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WO2021255021A1 - System for rapid analysis of a biological sample for distinguishing specific serological antibodies of an infectious agent present in the biological sample - Google Patents

System for rapid analysis of a biological sample for distinguishing specific serological antibodies of an infectious agent present in the biological sample Download PDF

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Publication number
WO2021255021A1
WO2021255021A1 PCT/EP2021/066103 EP2021066103W WO2021255021A1 WO 2021255021 A1 WO2021255021 A1 WO 2021255021A1 EP 2021066103 W EP2021066103 W EP 2021066103W WO 2021255021 A1 WO2021255021 A1 WO 2021255021A1
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Prior art keywords
antibodies
infectious agent
serological
capture
rapid analysis
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PCT/EP2021/066103
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French (fr)
Inventor
Milovan Stankov
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Ng Biotech
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Publication of WO2021255021A1 publication Critical patent/WO2021255021A1/en

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    • 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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • 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
    • 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
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present invention relates to the technical field of systems for rapid analysis of a biological sample, to discriminate serological antibodies specific to an infectious agent present in said biological sample.
  • a rapid diagnostic test also called a “rapid screening test” or “RDT” is a test that quickly establishes (within minutes) the presence of at least one analyte of interest in a biological sample.
  • This technique has the advantage of being simple, fast and inexpensive. Such tests can also be used in the doctor's office but also at the patient's bedside or in the field.
  • serological tests allow rapid and efficient detection of the presence of specific serological antibodies to an infectious agent present in a biological sample.
  • Such serological tests are conventionally used to determine, retrospectively, whether an individual has suffered an infection by an infectious agent (suspected infection, investigation of contact with an infected person, etc.).
  • the present invention provides a system for rapid analysis of a biological sample, in order to discriminate the specific serological antibodies of an infectious agent present in said biological sample.
  • the rapid analysis system comprises at least one chromatographic strip, advantageously an immunochromatographic strip, which comprises different successive zones, in the direction of upstream to downstream capillary migration, namely at least:
  • a deposit zone intended to receive said biological sample mixed with a buffer solution
  • a release zone which comprises at least one detection reagent conjugated with a visible and / or measurable marker, said detection reagent being able to move as a consequence of the migration of the buffer solution along the chromatographic strip
  • At least one capture zone which comprises at least one capture reagent, immobilized on the chromatographic strip.
  • the combination of detection reagent (s) / capture reagent (s) is chosen from the combinations of detection reagent (s) / capture reagent (s) suitable for discriminating at least two groups of serological antibodies. :
  • neutralizing antibodies comprising the specific serological antibodies of said infectious agent, capable of canceling, or at least reducing, the infectious power of said infectious agent
  • non-neutralizing antibodies comprising specific serological antibodies for said infectious agent, other than said antibodies belonging to said first group of serological antibodies.
  • said at least one capture zone comprises at least two capture lines: a first line, upstream, designed to detect said first group of serological antibodies (“neutralizing antibodies”), and a second line, downstream, designed to detect said second group of serological antibodies (“non-neutralizing antibodies”);
  • the rapid analysis system further comprises a reading device comprising optical capture means, adapted to capture an image of said at least one capture zone, and processing means comprising a first module designed to determine the intensity of each line and a second module configured to determine an intensity ratio between said lines;
  • infectious agent is chosen from viruses, in particular viruses responsible for pneumopathies, advantageously the Coronaviridae, more preferably Orthocoronavirinae or coronavirus;
  • Said rapid analysis system also advantageously comprises at least one sampling device, suitable for taking said biological sample from an individual, for example a capillary blood sample, and a buffer solution, intended to be mixed with the biological sample, for the implementation of the chromatographic technique.
  • the combination of detection reagent (s) / capture reagent (s) is chosen from combinations of detection reagent (s) / capture reagent (s) suitable for detecting the serological antibodies directed against the antigens. surface area of said infectious agent, including:
  • the first group of serological antibodies comprises serological antibodies directed against the surface antigens responsible for the infectious power of said infectious agent, preferably the proteins responsible for attachment and / or penetration and / or decapsidation, and
  • the second group of serological antibodies comprises serological antibodies directed against the surface antigens of said infectious agent other than those responsible for said infectious power.
  • said at least one detection reagent and / or said at least one capture reagent comprise a first surface antigen of said infectious agent, or fragment of said first antigen, chosen from the surface antigens responsible for infectivity, and a second antigen of surface of said infectious agent, or fragment of said second antigen, chosen from surface antigens other than said surface antigens responsible for infectivity;
  • the release zone comprises a combination of two detection reagents: a first surface antigen of the infectious agent, or fragment of said first antigen (surface antigens responsible for the infectious power), and a second surface antigen of the agent infectious, or fragment of said second antigen (surface antigens other than those responsible for infectivity);
  • the capture zone comprises two lines of capture reagents: a first line, upstream, of which the capture reagent comprises a first surface antigen of the infectious agent or fragment of said first antigen (surface antigens responsible for infectious power), and a second, downstream line, the capture reagent of which is chosen from anti-human antibody antibodies;
  • an infectious agent SARS-Cov-V2 (Covid 19) whose surface comprises NP core proteins and Spike S proteins, said S protein comprising an S1 subunit, an S2 subunit and an RBD domain (Receptor Binding Domain), said at least one detection reagent and / or said at least one capture reagent comprise a first surface antigen of the infectious agent chosen from S proteins and S protein fragments, preferably from the sub- S1 unit, the S2 subunit and the RBD domain (Receptor Binding Domain), and a second surface antigen of the infectious agent chosen from NP core proteins and N P core protein fragments.
  • the present invention also relates to the method of rapid analysis of a biological sample by the implementation of a rapid analysis system according to the invention, to discriminate the neutralizing serological antibodies and the non-neutralizing serological antibodies which are specific. an infectious agent.
  • the process includes:
  • the biological sample is a capillary blood sample or a serum / plasma sample
  • the reading step comprises a phase of determining the intensity of each line, then a phase of determining the intensity ratio between said lines.
  • FIG. 1 is a schematic, and functional, view of an immunochromatographic strip suitable for the rapid analysis system according to the invention
  • FIG. 2 is a schematic view of a rapid analysis system into which the immunochromatographic strip can be integrated.
  • the present invention relates, in general, to systems for rapid analysis of biological samples, also called “rapid diagnostic test” or “rapid screening test” or “RDT”.
  • the rapid analysis system according to the invention is in particular dedicated to discriminating serological antibodies specific for an infectious agent, present in a biological sample.
  • biological sample is meant any sample taken from an individual (human or animal in particular) and in which serological antibodies are sought.
  • the biological sample preferably consists of a liquid sample in which the serological antibodies are in solution or in suspension.
  • This liquid sample can in particular be any biological or bodily fluid.
  • the liquid sample may also have been obtained directly or indirectly from a biological or bodily fluid.
  • the sample can also be a liquid extract from a solid sample.
  • the biological sample is whole blood or serum.
  • whole blood is meant in particular capillary blood advantageously obtained by means of an auto-pricker at the level of the pulp of the finger, or of the heel, of a human individual.
  • serological antibodies specific to an infectious agent also called “serological antibodies directed against an infectious agent”
  • serological antibodies directed against an infectious agent is advantageously meant the antibodies developed by an individual and indicating an immune response to infection by an infectious agent.
  • the antibodies in question advantageously consist of polyclonal antibodies which consist of a mixture of antibodies recognizing different epitopes on a given antigen (each idiotype being secreted by a different plasma cell clone).
  • the antibodies sought are advantageously chosen from serum antibodies IgM (immunoglobulins M) and / or IgG (immunoglobulins G) and / or IgA (immunoglobulins A).
  • infectious agent preferably means viruses, in particular viruses responsible for pneumopathies, advantageously the Coronaviridae, more preferably Orthocoronavirinae or coronavirus.
  • coronavirus is meant SARS-CoV, MERS-CoV or SARS-CoV-2.
  • discriminate advantageously means the qualitative (advantageously the presence or absence), or even quantitative, determination of groups of serological antibodies specific to an infectious agent which are present in said biological sample and which have different immunological properties. for the same infectious agent (advantageously resulting from different antigenic specificities for the same infectious agent).
  • the present invention is thus designed to discriminate serological antibodies from at least two groups of the following serological antibodies: a first group of serological antibodies A1, called “neutralizing antibodies”, comprising the specific serological antibodies of said infectious agent which are capable of canceling, or at least reducing, the infectious power of said infectious agent, and
  • non-neutralizing antibodies or “immunizing antibodies”, comprising the specific serological antibodies of said infectious agent (directed against the infectious agent), other than said antibodies belonging to said first group of A1 serological antibodies (“non-neutralizing antibodies”, while being directed against the infectious agent, are not able to cancel, or at least reduce, the infectious power of said infectious agent).
  • the first group of serological antibodies A1 comprises the serological antibodies directed against the surface antigens responsible for the infectious power of the said infectious agent, preferably the proteins or fragments of proteins responsible for the attachment and / or the penetration and / or the decapsidation, and
  • the second group of serological antibodies A2 comprises serological antibodies directed against the surface antigens of said infectious agent other than those responsible for said infectious power.
  • the infectious agent is the SARS-Cov-V2 virus (Covid 19), advantageously exhibiting the GenBank accession MN908947 genomic sequence.
  • Such an infectious agent has a surface which comprises NP core proteins and Spike S proteins; the S protein advantageously comprises an S1 subunit, an S2 subunit and an RBD domain (Receptor Binding Domain) (Chen Z et al. Clin Chem 2004; 50: 988-95; Zhou P et al., Nature 2020; Lu R et a., Lancet, 2020; 395: 565-74).
  • S protein advantageously comprises an S1 subunit, an S2 subunit and an RBD domain (Receptor Binding Domain)
  • the first group of serological antibodies A1 comprises the serological antibodies directed advantageously against the surface antigens chosen from the S proteins and the fragments of S protein, preferably from the S1 subunit, the S2 subunit and the RBD domain (Receptor Binding Domain), and
  • the second group of serological antibodies A2 comprises the serological antibodies directed against the surface antigens chosen from the NP core proteins and the fragments of the N P core protein, and optionally against the fragments of S protein other than the RBD domain.
  • the first group of serological antibodies A1 comprises the serological antibodies directed advantageously against the surface antigens chosen from the RBD domain (Receptor Binding Domain)
  • the second group of serological antibodies A2 comprises the serological antibodies directed against the surface antigens chosen from the NP core proteins (and the NP core protein fragments) and against the regions of the S protein other than the RBD domain.
  • the rapid analysis system 1 comprises at least one chromatographic strip 2, advantageously an immunochromatographic strip, designed to discriminate, in a sample, the “neutralizing” serological antibodies, compared to the “non-neutralizing” antibodies. , Specific for an infectious agent.
  • Chromatographic strip 2 also called “capillary diffusion means” is advantageously chosen from chromatographic strips carrying reagents suitable for the discrimination, in a sample, of “neutralizing” / “non-neutralizing” serological antibodies specific for an agent. infectious.
  • This chromatographic strip 2 is formed from any means constituting or acting as a continuous capillary diffusion unit, by lateral migration (that is to say perpendicular to the thickness of the capillary material (s) used for the capillary diffusion. ).
  • This capillary diffusion means advantageously consists of a porous solid support allowing the migration of a liquid by simple capillary diffusion.
  • the porosity of this support allows capillary diffusion (or lateral migration) of the sample (in particular of serological antibodies) and / or of the reagents in the liquid or wet state.
  • capillary diffusion means are very widely used, in particular in all lateral migration immunochromatography techniques.
  • Such a chromatographic strip 2 here consists of a support elongated in the direction and / or the direction of capillary diffusion (lateral migration).
  • Chromatographic strip 2 can be made up of:
  • capillary or porous elements or materials suitably arranged with respect to each other (for example by overlapping), to obtain a continuity of capillary flow from one element or from one material to another, according to the direction of capillary diffusion.
  • Such a chromatographic strip 2 determines a direction and direction of capillary diffusion of any liquid which is received or deposited at an upstream end, and which then moves towards a downstream end of the chromatographic strip 2.
  • the chromatographic strip 2 can consist of various immunochromatographic supports, for example cellulose, nylon, nitrocellulose, polyethylene or glass fiber.
