solution into each compartment or in the presence of each set of balls.
• the wash solution may be 0.01 M Tris NaCl buffer, pH 7.4, supplemented with 0.1% Tween 20.
• the compartments of the solid support (s) are preferably identical, in particular in terms of the number and composition of the spots.
• the method can be implemented with at least one solid support comprising at least two distinct groups (or types) of compartments, each of the distinct groups having a number and / or a different spot composition (s).
• the sets of beads may be identical, particularly in terms of the number and composition of the beads.
• the method can be implemented with at least two distinct groups (or types) of different sets of beads, in particular each of separate groups of sets of beads having a different number and / or composition of beads. .
• Steps b), d), e) and f) are performed in each compartment for analyzing a sample or in each container containing a set of beads for analyzing a sample.
• a sample is added per compartment or per vessel containing a set of beads.
• An additive used in the process according to the invention is an additive recognized by the capture ligand (s) of the deposition control of an analyte detection ligand.
• a control compound present in the sample is a control compound recognized by the capture ligand (s) of the control of the deposition of a sample.
• Each analyte detection ligand is specific for an analyte recognized by at least one capture ligand present in an analyte detection spot or on an analyte detection ball.
• step b) When step b) is not performed simultaneously with one of steps c), d) or e), one or more washing steps can be performed after step b) and before said steps c), d ) and e) only when I is equal to 0.
• Steps b), c), d) and e), when present, can be performed in any desired order: successively, or one, two or three steps simultaneously.
• a step b) and a step d) respectively implement at least one additive and at least one detection ligand of said additive
• said step d) is always carried out simultaneously or subsequently in said step b).
• a step e), when it is present, is always carried out simultaneously or subsequently in step c), or before step c) provided that there is no step washing between step e) and c).
• step c) is carried out after a step b) in which I is greater than or equal to 1 and / or after a step d) in which ⁇ is greater than or equal to 1 and / or after step e ), no washing step is performed between this or these steps and step c).
• steps b) to e), when they are present, can be carried out simultaneously and / or successively, in the following orders: b) to e), b) c) e) d), c (b) (d) (e), (c) (b) (e) (d) or (c) (e) (b) (d).
• Steps c), d) and / or e) can be performed simultaneously in step b).
• steps b) and c) are performed simultaneously.
• steps c) and d) are carried out simultaneously. It may indeed be advantageous to bring the sample simultaneously with the detection ligands of the analytes which interact better in solution with the analyte of which they are specific than when the analyte is already bound to the capture antibody of the analyte. analyte.
• steps b), c) and d) are performed simultaneously.
• the process may comprise one or more steps b) (and one or more steps d)), each of the steps b) being possible independently before, after or simultaneously with step c).
• steps b) there may be at least two steps b), one being performed before or simultaneously with step c), the other being performed after or simultaneously with step c).
• the compounds of a given step may be provided in the form of a mixture, that is to say of a solution, which comprises all the compounds of this step and optionally the compounds of one or more stages performed simultaneously.
• the compounds of these different steps can be provided in the form of one or more mixtures, that is to say of one or more solutions, preferably in the form of a single mixture (that is, of a single solution).
• the additives (when present), the detection ligands of each of the p analytes to be detected, the detection ligand (s) or said additives (when present), the detection ligands of each of the m analytes to be detected can be provided as a single mixture.
• ⁇ is equal to n, i.e. at least n detection ligands of the n analytes are added in step d).
• step e) is performed subsequent to step c). This is particularly the case where the presence of one or one of the detection ligands of a compound controls at the same time as the sample may cause interference.
• step e) is performed subsequent to step c).
• step e) is carried out after step c), preferably at least one step of washing the compartments is carried out after step c) and before step e).
• step f When the method according to the invention comprises step f), this step is always carried out subsequently to steps a) to e) and it is generally preceded by at least one spot washing step of each compartment or each set of balls.
• the method may also comprise a plurality of steps f) if at least two reporter deposition controls are used, preferably a f) reporter step.