  • the rapid analysis system 1 comprises at least one chromatographic strip 2, advantageously an immunochromatographic strip, which comprises different successive zones, in the direction of upstream to downstream capillary migration, namely at least:
  • a deposit zone 3 intended to receive said biological sample mixed with a buffer solution
  • a release zone 4 which comprises / comprises at least one detection reagent 41, 42 conjugated with a visible and / or measurable marker 43, said detection reagent 41, 42 being able to move as a consequence of the migration of the solution buffer along chromatography strip 2, and
  • At least one capture zone 5 which comprises / comprises at least one capture reagent 51, 52, immobilized on the chromatographic strip 2.
  • the release and capture zones 4, 5 advantageously consist of a transverse line or strip (extending perpendicular to the direction of migration), for example having a width of between 1 and 2 mm, and an area of between 3 and 5 mm 2 .
  • the “detection reagent 41, 42” or the “capture reagent 51, 52” consists of any chemical, biochemical or biological entity, which is capable of binding specifically to form a complex allowing the detection of a serological antibody specific for (directed against) an infectious agent of interest (advantageously an antigen or an epitope of this infectious agent).
  • the detection reagent 41, 42 and / or the capture reagent 51, 52 also constitute so-called “binding” reagents.
  • binding reagents 41, 42, 51, 52, allowing the detection of at least one serological antibody in the liquid sample are well known and can be tailor-made for the implementation of the invention.
  • binding reagents 41, 42, 51, 52 are advantageously chosen from those which are capable of binding specifically with a serological antibody of interest and / or of binding specifically with one another.
  • the complementary binding reagents 41, 42, 51, 52 are advantageously intended to form different complexes:
  • the binding reagents 41, 42, 51, 52 are able to bind concomitantly to a serological antibody of interest, to form a test in sandwich format
  • binding reagents 41, 42, 51, 52 detection or capture
  • bind or “bond” is meant any strong bond, for example covalent, but also any weak bond, for example of the antigen / antibody type.
  • the binding reagents 41, 42, 51, 52 are advantageously chosen from antibodies and antigens.
  • antigen is understood to mean in particular proteins or protein fragments, advantageously recombinant proteins obtained by expression in a prokaryotic system.
  • binding reagents 41, 42, 51, 52 are advantageously chosen to exhibit, with regard to each group of serological antibodies sought, the following performances:
  • sensitivity of at least 80%, more preferably at least 90%, or even at least 95%.
  • the detection reagent (s) 41, 42 are advantageously combined with a visible and / or measurable label 43, advantageously a particulate label.
  • visible and / or measurable marker is meant any marking allowing direct or indirect detection with the naked eye, or with the aid of a device, due to the emission of a signal at said level. at least one capture area 5.
  • the signal is, for example, fluorescence, coloring, the presence of isotope or a magnetic signal.
  • colored particulate markers such as colloidal gold, or fluorescent, colored latex particles, fluorescent latex particles and particles conjugated with avidin and with streptavidin.
  • Particulate markers colored or fluorescent, thus consist of small particles which are insoluble in water and which therefore form suspensions, dispersions or solutions, in the liquid phase.
  • markers of the dextran type (Hansen T.M., IVD Technology 4, 35-40, 2003).
  • the binding reagent is then conjugated to a dextran (polysaccharide derivative) chain carrying fluorophores.
  • the markers can also consist of enzymes (alkaline phosphatase or AP, horseradish peroxidase or HRP, in particular), of dyes (or “dyes”) or of chemiluminescent compounds (in particular fluorescein isothiocyanate or FITC).
  • enzymes alkaline phosphatase or AP, horseradish peroxidase or HRP, in particular
  • dyes or “dyes”
  • chemiluminescent compounds in particular fluorescein isothiocyanate or FITC.
  • an antibody labeled according to techniques known to those skilled in the art for indirect detection such as for example a biotinylated antibody, indirectly allowing detection by the formation of avidin entities.
  • -biotin and streptavidin-biotin This labeled and biotinylated antibody can also either be directly deposited on a test line, in the capture zone 5, to increase the sensitivity, or be deposited with the specific detection antibody, to increase the contact time and again. sensitivity in particular, for example, due to the number of binding sites.
  • said at least one capture reagent 51, 52 specific for the analyte is immobilized on the solid support according to techniques known to those skilled in the art.
  • This capture reagent 51, 52 is immobilized in such a way that it is not mobile in the wet state.
  • This immobilization can take place, for example, by absorption or by covalent coupling.
  • neutralizing antibodies comprising the specific serological antibodies of said infectious agent, capable of canceling, or at least reducing, the infectious power of said infectious agent
  • non-neutralizing antibodies comprising serological antibodies specific for said infectious agent, other than said antibodies belonging to said first group of serological antibodies A1.
  • the non-neutralizing antibodies are directed against the infectious agent, but are not able to cancel (or to inhibit), or at least to reduce, the infectivity of said. infectious agent.
  • these non-neutralizing antibodies are directed against proteins, or protein fragments, which are not responsible for the infectivity of said infectious agent.
  • said at least one capture zone 5 advantageously comprises at least two capture lines:
  • the combination of detection reagent (s) 41, 42 / capture reagent (s) 51, 52 is chosen from the combinations of detection reagent (s) 41, 42 / capture reagent (s) 51 , 52 adapted to discriminate the serological antibodies directed against the surface antigens (also called “epitopes”) of said infectious agent, including: - the first group of serological antibodies A1 comprises the serological antibodies directed against the surface antigens responsible for the infectious power of said infectious agent, preferably the proteins (including glycoproteins) responsible for the attachment and / or the penetration and / or decapsidation, and
  • the second group of serological antibodies A2 comprises serological antibodies directed against the surface antigens of said infectious agent other than those responsible for said infectious power.
  • surface antigen advantageously means the proteins (including glycoproteins ”) on the surface of the infectious agent and comprising at least one epitope.
  • proteins responsible for attachment is meant in particular those proteins which attach to receptors located on the cytoplasmic membrane of the host cell.
  • proteins responsible for penetration is meant in particular the proteins which allow the entry of the virus into the interior of the host cell.
  • proteins responsible for decapsidation means in particular the proteins which allow the release of the genome relative to the capsid.
  • said at least one detection reagent 41, 42 and / or said at least one capture reagent 51, 52 comprise:
  • surface antigen advantageously means proteins (including glycoproteins ”), or protein fragments, which comprise at least one epitope.
  • the release zone 4 comprises a combination of two detection reagents:
  • a second surface antigen 42 of the infectious agent or fragment of said second antigen (surface antigens other than those responsible for infectivity), conjugated with a marker 43 (identical or different from the first surface antigen 41).
  • the capture zone 5 advantageously comprises the two lines of capture reagents 51, 52: - the first line 5a, upstream, of which the capture reagent 51 comprises a first surface antigen of the infectious agent or fragment of said first antigen (surface antigens responsible for the infectious power), and
  • the capture reagent 52 is chosen from anti-human antibody antibodies (monoclonal and / or polyclonal).
  • the first surface antigen 41 of the release zone 4 and the capture reagent 51 of the first line 5a may be the same, or different, from each other.
  • the first surface antigen 41 of the release zone 4 corresponds to the first surface antigen of the infectious agent (surface antigen responsible for infectivity), and
  • the capture reagent 51 of the first line 5a corresponds to a fragment of said first antigen (fragment responsible for the infectivity).
  • said at least one detection reagent 41, 42 and / or said at least one capture reagent 51, 52 comprise:
  • a first surface antigen 41 of the infectious agent chosen from S proteins and S protein fragments, preferably from the S1 subunit, the S2 subunit and the RBD domain (Receptor Binding Domain), and
  • the release zone 4 comprises a combination of two detection reagents:
  • a second antigen 42 chosen from the NP core proteins and the NP core protein fragments.
  • the first antigen 41 of the release zone 4 and / or the capture reagent 51 of the first line 5a is advantageously chosen from:
  • the first antigen 41 of the release zone 4 and the capture reagent 51 of the first line 5a are advantageously chosen from the following combination:
  • the first antigen 41 of the release zone 4 is chosen from protein S, any length, and the capture reagent 51 of the first line 5a is a fragment of protein S comprising the RBD domain, or even a fragment corresponding to the domain RBD, WHERE
  • the first antigen 41 of the release zone 4 and the capture reagent 51 of the first line 5a are among the S protein, any length.
  • non-neutralizing antibodies within the second line 5b, downstream.
  • the rapid analysis system 1 can further include a reading device 7 (electronic device) comprising:
  • optical capture means 71 adapted to capture an image of said at least one capture zone 5
  • the optical capture means 71 are suitable for acquiring at least one digital image of said at least one capture zone 5 of said at least one chromatographic strip 2.
  • the optical capture means 71 advantageously comprise:
  • processing module comprising a computer program which is advantageously adapted to generate a digital image from the data coming from the converter.
  • the optical sensor is a photosensitive electronic component used to convert electromagnetic radiation (UV, visible or IR) into an analog electrical signal.
  • the signal obtained is digitized by the analog-to-digital converter, then processed by the processing module to obtain a digital image of interest corresponding to said at least one capture zone 5.
  • digital image is understood to mean an image consisting of a set of pixels, each of which is defined by several parameters (for example color and brightness).
  • This digital image is advantageously in the form of an image file, that is to say a computer file whose content consists of at least one image.
  • the image file consists of:
  • the processing means 72 for example in the form of a microcontroller incorporating a computer program (firmware), are suitable for controlling and coordinating electronic components.
  • the processing means 72 include:
  • a second module 722 designed to determine an intensity ratio between said lines 5a, 5b.
  • the modules 721, 722 advantageously consist of computer programs, comprising program code means designed to determine the intensity of each line 5a, 5b (via a first suitable algorithm) then to determine an intensity ratio between said lines 5a, 5b (via a second adapted algorithm), when said computer programs are executed by the processing means 72.
  • the rapid analysis system 1 can still include:
  • At least one sampling device 8 suitable for taking said biological sample from an individual, for example a capillary blood sample, and
  • the sampling device 8 comprises for example:
  • an auto-pricker 81 classic in itself and disposable, suitable for generating a flow of a drop of capillary blood
  • a capillary blood collection member 82 disposable, for example in the form of a flexible pipette conventional per se.
  • Buffer solution 9 is intended to be mixed with the biological sample.
  • This buffer solution 9 is chosen from the buffer solutions suitable for the implementation of the chromatographic technique: it is in particular intended to migrate along the chromatographic strip 2 so as to cause, or at least to facilitate, the migration of the biological sample (and in particular serological antibodies).
  • the buffer solution 9 is for example chosen from diluents composed of a buffered saline solution. It can also comprise a detergent or any other component necessary in particular for the migration or for the reactions on the chromatographic strip 2.
  • the present invention also relates to the method of rapid analysis of the biological sample by the implementation of the rapid analysis system 1 according to the invention, to discriminate the neutralizing serological antibodies and the non-neutralizing serological antibodies which are specific for 'an infectious agent.
  • the method comprises the following steps for this:
  • the biological sample is a capillary blood sample or a serum / plasma sample.
  • the reading step comprises:
  • the first line 5a, upstream, is then indicative of the presence of neutralizing antibodies A1 of the IgG, IgM and / or IgA type.
  • the second line 5b downstream, is then indicative of the presence of the non-neutralizing antibodies A2 of the IgG, IgM and / or IgA type.
  • each line 5a, 5b is advantageously proportional to the respective concentrations of the "neutralizing” / “non-neutralizing” serological antibodies.
  • the two lines 5a, 5b have zero intensity
  • the second line 5b (downstream) has a non-zero intensity and the first line 5a (upstream) has a zero intensity;
  • the first line 5a (upstream) and the second line 5b (downstream) have a non-zero intensity.
  • the ratio of the two lines 5a, 5b is indicative of the prevalence of neutralizing antibodies relative to non-neutralizing antibodies.
  • This ratio can be identified directly and / or through the use of the reading device 7.
  • This ratio provides a percentage value (%) of the neutralizing antibodies, relative to the non-neutralizing antibodies.

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Abstract

The present invention relates to a system for rapid analysis of a biological sample for distinguishing specific serological antibodies of an infectious agent present in the biological sample. The rapid analysis system (1) comprises at least one chromatographic strip (2) comprising different consecutive zones, namely: - a release zone (4) comprising at least one detection reagent (41, 42) and - at least one capture zone (5) comprising at least one capture reagent (51, 52) immobilised on the chromatographic strip (2). The combination of detection reagents (41, 42)/capture reagents (51, 52) is selected from combinations of detection reagents (41, 42)/capture reagents (51, 52) suitable for distinguishing at least two groups of serological antibodies: - a first group of serological antibodies (A1), referred to as 'neutralising antibodies', which comprise the specific serological antibodies of the infectious agent and are capable of neutralising, or at least reducing, the infectivity of the infectious agent, and - a second group of serological antibodies (A2), referred to as 'non-neutralising antibodies', which comprise the specific serological antibodies of the infectious agent and are different from the antibodies belonging to the first group of serological antibodies (A1).

Description

Système d’analyse rapide d’un échantillon biologique, pour discriminer les anticorps sérologiques spécifiques d’un agent infectieux présents dans ledit échantillon biologique System for rapid analysis of a biological sample, to discriminate serological antibodies specific to an infectious agent present in said biological sample
Domaine technique de l'invention Technical field of the invention
La présente invention concerne le domaine technique des systèmes d’analyse rapide d’un échantillon biologique, pour discriminer les anticorps sérologiques spécifiques d’un agent infectieux présents dans ledit échantillon biologique. The present invention relates to the technical field of systems for rapid analysis of a biological sample, to discriminate serological antibodies specific to an infectious agent present in said biological sample.
Etat de la technique State of the art
Un test de diagnostic rapide, dit encore « test de dépistage rapide » ou « TDR », est un test qui permet d'établir rapidement (en quelques minutes) la présence d’au moins un analyte d’intérêt dans un échantillon biologique. A rapid diagnostic test, also called a "rapid screening test" or "RDT", is a test that quickly establishes (within minutes) the presence of at least one analyte of interest in a biological sample.
Cette approche utilise classiquement les phénomènes de réactions chimiques par immunochromatographie sur bandelettes (dit encore « latéral flow test ») qui font apparaître une coloration particulière permettant d'interpréter immédiatement le résultat. This approach conventionally uses the phenomena of chemical reactions by immunochromatography on strips (also called “lateral flow test”) which reveal a particular coloration making it possible to immediately interpret the result.
Cette technique a l’intérêt d’être simple, rapide et peu coûteuse. De tels tests sont en plus utilisables au cabinet du médecin mais aussi au chevet du patient ou sur le terrain. This technique has the advantage of being simple, fast and inexpensive. Such tests can also be used in the doctor's office but also at the patient's bedside or in the field.
Ces tests de diagnostic rapide ont monté leur intérêt dans le cadre des tests dits « sérologiques » : un tel test sérologique permet une détection, rapide et efficace, de la présence d'anticorps sérologiques spécifiques d’un agent infectieux présents dans un échantillon biologique. These rapid diagnostic tests have gained interest in the context of so-called "serological" tests: such a serological test allows rapid and efficient detection of the presence of specific serological antibodies to an infectious agent present in a biological sample.
De tels tests sérologiques sont classiquement employés pour rechercher, rétrospectivement, si un individu a subi une infection par un agent infectieux (infection suspectée, enquête de contact d'un infecté, etc.). Such serological tests are conventionally used to determine, retrospectively, whether an individual has suffered an infection by an infectious agent (suspected infection, investigation of contact with an infected person, etc.).
Outre cette information « rétroactive », il pourrait également être essentiel de pouvoir évaluer l’immunité d’un individu à l’égard de ce même agent infectieux. In addition to this "retroactive" information, it might also be essential to be able to assess an individual's immunity to the same infectious agent.
Or, les anticorps sérologiques recherchés dans les test sérologiques actuels ne fourniraient pas un résultat fiable quant à cette immunité. However, the serological antibodies sought in current serological tests would not provide a reliable result as to this immunity.
Présentation de l'invention Presentation of the invention
Afin de remédier à l’inconvénient précité de l’état de la technique, la présente invention propose un système d’analyse rapide d’un échantillon biologique, pour discriminer les anticorps sérologiques spécifiques d’un agent infectieux présents dans ledit échantillon biologique. In order to overcome the aforementioned drawback of the state of the art, the present invention provides a system for rapid analysis of a biological sample, in order to discriminate the specific serological antibodies of an infectious agent present in said biological sample.
Le système d’analyse rapide comprend au moins une bandelette chromatographique, avantageusement une bandelette immunochromatographique, qui comporte différentes zones successives, dans le sens de migration capillaire amont vers aval, à savoir au moins : The rapid analysis system comprises at least one chromatographic strip, advantageously an immunochromatographic strip, which comprises different successive zones, in the direction of upstream to downstream capillary migration, namely at least:
- une zone de dépôt, destinée à recevoir ledit échantillon biologique mélangé avec une solution tampon, - une zone de libération qui comprend au moins un réactif de détection conjugué avec un marqueur visible et/ou mesurable, ledit réactif de détection étant apte à se déplacer en conséquence de la migration de la solution tampon le long de la bandelette chromatographique, et - a deposit zone, intended to receive said biological sample mixed with a buffer solution, - a release zone which comprises at least one detection reagent conjugated with a visible and / or measurable marker, said detection reagent being able to move as a consequence of the migration of the buffer solution along the chromatographic strip, and
- au moins une zone de capture qui comprend au moins un réactif de capture, immobilisé sur la bandelette chromatographique. - at least one capture zone which comprises at least one capture reagent, immobilized on the chromatographic strip.
Et selon l’invention, la combinaison réactif(s) de détection / réactif(s) de capture est choisie parmi les combinaisons réactif(s) de détection / réactif(s) de capture adaptées à discriminer au moins deux groupes d’anticorps sérologiques : And according to the invention, the combination of detection reagent (s) / capture reagent (s) is chosen from the combinations of detection reagent (s) / capture reagent (s) suitable for discriminating at least two groups of serological antibodies. :
- un premier groupe d’anticorps sérologiques, dits « anticorps neutralisants », comprenant les anticorps sérologiques spécifiques dudit agent infectieux, aptes à annuler, ou au moins à réduire, le pouvoir infectieux dudit agent infectieux, et - a first group of serological antibodies, called "neutralizing antibodies", comprising the specific serological antibodies of said infectious agent, capable of canceling, or at least reducing, the infectious power of said infectious agent, and
- un second groupe d’anticorps sérologiques, dit « anticorps non-neutralisants », comprenant les anticorps sérologiques spécifiques dudit agent infectieux, autres que lesdits anticorps appartenant audit premier groupe d’anticorps sérologiques. - a second group of serological antibodies, called "non-neutralizing antibodies", comprising specific serological antibodies for said infectious agent, other than said antibodies belonging to said first group of serological antibodies.
Sans être limité par une quelconque théorie, cette détection des anticorps neutralisants et des anticorps non-neutralisants permet alors d’établir le statut immunitaire d’un individu et, en corolaire, le degré de protection de cet individu contre une réinfection. Without being limited by any theory, this detection of neutralizing antibodies and non-neutralizing antibodies then makes it possible to establish the immune status of an individual and, as a corollary, the degree of protection of that individual against reinfection.
D’autres caractéristiques non limitatives et avantageuses du produit conforme à l’invention, prises individuellement ou selon toutes les combinaisons techniquement possibles, sont les suivantes : Other non-limiting and advantageous characteristics of the product according to the invention, taken individually or in any technically possible combination, are as follows:
- ladite au moins une zone de capture comporte au moins deux lignes de capture : une première ligne, amont, conçue pour détecter ledit premier groupe d’anticorps sérologiques (« anticorps neutralisants »), et une seconde ligne, aval, conçue pour détecter ledit second groupe d’anticorps sérologiques (« anticorps non-neutralisants ») ; de préférence, le système d’analyse rapide comprend encore un dispositif de lecture comprenant des moyens de capture optique, adaptés à capter une image de ladite au moins une zone de capture, et des moyens de traitement comprenant un premier module conçu pour déterminer l’intensité de chaque ligne et un second module conçu pour déterminer un ratio d’intensité entre lesdits lignes ; said at least one capture zone comprises at least two capture lines: a first line, upstream, designed to detect said first group of serological antibodies (“neutralizing antibodies”), and a second line, downstream, designed to detect said second group of serological antibodies ("non-neutralizing antibodies"); preferably, the rapid analysis system further comprises a reading device comprising optical capture means, adapted to capture an image of said at least one capture zone, and processing means comprising a first module designed to determine the intensity of each line and a second module configured to determine an intensity ratio between said lines;
- l’agent infectieux est choisi parmi les virus, notamment les virus responsables de pneumopathies, avantageusement les Coronaviridae, de préférence encore Orthocoronavirinae ou coronavirus ; - the infectious agent is chosen from viruses, in particular viruses responsible for pneumopathies, advantageously the Coronaviridae, more preferably Orthocoronavirinae or coronavirus;
- ledit système d’analyse rapide comprend encore avantageusement au moins un dispositif de prélèvement, adapté au prélèvement dudit échantillon biologique chez un individu, par exemple un échantillon de sang capillaire, et une solution tampon, destinée à être mélangée avec l’échantillon biologique, pour la mise en œuvre de la technique chromatographique. Selon un mode de réalisation préféré, la combinaison réactif(s) de détection / réactif(s) de capture est choisie parmi les combinaisons réactif(s) de détection / réactif(s) de capture adaptées à détecter les anticorps sérologiques dirigés contre les antigènes de surface dudit agent infectieux, dont : - Said rapid analysis system also advantageously comprises at least one sampling device, suitable for taking said biological sample from an individual, for example a capillary blood sample, and a buffer solution, intended to be mixed with the biological sample, for the implementation of the chromatographic technique. According to a preferred embodiment, the combination of detection reagent (s) / capture reagent (s) is chosen from combinations of detection reagent (s) / capture reagent (s) suitable for detecting the serological antibodies directed against the antigens. surface area of said infectious agent, including:
- le premier groupe d’anticorps sérologiques comprend les anticorps sérologiques dirigés contre les antigènes de surface responsables du pouvoir infectieux dudit agent infectieux, de préférence les protéines responsables de l’attachement et/ou de la pénétration et/ou de la décapsidation, et - the first group of serological antibodies comprises serological antibodies directed against the surface antigens responsible for the infectious power of said infectious agent, preferably the proteins responsible for attachment and / or penetration and / or decapsidation, and
- le second groupe d’anticorps sérologiques comprend les anticorps sérologiques dirigés contre les antigènes de surface dudit agent infectieux autres que ceux responsables dudit pouvoir infectieux. - the second group of serological antibodies comprises serological antibodies directed against the surface antigens of said infectious agent other than those responsible for said infectious power.
D’autres caractéristiques non limitatives et avantageuses de ce mode de réalisation préféré, prises individuellement ou selon toutes les combinaisons techniquement possibles, sont les suivantes : Other non-limiting and advantageous characteristics of this preferred embodiment, taken individually or in any technically possible combination, are as follows:
- ledit au moins un réactif de détection et/ou ledit au moins un réactif de capture comprennent un premier antigène de surface dudit agent infectieux, ou fragment dudit premier antigène, choisi parmi les antigènes de surface responsables du pouvoir infectieux, et un second antigène de surface dudit agent infectieux, ou fragment dudit second antigène, choisi parmi les antigènes de surface autres que lesdits antigènes de surface responsables du pouvoir infectieux ; - said at least one detection reagent and / or said at least one capture reagent comprise a first surface antigen of said infectious agent, or fragment of said first antigen, chosen from the surface antigens responsible for infectivity, and a second antigen of surface of said infectious agent, or fragment of said second antigen, chosen from surface antigens other than said surface antigens responsible for infectivity;
- la zone de libération comprend une combinaison de deux réactifs de détection : un premier antigène de surface de l’agent infectieux, ou fragment dudit premier antigène (antigènes de surface responsables du pouvoir infectieux), et un second antigène de surface de l’agent infectieux, ou fragment dudit second antigène (antigènes de surface autres que ceux responsables du pouvoir infectieux) ; la zone de capture comprend deux lignes de réactifs de capture : une première ligne, amont, dont le réactif de capture comprend un premier antigène de surface de l’agent infectieux ou fragment dudit premier antigène (antigènes de surface responsables du pouvoir infectieux), et une deuxième ligne, aval, dont le réactif de capture est choisi parmi les anticorps anti-anticorps humain ; - the release zone comprises a combination of two detection reagents: a first surface antigen of the infectious agent, or fragment of said first antigen (surface antigens responsible for the infectious power), and a second surface antigen of the agent infectious, or fragment of said second antigen (surface antigens other than those responsible for infectivity); the capture zone comprises two lines of capture reagents: a first line, upstream, of which the capture reagent comprises a first surface antigen of the infectious agent or fragment of said first antigen (surface antigens responsible for infectious power), and a second, downstream line, the capture reagent of which is chosen from anti-human antibody antibodies;
- pour un agent infectieux SRAS-Cov-V2 (Covid 19) dont la surface comporte des protéines de nucléocapside NP et des protéines Spike S, ladite protéine S comportant une sous-unité S1, une sous-unité S2 et un domaine RBD (Receptor Binding Domain), ledit au moins un réactif de détection et/ou ledit au moins un réactif de capture comprennent un premier antigène de surface de l’agent infectieux choisi parmi les protéines S et les fragments de protéine S, de préférence parmi la sous-unité S1, la sous-unité S2 et le domaine RBD (Receptor Binding Domain), et un second antigène de surface de l’agent infectieux choisi parmi les protéines de nucléocapside N P et les fragments de protéine de nucléocapside N P. La présente invention concerne encore le procédé d’analyse rapide d’un échantillon biologique par la mise en œuvre d’un système d’analyse rapide selon l’invention, pour discriminer les anticorps sérologiques neutralisants et les anticorps sérologiques non- neutralisants qui sont spécifiques d’un agent infectieux. - for an infectious agent SARS-Cov-V2 (Covid 19) whose surface comprises NP core proteins and Spike S proteins, said S protein comprising an S1 subunit, an S2 subunit and an RBD domain (Receptor Binding Domain), said at least one detection reagent and / or said at least one capture reagent comprise a first surface antigen of the infectious agent chosen from S proteins and S protein fragments, preferably from the sub- S1 unit, the S2 subunit and the RBD domain (Receptor Binding Domain), and a second surface antigen of the infectious agent chosen from NP core proteins and N P core protein fragments. The present invention also relates to the method of rapid analysis of a biological sample by the implementation of a rapid analysis system according to the invention, to discriminate the neutralizing serological antibodies and the non-neutralizing serological antibodies which are specific. an infectious agent.