• this step is always carried out subsequently in a step f) and it is generally preceded by at least one spot washing step of each compartment or of each set of beads.
• the or at least one of the reporters of step f) is preferably both a reporter of an indirect detection marker coupled to at least one analyte detection ligand and that of a detection marker present in at least one control of the deposit of a protractor.
• the controls used for carrying out the multiplex analysis method according to the invention may mimic the different steps of the detection of the analytes.
• the analyte capture ligands and the analyte detection ligands are antibodies
• the additive detection ligand and, preferably, the control compound detection ligand are also antibodies.
• the additive detection ligand and, preferably, the detection ligand of the control compound are labeled with the same label as that of the analyte detection ligands.
• the method according to the invention generally comprises a step h) of detecting the signal corresponding to the detection markers of the control (s) (in particular of the control spot or spots or of the control ball or beads) and of the analyte (s).
• a check is positive if a positive signal is detected at the end of the process.
• a control is negative in the absence of a positive signal detected at the end of the process.
• the detection of a positive signal for each control used during the implementation of the method makes it possible to validate the entire process.
• a negative sample deposition check means that the sample has not been deposited and / or that said detection ligand of a control compound has not been deposited.
• control of the deposit of a sample the control of the deposition of an analyte detection ligand and, where appropriate, the control of the deposit of a protractor are complementary and make it possible to ensure the smooth running of each steps of the method and make it possible to understand the origin of a possible defect encountered in the analysis method.
• the method according to the invention may also comprise:
• the present invention also relates to the use of at least one control of the deposition of a sample and / or at least one control of the deposition of an analyte detection ligand, to secure a multiplex analysis of 'a sample.
• Said use may further include the use of at least one control of the deposition of a protractor.
• the present invention particularly relates to the use of at least one control selected from the group consisting of a control of the deposition of a sample, a control of the deposition of an analyte detection ligand and a control of the deposit a reporter, to secure a multiplexed analysis of a sample.
• 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.
• the present invention also relates to the use of at least two controls selected from the group consisting of a control of the deposition of a sample, a control of the deposition of an analyte detection ligand and a control of the deposit a reporter, to secure a multiplexed analysis of a sample.
• the present invention particularly relates to the use of at least one control of the deposition of a sample, at least one control of the deposition of an analyte detection ligand and at least one control of the deposition of a protractor , to secure a multiplex analysis of a sample.
• Said use is preferably carried out on a solid support comprising at least one compartment as defined above or on a set of beads as defined above.
• control of the deposition of a sample the control of the deposition of an analyte detection ligand, the control of the deposition of a protractor and the sample are in particular as defined above.
• 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 and / or at least one set of balls according to the invention and, where appropriate at least one composition or solution to be used to implement a multiplex analysis method according to the invention and / or a user guide.
• FIG. 1 Example of a solid microplate support for secure multiplex analysis.
• A is shown a schematic diagram of a microplate with 18 wells.
• B is shown schematically the bottom of a well of the microplate.
• Each well of the microplate comprises 9 spots, of which 6 spots are each intended for the detection of one or more analyte (s) for diagnosing an infection (respectively A1, A2, A3, A4, A5 and A6) and 3 control spots.
• SDC Control of the deposit of a sample
• PVC Control of the deposit of an analyte detection ligand
• RVC Control of the deposit of a Rapporteur
• C are schematically represented the three control spots comprising detection antibodies bound to the solid phase (PS).
• Ac CC Antibody for detection of the control compound.
• Ac Add Antibody for the detection of the additive.
• MS + B Support molecule labeled with biotin.
• FIG. 2 Schematic of a positive sample deposition (SDC) control in heterologous format.
• the control of the deposition of a sample comprises the capture antibody of the control compound attached to the solid phase (PS).
• the capture antibody is here specific for the soluble transferrin receptor (sTfR).
• the compound controls, here the complex 2: 2 soluble transferrin receptor (sTfR): transferrin (Tf) is attached to the capture antibody at the level of the control of the deposition of a sample and to the detection antibody (Ac 2) which is specific to transferrin.
• the detection antibody (Ac 2) is labeled with the biotin (B).
• S-POD streptavidin coupled to peroxidase
• the capture antibody and the detection antibody may be monoclonal or polyclonal antibodies. In any case, it is a heterologous sandwich.
• FIG 3 Schematic of a Positive Sample Deposition (SDC) Control in Peer Format.
• the control of the deposition of a sample comprises the capture antibody of the control compound attached to the solid phase (PS).
• the capture antibody is here specific for the soluble transferrin receptor (sTfR).
• the compound controls, here the complex 2: 2 soluble transferrin receptor (sTfR): transferrin (Tf) is attached to the capture antibody at the level of the control of the deposition of a sample and to the detection antibody (Ac 2) which is also specific for the soluble transferrin receptor (sTfR).
• the detection antibody (Ac 2) is labeled with biotin (B).
• S-POD streptavidin coupled to peroxidase
• the capture antibody and the detection antibody are, for example, monoclonal antibodies. This is a homologous sandwich.
• FIG 4 Schematic of a deposit control of a positive analyte (PVC) detection ligand.
• the process control includes the capture antibody of the additive attached to the solid phase (PS).
• the capture antibody here is specific for digoxigenin (Dig).
• the additive comprises digoxigenin and BSA, the digoxigenin being complexed with BSA.
• the additive is bound via digoxigenin to the capture antibody at the control of the deposition of an analyte detection ligand and the detection antibody (Ac 2).
• the detection antibody (Ac 2) is labeled with biotin (B).
• S-POD streptavidin coupled to peroxidase
• Both the capture antibody and the detection antibody may be monoclonal antibodies or both polyclonal antibodies, in which case it is a homologous sandwich.
• the capture antibody may be a monoclonal antibody and the detection antibody a polyclonal antibody, or conversely, in which case it is a heterologous sandwich.
• FIG. 5 Diagram of a positive reporter (RVC) deposition control.
• the control of the deposition of a reporter comprises a carrier molecule (MS) coupled to biotin (B) attached to the solid phase (PS).
• MS carrier molecule
• B biotin
• PS solid phase
• S-POD streptavidin coupled to peroxidase
• Figure 6 Spotting grid located at the bottom of a well of a microplate and comprising the spots of 5 analytes to be assayed (A1 to A5) and the three control spots SDC, PVC and RVC.
• Figure 7 Signal distribution of SDC control beads in simplex format of a population of 94 samples. The ordinate shows the number of samples and the abscissa of the Relative Fluorescence Intensity Units (RFI) intervals.
• RFI Relative Fluorescence Intensity Units
• FIG. 8 Signal distribution of PVC control beads in simplex format of a population of 38 samples. The ordinate shows the number of samples and the abscissa of the Relative Fluorescence Intensity Units (RFI) intervals.
• RFI Relative Fluorescence Intensity Unit
• sample Deposit Control the deposit of the sample and also step 2 (deposition of the conjugates 2, ie the detection ligands deposited in the step 2) and step 3 (deposition of the S-PE reporter: streptavidin coupled with Phycoerythrin) and
• stage 1 deposition of conjugates 1, ie the detection ligands step 1
• step 2 deposition of the conjugates 2, in dotted lines, as not illustrated in this example
• step 3 deposition of the S-PE reporter
• the BioPlex 200® analyzer (Bio-Rad, Marnes-la-Coquette, France) is used according to the manufacturer's instructions.
• This immunoassay automaton contains a flow cytometer and a Luminex 100 TM detector (Luminex Corp., Austin, Texas, USA) and uses heterogeneous sets of superparamagnetic particles.
• Each homogeneous group of particles, composed of polystyrene and methacrylic acid (COOH function), with a size of 8 ⁇ in diameter, is manufactured with different percentages of fluorochromes (CL1 and CL2) producing a unique identification code assigned to each particle group and detectable by the Luminex 100 TM detector laser (Luminex Corp., Austin, TX, USA).
• the balls of a game pass one by one through a flow cell, in the center of a liquid sheath, to be simultaneously excited and read by two separate lasers. Measurements are made at the passage of each ball.