Le procédé comprend : The process includes:
- une étape de dépôt d’un mélange solution tampon / échantillon biologique sur la zone de dépôt de ladite au moins une bandelette chromatographique, pour la mise en œuvre de la technique chromatographique, et - a step of depositing a buffer solution / biological sample mixture on the deposit zone of said at least one chromatographic strip, for the implementation of the chromatographic technique, and
- une étape de lecture de ladite au moins une bandelette chromatographique après migration de la solution tampon, au sein de la zone de capture, pour discriminer lesdites anticorps sérologiques neutralisants et les anticorps sérologiques non-neutralisants spécifiques d’un agent infectieux. - a step of reading said at least one chromatographic strip after migration of the buffer solution, within the capture zone, to discriminate said neutralizing serological antibodies and non-neutralizing serological antibodies specific for an infectious agent.
D’autres caractéristiques non limitatives et avantageuses du procédé conforme à l’invention, prises individuellement ou selon toutes les combinaisons techniquement possibles, sont les suivantes : Other non-limiting and advantageous characteristics of the process according to the invention, taken individually or in any technically possible combination, are as follows:
- l’échantillon biologique est un échantillon de sang capillaire ou un échantillon de sérum / plasma ; - the biological sample is a capillary blood sample or a serum / plasma sample;
- dans le cas d’au moins une zone de capture comportant au moins deux lignes de capture, l’étape de lecture comprend une phase de détermination de l’intensité de chaque ligne, puis une phase de détermination du ratio d’intensité entre lesdites lignes. - in the case of at least one capture zone comprising at least two capture lines, the reading step comprises a phase of determining the intensity of each line, then a phase of determining the intensity ratio between said lines.
Bien entendu, les différentes caractéristiques, variantes et formes de réalisation de l'invention peuvent être associées les unes avec les autres selon diverses combinaisons dans la mesure où elles ne sont pas incompatibles ou exclusives les unes des autres. Of course, the different characteristics, variants and embodiments of the invention can be associated with each other in various combinations insofar as they are not incompatible or mutually exclusive.
Description détaillée de l'invention Detailed description of the invention
De plus, diverses autres caractéristiques de l'invention ressortent de la description annexée effectuée en référence aux dessins qui illustrent des formes, non limitatives, de réalisation de l'invention et où : In addition, various other characteristics of the invention emerge from the appended description given with reference to the drawings which illustrate non-limiting embodiments of the invention and in which:
[Fig. 1] est une vue schématique, et fonctionnelle, d’une bandelette immunochromatographique adaptée au système d’analyse rapide selon l’invention ; [Fig. 1] is a schematic, and functional, view of an immunochromatographic strip suitable for the rapid analysis system according to the invention;
[Fig. 2] est une vue schématique d’un système d’analyse rapide dans lequel la bandelette immunochromatographique peut être intégrée. [Fig. 2] is a schematic view of a rapid analysis system into which the immunochromatographic strip can be integrated.
Il est à noter que, sur ces figures, les éléments structurels et/ou fonctionnels communs aux différentes variantes peuvent présenter les mêmes références. It should be noted that, in these figures, the structural and / or functional elements common to the different variants may have the same references.
La présente invention concerne, de manière générale, les systèmes d’analyse rapide des échantillons biologiques, dits encore « test de diagnostic rapide » ou « test de dépistage rapide » ou « TDR ». Le système d’analyse rapide selon l’invention est en particulier dédié à discriminer les anticorps sérologiques spécifiques d’un agent infectieux, présents dans un échantillon biologique. The present invention relates, in general, to systems for rapid analysis of biological samples, also called “rapid diagnostic test” or “rapid screening test” or “RDT”. The rapid analysis system according to the invention is in particular dedicated to discriminating serological antibodies specific for an infectious agent, present in a biological sample.
Par « échantillon biologique », on entend tout échantillon prélevé sur un individu (humain ou animal notamment) et dans lequel les anticorps sérologiques sont recherchés. By “biological sample” is meant any sample taken from an individual (human or animal in particular) and in which serological antibodies are sought.
L’échantillon biologique consiste de préférence en un échantillon liquide dans lequel les anticorps sérologiques sont en solution ou en suspension. The biological sample preferably consists of a liquid sample in which the serological antibodies are in solution or in suspension.
Cet échantillon liquide peut notamment être tout fluide biologique ou corporel. This liquid sample can in particular be any biological or bodily fluid.
L'échantillon liquide peut également avoir été obtenu directement ou indirectement à partir d'un fluide biologique ou corporel. The liquid sample may also have been obtained directly or indirectly from a biological or bodily fluid.
L'échantillon peut également être un extrait liquide d'un échantillon solide. The sample can also be a liquid extract from a solid sample.
De préférence, l'échantillon biologique est du sang total ou du sérum. Preferably, the biological sample is whole blood or serum.
Par « sang total », on entend en particulier du sang capillaire obtenu avantageusement au moyen d’un auto-piqueur au niveau de la pulpe du doigt, ou du talon, d’un individu humain. By "whole blood" is meant in particular capillary blood advantageously obtained by means of an auto-pricker at the level of the pulp of the finger, or of the heel, of a human individual.
Par « anticorps sérologiques spécifiques d’un agent infectieux » (dits encore « anticorps sérologiques dirigés contre un agent infectieux »), on entend avantageusement les anticorps développés par un individu et témoignant d’une réponse immunitaire à l’infection par un agent infectieux. By "serological antibodies specific to an infectious agent" (also called "serological antibodies directed against an infectious agent"), is advantageously meant the antibodies developed by an individual and indicating an immune response to infection by an infectious agent.
Les anticorps en question consistent avantageusement en des anticorps polyclonaux qui consistent en un mélange d'anticorps reconnaissant différents épitopes sur un antigène donné (chaque idiotype étant sécrété par un clone de plasmocytes différent). The antibodies in question advantageously consist of polyclonal antibodies which consist of a mixture of antibodies recognizing different epitopes on a given antigen (each idiotype being secreted by a different plasma cell clone).
Les anticorps recherchés sont avantageusement choisis parmi les anticorps sériques IgM (immunoglobulines M) et/ou IgG (immunoglobulines G) et/ou IgA (immunoglobulines A). The antibodies sought are advantageously chosen from serum antibodies IgM (immunoglobulins M) and / or IgG (immunoglobulins G) and / or IgA (immunoglobulins A).
Par « agent infectieux », on entend de préférence les virus, notamment les virus responsables de pneumopathies, avantageusement les Coronaviridae, de préférence encore Orthocoronavirinae ou coronavirus. The term “infectious agent” preferably means viruses, in particular viruses responsible for pneumopathies, advantageously the Coronaviridae, more preferably Orthocoronavirinae or coronavirus.
Par « coronavirus », on englobe le SARS-CoV, le MERS-CoV ou le SARS-CoV-2. By "coronavirus" is meant SARS-CoV, MERS-CoV or SARS-CoV-2.
Par « discriminer », on entend avantageusement la détermination qualitative (avantageusement la présence ou l'absence), voire quantitative, de groupes d’anticorps sérologiques spécifiques d’un agent infectieux qui sont présents dans ledit échantillon biologique et qui possèdent des propriétés immunologiques différentes pour un même agent infectieux (découlant avantageusement de spécificités antigéniques différentes pour un même agent infectieux). The term “discriminate” advantageously means the qualitative (advantageously the presence or absence), or even quantitative, determination of groups of serological antibodies specific to an infectious agent which are present in said biological sample and which have different immunological properties. for the same infectious agent (advantageously resulting from different antigenic specificities for the same infectious agent).
La présente invention est ainsi conçue pour discriminer les anticorps sérologiques parmi au moins deux groupes d’anticorps sérologiques suivants : - un premier groupe d’anticorps sérologiques A1, dits « anticorps neutralisants », comprenant les anticorps sérologiques spécifiques dudit agent infectieux qui sont aptes à annuler, ou au moins à réduire, le pouvoir infectieux dudit agent infectieux, et The present invention is thus designed to discriminate serological antibodies from at least two groups of the following serological antibodies: a first group of serological antibodies A1, called “neutralizing antibodies”, comprising the specific serological antibodies of said infectious agent which are capable of canceling, or at least reducing, the infectious power of said infectious agent, and
- un second groupe d’anticorps sérologiques A2, dit « anticorps non-neutralisants » ou « anticorps immunisants », comprenant les anticorps sérologiques spécifiques dudit agent infectieux (dirigés contre l’agent infectieux), autres que lesdits anticorps appartenant audit premier groupe d’anticorps sérologiques A1 (les « anticorps non-neutralisants », tout en étant dirigés contre l’agent infectieux, ne sont pas aptes à annuler, ou au moins à réduire, le pouvoir infectieux dudit agent infectieux). a second group of serological antibodies A2, called “non-neutralizing antibodies” or “immunizing antibodies”, comprising the specific serological antibodies of said infectious agent (directed against the infectious agent), other than said antibodies belonging to said first group of A1 serological antibodies (“non-neutralizing antibodies”, while being directed against the infectious agent, are not able to cancel, or at least reduce, the infectious power of said infectious agent).
Selon un mode de réalisation préféré : According to a preferred embodiment:
- le premier groupe d’anticorps sérologiques A1 comprend les anticorps sérologiques dirigés contre les antigènes de surface responsables du pouvoir infectieux dudit agent infectieux, de préférence les protéines ou fragments de protéines responsables de l’attachement et/ou de la pénétration et/ou de la décapsidation, et - the first group of serological antibodies A1 comprises the serological antibodies directed against the surface antigens responsible for the infectious power of the said infectious agent, preferably the proteins or fragments of proteins responsible for the attachment and / or the penetration and / or the decapsidation, and
- le second groupe d’anticorps sérologiques A2 comprend les anticorps sérologiques dirigés contre les antigènes de surface dudit agent infectieux autres que ceux responsables dudit pouvoir infectieux. - the second group of serological antibodies A2 comprises serological antibodies directed against the surface antigens of said infectious agent other than those responsible for said infectious power.
Selon un mode de réalisation particulier, l’agent infectieux est le virus SRAS-Cov-V2 (Covid 19), présentant avantageusement la séquence génomique GenBank accession MN908947. According to a particular embodiment, the infectious agent is the SARS-Cov-V2 virus (Covid 19), advantageously exhibiting the GenBank accession MN908947 genomic sequence.
Un tel agent infectieux possède une surface qui comporte des protéines de nucléocapside NP et des protéines Spike S ; la protéine S comporte avantageusement une sous-unité S1, une sous-unité S2 et un domaine RBD (Receptor Binding Domain) (Chen Z et al. Clin Chem 2004; 50:988-95 ; Zhou P et al., Nature 2020 ; Lu R et a., Lancet, 2020; 395:565-74). Such an infectious agent has a surface which comprises NP core proteins and Spike S proteins; the S protein advantageously comprises an S1 subunit, an S2 subunit and an RBD domain (Receptor Binding Domain) (Chen Z et al. Clin Chem 2004; 50: 988-95; Zhou P et al., Nature 2020; Lu R et a., Lancet, 2020; 395: 565-74).
Dans ce mode de réalisation particulier : In this particular embodiment:
- le premier groupe d’anticorps sérologiques A1 comprend les anticorps sérologiques dirigés avantageusement contre les antigènes de surface choisis parmi les protéines S et les fragments de protéine S, de préférence parmi la sous-unité S1, la sous-unité S2 et le domaine RBD (Receptor Binding Domain), et - the first group of serological antibodies A1 comprises the serological antibodies directed advantageously against the surface antigens chosen from the S proteins and the fragments of S protein, preferably from the S1 subunit, the S2 subunit and the RBD domain (Receptor Binding Domain), and
- le second groupe d’anticorps sérologiques A2 comprend les anticorps sérologiques dirigés contre les antigènes de surface choisis parmi les protéines de nucléocapside NP et les fragments de protéine de nucléocapside N P, et éventuellement contre les fragments de protéine S autre que le domaine RBD. - the second group of serological antibodies A2 comprises the serological antibodies directed against the surface antigens chosen from the NP core proteins and the fragments of the N P core protein, and optionally against the fragments of S protein other than the RBD domain.
De préférence encore : More preferably:
- le premier groupe d’anticorps sérologiques A1 comprend les anticorps sérologiques dirigés avantageusement contre les antigènes de surface choisis parmi le domaine RBD (Receptor Binding Domain), et - le second groupe d’anticorps sérologiques A2 comprend les anticorps sérologiques dirigés contre les antigènes de surface choisis parmi les protéines de nucléocapside NP (et les fragments de protéine de nucléocapside N P) et contre les régions de la protéine S autres que le domaine RBD. - the first group of serological antibodies A1 comprises the serological antibodies directed advantageously against the surface antigens chosen from the RBD domain (Receptor Binding Domain), and the second group of serological antibodies A2 comprises the serological antibodies directed against the surface antigens chosen from the NP core proteins (and the NP core protein fragments) and against the regions of the S protein other than the RBD domain.
Bandelette chromatographique Chromatographic strip
Pour cela et de manière générale, le système d’analyse rapide 1 comprend au moins une bandelette chromatographique 2, avantageusement une bandelette immunochromatographique, conçue pour discriminer, dans un échantillon, les anticorps sérologiques « neutralisants », par rapport aux anticorps « non-neutralisants », spécifiques d’un agent infectieux. For this and in general, the rapid analysis system 1 comprises at least one chromatographic strip 2, advantageously an immunochromatographic strip, designed to discriminate, in a sample, the “neutralizing” serological antibodies, compared to the “non-neutralizing” antibodies. , Specific for an infectious agent.
La bandelette chromatographique 2, dite encore « moyens de diffusion capillaire », est avantageusement choisies parmi les bandelettes chromatographiques portant les réactifs adaptés à la discrimination, dans un échantillon, des anticorps sérologiques « neutralisants » / « non-neutralisants » spécifiques d’un agent infectieux. Chromatographic strip 2, also called “capillary diffusion means”, is advantageously chosen from chromatographic strips carrying reagents suitable for the discrimination, in a sample, of “neutralizing” / “non-neutralizing” serological antibodies specific for an agent. infectious.
Cette bandelette chromatographique 2 est formée de tout moyen constituant ou agissant en tant qu'unité de diffusion capillaire continue, par migration latérale (c'est-à-dire perpendiculairement à l'épaisseur du ou des matériaux capillaires mis en œuvre pour la diffusion capillaire). This chromatographic strip 2 is formed from any means constituting or acting as a continuous capillary diffusion unit, by lateral migration (that is to say perpendicular to the thickness of the capillary material (s) used for the capillary diffusion. ).
Ce moyen de diffusion capillaire consiste avantageusement en un support solide poreux permettant la migration d'un liquide par simple diffusion capillaire. This capillary diffusion means advantageously consists of a porous solid support allowing the migration of a liquid by simple capillary diffusion.
La porosité de ce support permet la diffusion capillaire (ou migration latérale) de l'échantillon (en particulier d’anticorps sérologiques) et/ou des réactifs à l'état liquide ou humide. The porosity of this support allows capillary diffusion (or lateral migration) of the sample (in particular of serological antibodies) and / or of the reagents in the liquid or wet state.
De tels moyens de diffusion capillaire sont très largement utilisés, notamment dans toutes les techniques d'immunochromatographie à migration latérale. Such capillary diffusion means are very widely used, in particular in all lateral migration immunochromatography techniques.
Une telle bandelette chromatographique 2 consiste ici en un support allongé selon la direction et/ou le sens de la diffusion capillaire (migration latérale). Such a chromatographic strip 2 here consists of a support elongated in the direction and / or the direction of capillary diffusion (lateral migration).
La bandelette chromatographique 2 peut être constituée par : Chromatographic strip 2 can be made up of:
- un seul et même matériau capillaire ou poreux, ou - one and the same capillary or porous material, or
- plusieurs éléments ou matériaux capillaires ou poreux différents, convenablement agencés les uns par rapport aux autres (par exemple par chevauchement), pour obtenir une continuité d'écoulement capillaire d'un élément ou d'un matériau à un autre, selon la direction de diffusion capillaire. - several different capillary or porous elements or materials, suitably arranged with respect to each other (for example by overlapping), to obtain a continuity of capillary flow from one element or from one material to another, according to the direction of capillary diffusion.
Une telle bandelette chromatographique 2 détermine une direction et sens de diffusion capillaire de tout liquide qui est reçu ou déposé à une extrémité amont, et qui se déplace alors vers une extrémité aval de la bandelette chromatographique 2. Such a chromatographic strip 2 determines a direction and direction of capillary diffusion of any liquid which is received or deposited at an upstream end, and which then moves towards a downstream end of the chromatographic strip 2.
A titre d'exemple, la bandelette chromatographique 2 peut être constituée de divers supports immunochromatographiques, par exemple de cellulose, de nylon, de nitrocellulose, de polyéthylène ou de fibre de verre. Pour discriminer les anticorps sérologiques « neutralisants » / « non-neutralisants » dirigés contre un agent infectieux, le système d’analyse rapide 1 comprend au moins une bandelette chromatographique 2, avantageusement une bandelette immunochromatographique, qui comporte différentes zones successives, dans le sens de migration capillaire amont vers aval, à savoir au moins : By way of example, the chromatographic strip 2 can consist of various immunochromatographic supports, for example cellulose, nylon, nitrocellulose, polyethylene or glass fiber. To discriminate the “neutralizing” / “non-neutralizing” serological antibodies directed against an infectious agent, the rapid analysis system 1 comprises at least one chromatographic strip 2, advantageously an immunochromatographic strip, which comprises different successive zones, in the direction of upstream to downstream capillary migration, namely at least:
- une zone de dépôt 3, destinée à recevoir ledit échantillon biologique mélangé avec une solution tampon, - a deposit zone 3, intended to receive said biological sample mixed with a buffer solution,
- une zone de libération 4 qui comprend / comporte au moins un réactif de détection 41, 42 conjugué avec un marqueur 43 visible et/ou mesurable, ledit réactif de détection 41, 42 étant apte à se déplacer en conséquence de la migration de la solution tampon le long de la bandelette chromatographique 2, et - a release zone 4 which comprises / comprises at least one detection reagent 41, 42 conjugated with a visible and / or measurable marker 43, said detection reagent 41, 42 being able to move as a consequence of the migration of the solution buffer along chromatography strip 2, and
- au moins une zone de capture 5 qui comprend / comporte au moins un réactif de capture 51, 52, immobilisé sur la bandelette chromatographique 2. - at least one capture zone 5 which comprises / comprises at least one capture reagent 51, 52, immobilized on the chromatographic strip 2.
Les zones de libération et de capture 4, 5 consistent avantageusement en une ligne ou bande transversale (s’étendant perpendiculairement à la direction de migration), présentant par exemple une largeur comprise entre 1 et 2 mm, et une surface comprise entre 3 et 5 mm2. The release and capture zones 4, 5 advantageously consist of a transverse line or strip (extending perpendicular to the direction of migration), for example having a width of between 1 and 2 mm, and an area of between 3 and 5 mm 2 .
De manière générale, le « réactif de détection 41, 42 » ou le « réactif de capture 51, 52 » consiste en toute entité chimique, biochimique ou biologique, qui est apte à se lier spécifiquement pour former un complexe permettant la détection d’un anticorps sérologique spécifique de (dirigé contre) un agent infectieux d’intérêt (avantageusement un antigène ou un épitope de cet agent infectieux). In general, the “detection reagent 41, 42” or the “capture reagent 51, 52” consists of any chemical, biochemical or biological entity, which is capable of binding specifically to form a complex allowing the detection of a serological antibody specific for (directed against) an infectious agent of interest (advantageously an antigen or an epitope of this infectious agent).
Le réactif de détection 41, 42 et/ou le réactif de capture 51, 52 constituent encore des réactifs dits de « liaison ». The detection reagent 41, 42 and / or the capture reagent 51, 52 also constitute so-called “binding” reagents.
De tels réactifs de liaison 41, 42, 51, 52, permettant la détection d’au moins un anticorps sérologique dans l'échantillon liquide, sont bien connus et peuvent être choisis à façon pour la mise en œuvre de l’invention. Such binding reagents 41, 42, 51, 52, allowing the detection of at least one serological antibody in the liquid sample, are well known and can be tailor-made for the implementation of the invention.
Ces réactifs de liaison 41, 42, 51, 52 sont choisis avantageusement parmi ceux qui sont aptes à se lier spécifiquement avec un anticorps sérologique d’intérêt et/ou à se lier spécifiquement l’un avec l’autre. These binding reagents 41, 42, 51, 52 are advantageously chosen from those which are capable of binding specifically with a serological antibody of interest and / or of binding specifically with one another.
Selon le format de test mis en œuvre, les réactifs de liaison 41, 42, 51, 52 complémentaires sont avantageusement destinés à former des complexes différents : Depending on the test format used, the complementary binding reagents 41, 42, 51, 52 are advantageously intended to form different complexes:
- les réactifs de liaison 41, 42, 51, 52 sont aptes à se fixer concomitamment à un anticorps sérologique d’intérêt, pour former un test au format sandwich, - the binding reagents 41, 42, 51, 52 are able to bind concomitantly to a serological antibody of interest, to form a test in sandwich format,
- l’un des réactifs de liaison 41, 42, 51, 52 (détection ou capture) est apte à se fixer à un anticorps sérologique d’intérêt mais aussi à l’autre réactif de liaison (respectivement capture ou détection), pour former un test au format compétition. Par « lier » ou « liaison », on entend toute liaison forte, par exemple covalente, mais aussi toute liaison faible, par exemple du type antigène / anticorps. - one of the binding reagents 41, 42, 51, 52 (detection or capture) is able to bind to a serological antibody of interest but also to the other binding reagent (respectively capture or detection), to form a test in competition format. By “bind” or “bond” is meant any strong bond, for example covalent, but also any weak bond, for example of the antigen / antibody type.
Les réactifs de liaison 41, 42, 51, 52 sont avantageusement choisis parmi les anticorps et les antigènes. The binding reagents 41, 42, 51, 52 are advantageously chosen from antibodies and antigens.
Par « antigène », on entend en particulier les protéines ou les fragments de protéine, avantageusement des protéines recombinantes obtenues par expression en système procary ote. The term “antigen” is understood to mean in particular proteins or protein fragments, advantageously recombinant proteins obtained by expression in a prokaryotic system.
Les réactifs de liaison 41, 42, 51, 52 sont avantageusement choisis pour présenter, à l’égard de chaque groupe d’anticorps sérologiques recherchés, les performances suivantes : The binding reagents 41, 42, 51, 52 are advantageously chosen to exhibit, with regard to each group of serological antibodies sought, the following performances:
- une spécificité d’au moins 90%, de préférence encore d’au moins 95%, de préférence encore d’au moins 98%, voire d’au moins 99%, voire de 100%, et - a specificity of at least 90%, more preferably at least 95%, more preferably at least 98%, or even at least 99%, or even 100%, and
- une sensibilité d’au moins 80%, de préférence encore d’au moins 90%, voire encore d’au moins 95%. - a sensitivity of at least 80%, more preferably at least 90%, or even at least 95%.