• the 638 nm red laser excites the identification fluorochromes (CL1 and CL2) embedded on the surface of each particle and the composite signal is interpreted to identify the analyte detected by the particle. This laser therefore serves, by identifying the particle category, to identify the current test.
• the 532 nm green-beam laser excites the S-PE reporter (streptavidin coupled with Phycoerythrin) and the emitted fluorescence is proportional to the amount of reporter attached to the particle. This laser is therefore used to measure the reactivity of the immobilized analyte on said particle.
• the system software converts the signal related to the presence of the detection ligand into a relative fluorescence intensity value (RFI in English or RFI).
• RFI relative fluorescence intensity value
• a ratio can be calculated to classify the result qualitatively into positive or negative.
• a set of beads with six distinct groups of Luminex TM superparamagnetic particles (Luminex Corp., Austin, Texas, USA) is used.
• This set of beads comprises a group of beads to control the deposition of a sample (SDC beads), a group of beads to control the deposition of an analyte detection ligand (PVC beads) and 4 groups of beads. detection of an analyte (analytes to be assayed: A1, A2, A3 and A4).
• Each group of particles is coated with a specific capture ligand of a particular test.
• Each capture ligand is coupled with a hetero-bifunctional reagent.
• the SDC beads are coated with a transferrin-soluble, anti-transferrant mouse monoclonal antibody (Fitzgerald, USA) immobilized at 1 mg of particles.
• the PVC beads are covered with a polyclonal anti-Digoxigenin sheep antibody (Abcam, USA) immobilized at 5 ⁇ g / mg of particles.
• the detection beads of the analytes A1, A2, A3 and A4 are coated with one or more specific capture ligands of the analytes to be detected.
• the detection ligand of the control compound (of the SDC control), pAb-anti-Tf-biot, is a polyclonal sheep anti-Transferrin antibody (Bio-Rad, Marnes la Coquette, France) coupled to biotin (Thermo Scientific, France ) using a heterobifunctional reagent per se known to those skilled in the art.
• the additive detection (PVC control) ligand is a polyclonal sheep anti-Digoxigenin antibody (Abcam, France) coupled to biotin (Pierce, USA) at the same time. using a hetero-bifunctional reagent in itself known to those skilled in the art.
• the BSA-DIG additive is digoxigenin (Sigma, France) grafted onto a carrier molecule, in this example the Bovine Serum Albumin (Millipore, France) using a hetero-bifunctional reagent, itself known from the skilled person.
• the S-PE reporter is streptavidin (Roche, Germany) coupled with Phycoerythrin (Cyanotech, Hawai, USA) using a heterobifunctional reagent per se known to those skilled in the art.
• Phosphate buffer pH 7.4, containing: 150 mM NaCl, Tween 20 TM (Sigma brand) 0.1%, Proclin 300 TM (Supelco brand) 0.5%, PEG 6000 2.75 %, 1% Bovine Serum Albumin, 1% Sheep Normal Serum, 0.095% NaN3. vi.5. Wash solution
• the immunological reactions take place in the 96-well polypropylene microplate wells having a maximum volume of 355 ⁇ l per well.
• the negative samples (serum or plasma) used come from the French blood establishment of Lane.
• the test protocol in multiplex format comprises the assay of 4 different analytes, the assay of the A1 and A2 analytes being carried out in an immunological time, the assay of the analytes A3 and A4 being carried out in two immunological times.
• Step 1
• the mixture is incubated for 40 minutes at 37 ° C with shaking. 3.
• the following washing steps are performed: separation of the solid and liquid phases by magnetization and 3 successive washes with at least 300 ⁇ of washing solution. At the last wash the particles are resuspended.
• the mixture is incubated for 15 minutes at 37 ° C with stirring.
• the particles are resuspended in each reaction well by adding 120 ⁇ l of wash solution and the microplate is agitated.
• the suspension of particles from each well is read using the two laser beams.