Le ou les réactifs de détection 41, 42 sont avantageusement conjugués à un marqueur 43 visible et/ou mesurable, avantageusement un marqueur particulaire. The detection reagent (s) 41, 42 are advantageously combined with a visible and / or measurable label 43, advantageously a particulate label.
Par « marqueur visible et/ou mesurable », on entend tout marquage permettant une détection directe ou indirecte à l'œil nu, ou à l'aide d'un appareil, en raison de l'émission d'un signal au niveau de ladite au moins une zone de capture 5. By “visible and / or measurable marker” is meant any marking allowing direct or indirect detection with the naked eye, or with the aid of a device, due to the emission of a signal at said level. at least one capture area 5.
Le signal est par exemple une fluorescence, une coloration, une présence d'isotope ou un signal magnétique. The signal is, for example, fluorescence, coloring, the presence of isotope or a magnetic signal.
On citera par exemple les marqueurs particulaires colorés comme l'or colloïdal, ou fluorescents, les particules de latex colorées, les particules de latex fluorescentes et les particules conjuguées à l'avidine et à la streptavidine. Mention will be made, for example, of colored particulate markers such as colloidal gold, or fluorescent, colored latex particles, fluorescent latex particles and particles conjugated with avidin and with streptavidin.
Les marqueurs particulaires, colorés ou fluorescents, consistent ainsi en des particules de petite taille insolubles dans l'eau et qui forment donc des suspensions, dispersions ou solutions, en phase liquide. Particulate markers, colored or fluorescent, thus consist of small particles which are insoluble in water and which therefore form suspensions, dispersions or solutions, in the liquid phase.
Parmi les marqueurs permettant une observation directe à l'œil nu, on citera aussi les marqueurs de type dextran (Hansen T.M., IVD Technology 4, 35-40, 2003). Le réactif de liaison est alors conjugué à une chaîne de dextran (dérivé de polysaccharide) portant des fluorophores. Among the markers allowing direct observation with the naked eye, mention will also be made of markers of the dextran type (Hansen T.M., IVD Technology 4, 35-40, 2003). The binding reagent is then conjugated to a dextran (polysaccharide derivative) chain carrying fluorophores.
Les marqueurs peuvent également consister en des enzymes (la phosphatase alcaline ou AP, la peroxydase de raifort ou HRP, notamment), en des colorants (ou « dyes ») ou en des composés chimiluminescents (notamment l’isothiocyanate de fluorescéine ou FITC). The markers can also consist of enzymes (alkaline phosphatase or AP, horseradish peroxidase or HRP, in particular), of dyes (or "dyes") or of chemiluminescent compounds (in particular fluorescein isothiocyanate or FITC).
Pour augmenter la sensibilité, on peut avoir recours par exemple, à un anticorps marqué selon des techniques connues de l'homme de l'art pour une détection indirecte, comme par exemple un anticorps biotinylé, permettant indirectement une détection par la formation des entités avidine-biotine et streptavidine-biotine. Cet anticorps marqué et biotinylé peut également, soit être directement déjà déposé sur une ligne-test, dans la zone de capture 5, pour augmenter la sensibilité, soit être déposé avec l’anticorps de détection spécifique, pour augmenter le temps de contact et encore la sensibilité notamment, par exemple, en raison du nombre de sites de fixation. To increase the sensitivity, recourse may be had, for example, to an antibody labeled according to techniques known to those skilled in the art for indirect detection, such as for example a biotinylated antibody, indirectly allowing detection by the formation of avidin entities. -biotin and streptavidin-biotin. This labeled and biotinylated antibody can also either be directly deposited on a test line, in the capture zone 5, to increase the sensitivity, or be deposited with the specific detection antibody, to increase the contact time and again. sensitivity in particular, for example, due to the number of binding sites.
De son côté, dans la zone de capture 5, ledit au moins un réactif de capture 51, 52 spécifique de l'analyte est immobilisé sur le support solide selon des techniques connues de l'homme du métier. For its part, in the capture zone 5, said at least one capture reagent 51, 52 specific for the analyte is immobilized on the solid support according to techniques known to those skilled in the art.
Ce réactif de capture 51, 52 est immobilisé de telle façon qu'il ne soit pas mobile à l'état humide. This capture reagent 51, 52 is immobilized in such a way that it is not mobile in the wet state.
Cette immobilisation peut s'effectuer par exemple par absorption ou par un couplage covalent. This immobilization can take place, for example, by absorption or by covalent coupling.
Selon l’invention, la combinaison réactif(s) de détection 41, 42 / réactif(s) de capture 51,According to the invention, the combination of detection reagent (s) 41, 42 / capture reagent (s) 51,
52 est choisie parmi les combinaisons réactif(s) de détection 41, 42 / réactif(s) de capture 51,52 is chosen from the combinations of detection reagent (s) 41, 42 / capture reagent (s) 51,
52 adaptées à discriminer au moins les deux groupes d’anticorps sérologiques précités : 52 adapted to discriminate at least the two groups of serological antibodies mentioned above:
- le premier groupe d’anticorps sérologiques A1, dits « anticorps neutralisants », comprenant les anticorps sérologiques spécifiques dudit agent infectieux, aptes à annuler, ou au moins à réduire, le pouvoir infectieux dudit agent infectieux, et - the first group of serological antibodies A1, called "neutralizing antibodies", comprising the specific serological antibodies of said infectious agent, capable of canceling, or at least reducing, the infectious power of said infectious agent, and
- un second groupe d’anticorps sérologiques A2, dit « anticorps non-neutralisants », comprenant les anticorps sérologiques spécifiques dudit agent infectieux, autres que lesdits anticorps appartenant audit premier groupe d’anticorps sérologiques A1. - a second group of serological antibodies A2, called "non-neutralizing antibodies", comprising serological antibodies specific for said infectious agent, other than said antibodies belonging to said first group of serological antibodies A1.
En d’autres termes et sans être limités par une quelconque théorie, les anticorps non- neutralisants sont dirigés contre l’agent infectieux, mais ne sont pas aptes à annuler (ou à inhiber), ou au moins à réduire, le pouvoir infectieux dudit agent infectieux. In other words and without being limited by any theory, the non-neutralizing antibodies are directed against the infectious agent, but are not able to cancel (or to inhibit), or at least to reduce, the infectivity of said. infectious agent.
Sans être limité par une quelconque théorie, ces anticorps non-neutralisants sont dirigés contre des protéines, ou fragments de protéine, qui ne sont pas responsables du pouvoir infectieux dudit agent infectieux. Without being limited by any theory, these non-neutralizing antibodies are directed against proteins, or protein fragments, which are not responsible for the infectivity of said infectious agent.
Pour cela, ladite au moins une zone de capture 5 comporte avantageusement au moins deux lignes de capture : For this, said at least one capture zone 5 advantageously comprises at least two capture lines:
- une première ligne 5a, amont, conçue pour détecter ledit premier groupe d’anticorps sérologiques A1 (« anticorps neutralisants »), et - a first line 5a, upstream, designed to detect said first group of serological antibodies A1 ("neutralizing antibodies"), and
- une seconde ligne 5b, aval, conçue pour détecter ledit second groupe d’anticorps sérologiques A2 (« anticorps non-neutralisants »). - a second line 5b, downstream, designed to detect said second group of serological antibodies A2 ("non-neutralizing antibodies").
Selon un mode de réalisation préféré, la combinaison réactif(s) de détection 41, 42 / réactif(s) de capture 51, 52 est choisie parmi les combinaisons réactif(s) de détection 41, 42 / réactif(s) de capture 51, 52 adaptées à discriminer les anticorps sérologiques dirigés contre les antigènes de surface (dits encore « épitopes ») dudit agent infectieux, dont : - le premier groupe d’anticorps sérologiques A1 comprend les anticorps sérologiques dirigés contre les antigènes de surface responsables du pouvoir infectieux dudit agent infectieux, de préférence les protéines (y compris les glycoprotéines) responsables de l’attachement et/ou de la pénétration et/ou de la décapsidation, et According to a preferred embodiment, the combination of detection reagent (s) 41, 42 / capture reagent (s) 51, 52 is chosen from the combinations of detection reagent (s) 41, 42 / capture reagent (s) 51 , 52 adapted to discriminate the serological antibodies directed against the surface antigens (also called “epitopes”) of said infectious agent, including: - the first group of serological antibodies A1 comprises the serological antibodies directed against the surface antigens responsible for the infectious power of said infectious agent, preferably the proteins (including glycoproteins) responsible for the attachment and / or the penetration and / or decapsidation, and
- le second groupe d’anticorps sérologiques A2 comprend les anticorps sérologiques dirigés contre les antigènes de surface dudit agent infectieux autres que ceux responsables dudit pouvoir infectieux. - the second group of serological antibodies A2 comprises serological antibodies directed against the surface antigens of said infectious agent other than those responsible for said infectious power.
Par « antigène de surface », on entend avantageusement les protéines (y compris les glycoprotéines ») en surface de l’agent infectieux et comportant au moins un épitope. The term "surface antigen" advantageously means the proteins (including glycoproteins ") on the surface of the infectious agent and comprising at least one epitope.
Par « protéines responsables de l’attachement », on entend en particulier les protéines qui s’attachent à des récepteurs situés sur la membrane cytoplasmique de la cellule hôte. By "proteins responsible for attachment" is meant in particular those proteins which attach to receptors located on the cytoplasmic membrane of the host cell.
Par « protéines responsables de la pénétration », on entend en particulier les protéines qui permettent l’entrée du virus à l'intérieur de la cellule hôte. By "proteins responsible for penetration" is meant in particular the proteins which allow the entry of the virus into the interior of the host cell.
Par « protéines responsables de la décapsidation », on entend en particulier les protéines qui permettent la libération du génome par rapport à la capside. The term “proteins responsible for decapsidation” means in particular the proteins which allow the release of the genome relative to the capsid.
De préférence encore, ledit au moins un réactif de détection 41, 42 et/ou ledit au moins un réactif de capture 51, 52 comprennent : More preferably, said at least one detection reagent 41, 42 and / or said at least one capture reagent 51, 52 comprise:
- un premier antigène de surface 41 dudit agent infectieux, ou fragment dudit premier antigène, choisi parmi les antigènes de surface responsables du pouvoir infectieux, et - a first surface antigen 41 of said infectious agent, or fragment of said first antigen, chosen from the surface antigens responsible for infectivity, and
- un second antigène de surface 42 dudit agent infectieux, ou fragment dudit second antigène, choisi parmi les antigènes de surface autres que lesdits antigènes de surface responsables du pouvoir infectieux. a second surface antigen 42 of said infectious agent, or fragment of said second antigen, chosen from surface antigens other than said surface antigens responsible for infectivity.
Là encore, par « antigène de surface », on entend avantageusement les protéines (y compris les glycoprotéines »), ou les fragments de protéine, qui comportent au moins un épitope. Here again, the term “surface antigen” advantageously means proteins (including glycoproteins ”), or protein fragments, which comprise at least one epitope.
De préférence encore, la zone de libération 4 comprend une combinaison de deux réactifs de détection : More preferably, the release zone 4 comprises a combination of two detection reagents:
- un premier antigène de surface 41 de l’agent infectieux, ou fragment dudit premier antigène (antigènes de surface responsables du pouvoir infectieux) conjugué avec un marqueur 43, et - a first surface antigen 41 of the infectious agent, or fragment of said first antigen (surface antigens responsible for infectivity) conjugated with a marker 43, and
- un second antigène de surface 42 de l’agent infectieux, ou fragment dudit second antigène (antigènes de surface autres que ceux responsables du pouvoir infectieux), conjugué avec un marqueur 43 (identique ou différent du premier antigène de surface 41). - a second surface antigen 42 of the infectious agent, or fragment of said second antigen (surface antigens other than those responsible for infectivity), conjugated with a marker 43 (identical or different from the first surface antigen 41).
Dans ce cas, la zone de capture 5 comprend avantageusement les deux lignes de réactifs de capture 51, 52 : - la première ligne 5a, amont, dont le réactif de capture 51 comprend un premier antigène de surface de l’agent infectieux ou fragment dudit premier antigène (antigènes de surface responsables du pouvoir infectieux), et In this case, the capture zone 5 advantageously comprises the two lines of capture reagents 51, 52: - the first line 5a, upstream, of which the capture reagent 51 comprises a first surface antigen of the infectious agent or fragment of said first antigen (surface antigens responsible for the infectious power), and
- la deuxième ligne 5b, aval, dont le réactif de capture 52 est choisi parmi les anticorps anti-anticorps humain (monoclonal et/ou polyclonal). - the second line 5b, downstream, in which the capture reagent 52 is chosen from anti-human antibody antibodies (monoclonal and / or polyclonal).