• the SDC ratio of the samples is calculated as follows:
• the PVC ratio of the samples is calculated as follows RFI signal of the sample
• the threshold value SDC and PVC was established according to a statistical study described in the results below.
• the analyte detection beads A1 to A4 and their detection ligands are not used.
• the study of 94 samples makes it possible to define the threshold value of the SDC system (see FIG.
• the threshold value of the SDC system is calculated by subtracting 3 times the standard deviation of the signal from the sample population to the average value of the population signal.
• Table 2 CDS signal statistics of the population of 94 samples and calculation of the threshold value
• the mixture comprising "conjugates 2" comprises 90 ⁇ l of conjugate diluent 2 containing the pAb-anti-Tf-biot control compound detection ligand.
• Table 3 Summary of SDC ratios obtained in a nominal process (case 1) and degraded processes (cases 2, 3 and 4)
• the analyte detection beads A1 to A4 and the corresponding detection ligands are used.
• the analyte detection beads A1 to A4 and the corresponding detection ligands are not used.
• the threshold value of the PVC system is calculated by subtracting 3 times the standard deviation of the signal from the population of samples from the average value of the signal of the population (see Table 5).
• Table 5 Statistics of the PVC signal of the population of 38 samples and calculation of the threshold value.
• Threshold value Mean - 3 ⁇ (RFI) 143.4
• the analyte detection beads A1 to A4 and the corresponding detection ligands are used.
• the performance of the PVC system is similar in simplex mode and in multiplex mode (see Table 7).
• Threshold value Mean - 3 ⁇ (RFI) 143.4 1 21, 7 Moreover, by comparing the RFI signals obtained between an MPX format without SDC and PVC versus an MPX format with SDC and PVC, it turns out that the addition of the SDC and PVC tests does not impact the performance of a multiplex comprising 4 analytes to be assayed (ie deviations in% between RFI signals of MPX format without SDC and PVC versus MPX format with SDC and PVC are within +/- 20%, which is considered statistically acceptable) .
• step 2 deposit of the sample and also step 2 (deposit of the conjugates 2), step 3 (deposit of the protractor S -POD) and step 4 (deposit of Luminol substrate),
• step 1 deposition of the additive and deposition of the conjugates 1
• step 3 deposit of the S-POD reporter
• step 4 deposit of Luminol substrate
• step 3 S-POD reporter deposit of Luminol substrate
• the technology used for this system is an innovative multiplex technology nanospotting on biochip (see definition below), with revelation by chemiluminescence thanks to a reporter labeled by the horseradish peroxidase enzyme and revealed by a luminol-type substrate.
• biochip is a collection of miniaturized test sites (or “micro-array”) arranged on a solid support that allows for many tests simultaneously to obtain a higher rate high.
• each well of a microplate (Greiner, Germany) are deposited in rows using a robot spotteur, drops of 50 nL of a solution of proteins or antibodies specific to the analyte to be assayed (A1, A2, A3, A4, A5, SDC, RVC and PVC) (cf. Figure 6).
• the bottom of each well of these microplates has adsorption capabilities of proteins and peptides per se 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 addition of the revealing substrate causes a light emission.
• the oxidation of luminol by enzymatic catalysis leads to a light emission proportional to the amount of Streptavidin-peroxidase reporter fixed by the spot.
• the signal is acquired by a scientific camera.
• the resulting image is then analyzed in order to determine the intensity of the luminescence produced by each geographic area of the bottom of the well corresponding to each spot (addressing of the information).
• the system software converts the signal measured by spot into a value of Luminescence Relative Units ("URL” in French or "RLU” in English). A ratio can be calculated to classify the result qualitatively into positive or negative, as detailed below.
• the different control spots are:
• the SDC spot comprising a mouse monoclonal antibody anti-soluble transferrin receptor (Fitzgerald, USA) immobilized at 50 ⁇ g / mL,
• the PVC spot comprising a mouse monoclonal antibody anti-Digoxigenin (Covalab, France) immobilized at 25 ⁇ g / mL, and
• the RVC spot comprising a mouse monoclonal anti-KLH ("Keyhole Limpet Hemocyanin”) antibody (Genway, USA) coupled to biotin (Thermo Scientific, France) using a hetero-bifunctional reagent in itself known to those skilled in the art and immobilized at 1 ⁇ 9 ⁇ _.
• KLH Keyhole Limpet Hemocyanin
• the detection ligand of the control compound (relating to the SDC control), pAb-anti-Tf-biot, is a polyclonal sheep anti-Transferrin antibody (Bio-Rad, Marnes la Coquette, France) coupled to biotin (Thermo Scientific, France) using a hetero-bifunctional reagent per se known to those skilled in the art.
• the additive detection (PVC control) ligand is a mouse monoclonal anti-digoxigenin antibody coupled to biotin (Covalab, France).
• the additive BSA-DIG is Digoxigenin (Sigma, France) grafted onto a carrier molecule, in this example the Bovine Serum Albumin (Millipore, France).
• the coupling is carried out using a hetero-bifunctional reagent, itself known to those skilled in the art.
• the S-POD reporter is streptavidin (Roche, Germany) coupled to peroxidase (Roche Germany) by the method described by P. Nakane and A. Kawaoi (J Histochem Cytochem (1974) Vol 22, No. 12. pp 1084-1091), as known to those skilled in the art.
• the ELISTAR ETA C Ultra ELISA revealing substrate (Cyanagen, Italy) consists of two solutions: XLSE024L Luminol enhancer solution (A) and XLSE024P Peroxide solution (B).
• the negative samples (serum or plasma) used come from the French blood establishment of Lane.
• the test protocol includes the following steps.
• Step 1
• conjugate diluent 1 comprising:
• the mixture is incubated for 40 minutes at 37 ° C with shaking.
• the mixture is incubated for 15 minutes at 37 ° C with stirring.
• the mixture is incubated for 1 minute at 37 ° C with stirring.
• the SDC ratio of the samples is calculated as follows
• Ratio- RVC sample on -
• the multiplex described in this example comprises 5 analytes to be assayed 3 analytes whose detection ligands are added in step 1 (A1, A2 and A3), 2 analytes whose detection ligands are added in step 2 (A4 and A5) and the three SDC, PVC and RVC controls.
• Table 9 lists the scenarios that can be encountered during an isolated or cumulative invalidation of the SDC, PVC and RVC controls.
• detection ligands of step 2 can not be validated or invalidated.
• Table 10a Response of SDC, PVC and RVC controls (average response, standard deviation, minimum and maximum value, threshold values).
• Table 10b Responses of the SDC, PVC and RVC checks (average response, standard deviation, minimum and maximum value, threshold values).
• Table 11a Responses of SDC, PVC and RVC controls in RLU and ratios of a population of 22 samples.
• Table 1 1b Responses of SDC, PVC and RVC controls in RLU and ratios (threshold +), ratios (threshold-) of a population of 22 samples.
• the status of SDC and PVC and RVC controls interpretations is "Valid” for all 22 samples.
• Cases 5 and 6 lead to SDC, PVC and RVC ratios of less than 1, which invalidates the measurements resulting from these degraded processes.
• the RVC signal specifically invalidates the revealing step (S-POD deposit and Luminol deposit).
• the invalidation of the process by a RVC ratio of less than 1 implies an absence of the systematic signal of the SDC and PVC controls (see Gray zone of Table 9). In this case, it is not possible to determine whether the sample deposits and conjugates 2 (SDC-controlled deposits) or additive deposits and conjugates 1 (PVC-controlled depositions) occurred correctly. or not.
• Cases 1 and 4 lead to SVC (threshold) ratios lower than 1, which invalidates the measurements resulting from these degraded processes. Moreover, in case 1, the ratio (threshold +) PVC is greater than 1 which reflects an indirect impact of the absence of sample on the PVC control.
• Table 13a Response of SDC, PVC and RVC Controls to RLU and Ratio of a Population of 22 Samples
• Table 13b Response of SDC, PVC and RVC controls to RLU and ratio of a population of 22 samples

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