Le premier antigène de surface 41 de la zone de libération 4 et le réactif de capture 51 de la première ligne 5a peuvent être identiques, ou différents, l’un par rapport à l’autre. The first surface antigen 41 of the release zone 4 and the capture reagent 51 of the first line 5a may be the same, or different, from each other.
Par exemple, s’ils sont différents, de manière avantageuse : For example, if they are different, advantageously:
- le premier antigène de surface 41 de la zone de libération 4 correspond au premier antigène de surface de l’agent infectieux (antigène de surface responsable du pouvoir infectieux), et - the first surface antigen 41 of the release zone 4 corresponds to the first surface antigen of the infectious agent (surface antigen responsible for infectivity), and
- le réactif de capture 51 de la première ligne 5a correspond à un fragment dudit premier antigène (fragment responsable du pouvoir infectieux). the capture reagent 51 of the first line 5a corresponds to a fragment of said first antigen (fragment responsible for the infectivity).
Selon un mode de réalisation particulier, pour l’agent infectieux SRAS-Cov-V2 (Covid 19), ledit au moins un réactif de détection 41, 42 et/ou ledit au moins un réactif de capture 51, 52 comprennent : According to a particular embodiment, for the infectious agent SARS-Cov-V2 (Covid 19), said at least one detection reagent 41, 42 and / or said at least one capture reagent 51, 52 comprise:
- un premier antigène de surface 41 de l’agent infectieux choisi parmi les protéines S et les fragments de protéine S, de préférence parmi la sous-unité S1, la sous-unité S2 et le domaine RBD (Receptor Binding Domain), et - a first surface antigen 41 of the infectious agent chosen from S proteins and S protein fragments, preferably from the S1 subunit, the S2 subunit and the RBD domain (Receptor Binding Domain), and
- un second antigène de surface 42 de l’agent infectieux choisi parmi les protéines de nucléocapside N P et les fragments de protéine de nucléocapside N P. - a second surface antigen 42 of the infectious agent chosen from N P core proteins and N P core protein fragments.
Dans un exemple de réalisation, la zone de libération 4 comprend une combinaison de deux réactifs de détection : In an exemplary embodiment, the release zone 4 comprises a combination of two detection reagents:
- un premier antigène 41 comprenant le domaine RBD (Receptor Binding Domain) et,- a first antigen 41 comprising the RBD domain (Receptor Binding Domain) and,
- un second antigène 42 choisi parmi les protéines de nucléocapside NP et les fragments de protéine de nucléocapside NP. a second antigen 42 chosen from the NP core proteins and the NP core protein fragments.
Le premier antigène 41 de la zone de libération 4 et/ou le réactif de capture 51 de la première ligne 5a est avantageusement choisi parmi : The first antigen 41 of the release zone 4 and / or the capture reagent 51 of the first line 5a is advantageously chosen from:
- la protéine S, toute longueur, ou - protein S, any length, or
- un fragment de la protéine S comprenant le domaine RBD ou un fragment constitué par le domaine RBD. - a fragment of protein S comprising the RBD domain or a fragment consisting of the RBD domain.
Selon des modes de réalisation, le premier antigène 41 de la zone de libération 4 et le réactif de capture 51 de la première ligne 5a sont avantageusement choisis parmi le combinaison suivante : According to embodiments, the first antigen 41 of the release zone 4 and the capture reagent 51 of the first line 5a are advantageously chosen from the following combination:
- le premier antigène 41 de la zone de libération 4 est choisi parmi la protéine S, toute longueur, et le réactif de capture 51 de la première ligne 5a est un fragment de la protéine S comprenant le domaine RBD, voire un fragment correspondant au domaine RBD, OU - the first antigen 41 of the release zone 4 is chosen from protein S, any length, and the capture reagent 51 of the first line 5a is a fragment of protein S comprising the RBD domain, or even a fragment corresponding to the domain RBD, WHERE
- le premier antigène 41 de la zone de libération 4 et le réactif de capture 51 de la première ligne 5a sont parmi la protéine S, toute longueur. the first antigen 41 of the release zone 4 and the capture reagent 51 of the first line 5a are among the S protein, any length.
La combinaison protéine S / fragment de protéine S, développée ci-dessus, permet encore d’affiner l’analyse sérologique avec : The S protein / S protein fragment combination, developed above, allows further refinement of the serological analysis with:
- une capture et une détection des anticorps dirigés contre le domaine RBD (« anticorps neutralisants ») au sein de la première ligne 5a, amont, et - capture and detection of antibodies directed against the RBD domain (“neutralizing antibodies”) within the first line 5a, upstream, and
- une capture et une détection des anticorps dirigés contre les protéines de nucléocapside NP et les fragments de protéine de nucléocapside NP, et aussi des anticorps dirigés contre les épitopes de la protéine S, autres que le domaine RBD (« anticorps non-neutralisants »), au sein de la deuxième ligne 5b, aval. - capture and detection of the antibodies directed against the NP core proteins and the fragments of NP core protein, and also of the antibodies directed against the epitopes of the S protein, other than the RBD domain (“non-neutralizing antibodies”) , within the second line 5b, downstream.
Autres composants du système d’analyse rapide Other components of the rapid analysis system
De manière générale, le système d’analyse rapide 1 peut comprendre encore un dispositif de lecture 7 (appareil électronique) comprenant : In general, the rapid analysis system 1 can further include a reading device 7 (electronic device) comprising:
- des moyens de capture optique 71, adaptés à capter une image de ladite au moins une zone de capture 5, et - optical capture means 71, adapted to capture an image of said at least one capture zone 5, and
- des moyens de traitement 72, pour déterminer l’intensité de chaque ligne 5a, 5b et pour déterminer un ratio d’intensité entre lesdits lignes 5a, 5b. - processing means 72, to determine the intensity of each line 5a, 5b and to determine an intensity ratio between said lines 5a, 5b.
Les moyens de capture optique 71 sont adaptés à l’acquisition d’au moins une image numérique de ladite au moins une zone de capture 5 de ladite au moins une bandelette chromatographique 2. The optical capture means 71 are suitable for acquiring at least one digital image of said at least one capture zone 5 of said at least one chromatographic strip 2.
Les moyens de capture optique 71 comprennent avantageusement : The optical capture means 71 advantageously comprise:
- un capteur optique (ou photographique), - an optical (or photographic) sensor,
- un convertisseur analogique-numérique, - an analog-to-digital converter,
- un module de traitement, comprenant un programme d’ordinateur qui est avantageusement adapté à générer une image numérique à partir des données provenant du convertisseur. - a processing module, comprising a computer program which is advantageously adapted to generate a digital image from the data coming from the converter.
Le capteur optique est un composant électronique photosensible servant à convertir un rayonnement électromagnétique (UV, visible ou IR) en un signal électrique analogique. The optical sensor is a photosensitive electronic component used to convert electromagnetic radiation (UV, visible or IR) into an analog electrical signal.
Ce capteur optique est avantageusement choisi parmi : This optical sensor is advantageously chosen from:
- les capteurs CCD (pour « Charge-Coupled Device » ou « dispositif à transfert de charge » (DTC)) et - CCD sensors (for "Charge-Coupled Device" or "charge transfer device" (DTC)) and
- les capteurs CMOS (pour « Complementarity metal-oxide-semiconductor »). - CMOS sensors (for “Complementarity metal-oxide-semiconductor”).
Le signal obtenu est numérisé par le convertisseur analogique-numérique, puis traité par le module de traitement pour obtenir une image numérique d’intérêt correspondant à ladite au moins une zone de capture 5. De manière générale, par « image numérique », on entend une image constituée d'un ensemble de pixels, dont chacun est défini par plusieurs paramètres (par exemple la couleur et la luminosité). The signal obtained is digitized by the analog-to-digital converter, then processed by the processing module to obtain a digital image of interest corresponding to said at least one capture zone 5. In general, the term “digital image” is understood to mean an image consisting of a set of pixels, each of which is defined by several parameters (for example color and brightness).
Cette image numérique se présente avantageusement sous la forme d’un fichier image, c’est-à-dire un fichier informatique dont le contenu est constitué d’au moins une image. This digital image is advantageously in the form of an image file, that is to say a computer file whose content consists of at least one image.
Selon le type de codage utilisé, le fichier image consiste en : Depending on the type of encoding used, the image file consists of:
- un fichier image en mode vectoriel, ou - an image file in vector mode, or
- un fichier image en mode matriciel ou mode points (bitmap image). - an image file in matrix mode or point mode (bitmap image).
Les moyens de traitement 72, par exemple sous la forme d’un microcontrôleur intégrant un programme d’ordinateur (firmware), sont adaptés au pilotage et la coordination des composants électroniques. The processing means 72, for example in the form of a microcontroller incorporating a computer program (firmware), are suitable for controlling and coordinating electronic components.
Les moyens de traitement 72 comprennent : The processing means 72 include:
- un premier module 721 conçu pour déterminer l’intensité de chaque ligne 5a, 5b de la zone de capture 5, - a first module 721 designed to determine the intensity of each line 5a, 5b of the capture zone 5,
- avantageusement, un second module 722 conçu pour déterminer un ratio d’intensité entre lesdits lignes 5a, 5b. - Advantageously, a second module 722 designed to determine an intensity ratio between said lines 5a, 5b.
Les modules 721, 722 consistent avantageusement en des programmes d’ordinateur, comprenant des moyens de code de programme conçus pour déterminer l’intensité de chaque ligne 5a, 5b (via un premier algorithme adapté) puis pour déterminer un ratio d’intensité entre lesdites lignes 5a, 5b (via un second algorithme adapté), lorsque lesdits programmes d’ordinateur sont exécutés par les moyens de traitement 72. The modules 721, 722 advantageously consist of computer programs, comprising program code means designed to determine the intensity of each line 5a, 5b (via a first suitable algorithm) then to determine an intensity ratio between said lines 5a, 5b (via a second adapted algorithm), when said computer programs are executed by the processing means 72.
Encore de manière générale, le système d’analyse rapide 1 peut encore comprendre :Still generally, the rapid analysis system 1 can still include:
- au moins un dispositif de prélèvement 8, adapté au prélèvement dudit échantillon biologique chez un individu, par exemple un échantillon de sang capillaire, et - at least one sampling device 8, suitable for taking said biological sample from an individual, for example a capillary blood sample, and
- une solution tampon 9, destinée à être mélangée avec l’échantillon biologique, pour la mise en œuvre de la technique chromatographique. - a buffer solution 9, intended to be mixed with the biological sample, for the implementation of the chromatographic technique.
Le dispositif de prélèvement 8 comprend par exemple : The sampling device 8 comprises for example:
- un auto-piqueur 81 , classique en soi et à usage unique, adapté à générer un écoulement d’une goutte de sang capillaire, et - an auto-pricker 81, classic in itself and disposable, suitable for generating a flow of a drop of capillary blood, and
- un organe de prélèvement du sang capillaire 82, à usage unique, par exemple sous la forme d’une pipette souple classique en soi. - A capillary blood collection member 82, disposable, for example in the form of a flexible pipette conventional per se.
La solution tampon 9 est destinée à être mélangée avec l’échantillon biologique. Buffer solution 9 is intended to be mixed with the biological sample.
Cette solution tampon 9 est choisie parmi les solutions tampon adaptées à la mise en œuvre de technique chromatographique : elle est en particulier destinée à migrer le long de la bandelette chromatographique 2 de sorte à entraîner, ou au moins à faciliter, la migration de l'échantillon biologique (et en particulier des anticorps sérologiques). La solution tampon 9 est par exemple choisie parmi les diluants composés d'une solution saline tamponnée. Il peut également comprendre un détergent ou tout autre composant nécessaire notamment à la migration ou aux réactions sur la bandelette chromatographique 2. This buffer solution 9 is chosen from the buffer solutions suitable for the implementation of the chromatographic technique: it is in particular intended to migrate along the chromatographic strip 2 so as to cause, or at least to facilitate, the migration of the biological sample (and in particular serological antibodies). The buffer solution 9 is for example chosen from diluents composed of a buffered saline solution. It can also comprise a detergent or any other component necessary in particular for the migration or for the reactions on the chromatographic strip 2.
Procédé d’analyse rapide Rapid analysis process
La présente invention concerne encore le procédé d’analyse rapide de l'échantillon biologique par la mise en œuvre du système d’analyse rapide 1 selon l’invention, pour discriminer les anticorps sérologiques neutralisants et les anticorps sérologiques non- neutralisants qui sont spécifiques d’un agent infectieux. The present invention also relates to the method of rapid analysis of the biological sample by the implementation of the rapid analysis system 1 according to the invention, to discriminate the neutralizing serological antibodies and the non-neutralizing serological antibodies which are specific for 'an infectious agent.
Le procédé comprend pour cela les étapes suivantes : The method comprises the following steps for this:
- une étape de dépôt d’un mélange solution tampon 9 / échantillon biologique sur la zone de dépôt 3 de ladite au moins une bandelette chromatographique 2, pour la mise en œuvre de la technique chromatographique, et - a step of depositing a buffer solution 9 / biological sample mixture on the deposit zone 3 of said at least one chromatographic strip 2, for the implementation of the chromatographic technique, and
- une étape de lecture de ladite au moins une bandelette chromatographique 2 après migration de la solution tampon 9, au sein de la zone de capture 5, pour discriminer les anticorps sérologiques neutralisants A1 et les anticorps sérologiques non-neutralisants A2 spécifiques de (dirigés contre) l’agent infectieux d’intérêt. a step of reading said at least one chromatographic strip 2 after migration of the buffer solution 9, within the capture zone 5, to discriminate the neutralizing serological antibodies A1 and the non-neutralizing serological antibodies A2 specific for (directed against ) the infectious agent of interest.
De préférence, l’échantillon biologique est un échantillon de sang capillaire ou un échantillon de sérum / plasma. Preferably, the biological sample is a capillary blood sample or a serum / plasma sample.
En présence de deux lignes 5a, 5b de capture sur ladite au moins une zone de capture 5, l’étape de lecture comprend : In the presence of two capture lines 5a, 5b on said at least one capture zone 5, the reading step comprises:
- une phase de détermination de l’intensité de chaque ligne 5a, 5b, puis - a phase of determining the intensity of each line 5a, 5b, then
- une phase de détermination du ratio d’intensité entre les lignes 5a, 5b. - a phase of determining the intensity ratio between lines 5a, 5b.
La première ligne 5a, amont, est alors indicative de la présence des anticorps neutralisants A1 de type IgG, IgM et/ou IgA. The first line 5a, upstream, is then indicative of the presence of neutralizing antibodies A1 of the IgG, IgM and / or IgA type.
La deuxième ligne 5b, aval, est alors indicative de la présence des anticorps non- neutralisants A2 de type IgG, IgM et/ou IgA. The second line 5b, downstream, is then indicative of the presence of the non-neutralizing antibodies A2 of the IgG, IgM and / or IgA type.
L’intensité de chaque ligne 5a, 5b est avantageusement proportionnelle aux concentrations respectives des anticorps sérologiques « neutralisants » / « non-neutralisants ». The intensity of each line 5a, 5b is advantageously proportional to the respective concentrations of the "neutralizing" / "non-neutralizing" serological antibodies.
Sans être limité par une quelconque théorie, dans une approche « statut immunitaire général » à l’égard d’un agent infectieux, seule l’intensité des lignes 5a, 5b est prise en compte : Without being limited by any theory, in a "general immune status" approach to an infectious agent, only the intensity of lines 5a, 5b is taken into account:
- une personne n’ayant pas été infectée par l’agent infectieux : les deux lignes 5a, 5b ont une intensité nulle ; - a person who has not been infected with the infectious agent: the two lines 5a, 5b have zero intensity;
- une personne ayant été infectée, sans protection : la seconde ligne 5b (aval) présente une intensité non-nulle et la première ligne 5a (amont) présente une intensité nulle ; - a person who has been infected, without protection: the second line 5b (downstream) has a non-zero intensity and the first line 5a (upstream) has a zero intensity;
- une personne ayant été infectée, avec protection : la première ligne 5a (amont) et la seconde ligne 5b (aval) présentent une intensité non-nulle. Le ratio des deux lignes 5a, 5b est indicatif de prévalence des anticorps neutralisants par rapport aux anticorps non-neutralisants. - a person who has been infected, with protection: the first line 5a (upstream) and the second line 5b (downstream) have a non-zero intensity. The ratio of the two lines 5a, 5b is indicative of the prevalence of neutralizing antibodies relative to non-neutralizing antibodies.
Ce ratio peut être identifié directement et/ou par l’utilisation du dispositif de lecture 7.This ratio can be identified directly and / or through the use of the reading device 7.
Ce ratio apporte une valeur en pourcentage (%) des anticorps neutralisants, par rapport aux anticorps non-neutralisants. This ratio provides a percentage value (%) of the neutralizing antibodies, relative to the non-neutralizing antibodies.
Sans être limité par une quelconque théorie, cette détection des anticorps neutralisants et des anticorps non-neutralisants permet alors d’établir le statut immunitaire d’un individu et, en corolaire, le degré de protection de cet individu contre une réinfection (« statut immunitaire affiné »). Bien entendu, diverses autres modifications peuvent être apportées à l’invention dans le cadre des revendications annexées. Without being limited by any theory whatsoever, this detection of neutralizing antibodies and non-neutralizing antibodies then makes it possible to establish the immune status of an individual and, as a corollary, the degree of protection of this individual against reinfection ("immune status refined ”). Of course, various other modifications can be made to the invention within the scope of the appended claims.

Claims

Revendications Claims
[Revendication 1] Système d’analyse rapide d’un échantillon biologique, pour discriminer les anticorps sérologiques spécifiques d’un agent infectieux présents dans ledit échantillon biologique, lequel système d’analyse rapide (1) comprend au moins une bandelette chromatographique (2), avantageusement une bandelette immunochromatographique, qui comporte différentes zones successives, dans le sens de migration capillaire amont vers aval, à savoir au moins : [Claim 1] System for rapid analysis of a biological sample, for discriminating serological antibodies specific for an infectious agent present in said biological sample, which rapid analysis system (1) comprises at least one chromatographic strip (2) , advantageously an immunochromatographic strip, which comprises different successive zones, in the direction of upstream to downstream capillary migration, namely at least:
- une zone de dépôt (3), destinée à recevoir ledit échantillon biologique mélangé avec une solution tampon (9), - a deposit zone (3), intended to receive said biological sample mixed with a buffer solution (9),
- une zone de libération (4) qui comprend au moins un réactif de détection (41, 42) conjugué avec un marqueur visible et/ou mesurable, ledit réactif de détection (41, 42) étant apte à se déplacer en conséquence de la migration de la solution tampon (9) le long de la bandelette chromatographique (2), et - a release zone (4) which comprises at least one detection reagent (41, 42) conjugated with a visible and / or measurable marker, said detection reagent (41, 42) being able to move as a consequence of the migration buffer solution (9) along the chromatographic strip (2), and
- au moins une zone de capture (5) qui comprend au moins un réactif de capture (51, 52), immobilisé sur la bandelette chromatographique (2), caractérisé en ce que la combinaison réactif(s) de détection (41, 42) / réactif(s) de capture (51, 52) est choisie parmi les combinaisons réactif(s) de détection (41, 42) / réactif(s) de capture (51, 52) adaptées à discriminer au moins deux groupes d’anticorps sérologiques : - at least one capture zone (5) which comprises at least one capture reagent (51, 52), immobilized on the chromatographic strip (2), characterized in that the combination of detection reagent (s) (41, 42) / capture reagent (s) (51, 52) is chosen from the combinations of detection reagent (s) (41, 42) / capture reagent (s) (51, 52) adapted to discriminate at least two groups of antibodies serological:
- un premier groupe d’anticorps sérologiques (A1), dits « anticorps neutralisants », comprenant les anticorps sérologiques spécifiques dudit agent infectieux, aptes à annuler, ou au moins à réduire, le pouvoir infectieux dudit agent infectieux, et - a first group of serological antibodies (A1), called "neutralizing antibodies", comprising the specific serological antibodies of said infectious agent, capable of canceling, or at least reducing, the infectious power of said infectious agent, and
- un second groupe d’anticorps sérologiques (A2), dit « anticorps non-neutralisants », comprenant les anticorps sérologiques spécifiques dudit agent infectieux, autres que lesdits anticorps appartenant audit premier groupe d’anticorps sérologiques (A1). - a second group of serological antibodies (A2), called "non-neutralizing antibodies", comprising specific serological antibodies for said infectious agent, other than said antibodies belonging to said first group of serological antibodies (A1).
[Revendication 2] Système d’analyse rapide selon la revendication 1, caractérisé en ce que ladite au moins une zone de capture (5) comporte au moins deux lignes (5a, 5b) de capture :[Claim 2] A rapid analysis system according to claim 1, characterized in that said at least one capture zone (5) comprises at least two capture lines (5a, 5b):
- une première ligne (5a), amont, conçue pour détecter ledit premier groupe d’anticorps sérologiques (A1) (« anticorps neutralisants »), et - a first line (5a), upstream, designed to detect said first group of serological antibodies (A1) ("neutralizing antibodies"), and
- une seconde ligne (5b), aval, conçue pour détecter ledit second groupe d’anticorps sérologiques (A2) (« anticorps non-neutralisants »). - a second line (5b), downstream, designed to detect said second group of serological antibodies (A2) ("non-neutralizing antibodies").
[Revendication 3] Système d’analyse rapide selon la revendication 2, caractérisé en ce qu’il comprend encore un dispositif de lecture (7) comprenant : - des moyens de capture optique (71), adaptés à capter une image de ladite au moins une zone de capture (5), et [Claim 3] A rapid analysis system according to claim 2, characterized in that it further comprises a reading device (7) comprising: - optical capture means (71), adapted to capture an image of said at least one capture zone (5), and
- des moyens de traitement (72) comprenant : - processing means (72) comprising:
-- un premier module (721) conçu pour déterminer l’intensité de chaque ligne (5a, 5b), - a first module (721) designed to determine the intensity of each line (5a, 5b),
-- un second module (722) conçu pour déterminer un ratio d’intensité entre lesdits lignes (5a, 5b). - a second module (722) designed to determine an intensity ratio between said lines (5a, 5b).
[Revendication 4] Système d’analyse rapide selon l’une quelconque des revendications 1 à 3, caractérisé en ce que la combinaison réactif(s) de détection (41, 42) / réactif(s) de capture (51, 52) est choisie parmi les combinaisons réactif(s) de détection (41, 42) / réactif(s) de capture (51, 52) adaptées à détecter les anticorps sérologiques dirigés contre les antigènes de surface dudit agent infectieux, dont : [Claim 4] A rapid analysis system according to any one of claims 1 to 3, characterized in that the combination of detection reagent (s) (41, 42) / capture reagent (s) (51, 52) is chosen from the detection reagent (s) (41, 42) / capture reagent (s) (51, 52) suitable for detecting serological antibodies directed against the surface antigens of said infectious agent, including:
- le premier groupe d’anticorps sérologiques (A1) comprend les anticorps sérologiques dirigés contre les antigènes de surface responsables du pouvoir infectieux dudit agent infectieux, de préférence les protéines responsables de l’attachement et/ou de la pénétration et/ou de la décapsidation, et - the first group of serological antibodies (A1) comprises the serological antibodies directed against the surface antigens responsible for the infectious power of said infectious agent, preferably the proteins responsible for the attachment and / or the penetration and / or the decapsidation , and
- le second groupe d’anticorps sérologiques (A2) comprend les anticorps sérologiques dirigés contre les antigènes de surface dudit agent infectieux autres que ceux responsables dudit pouvoir infectieux. - the second group of serological antibodies (A2) comprises serological antibodies directed against the surface antigens of said infectious agent other than those responsible for said infectious power.
[Revendication 5] Système d’analyse rapide selon la revendication 4, caractérisé en ce que ledit au moins un réactif de détection (41, 42) et/ou ledit au moins un réactif de capture (51, 52) comprennent : [Claim 5] A rapid analysis system according to claim 4, characterized in that said at least one detection reagent (41, 42) and / or said at least one capture reagent (51, 52) comprises:
- un premier antigène de surface (41) dudit agent infectieux, ou fragment dudit premier antigène- a first surface antigen (41) of said infectious agent, or fragment of said first antigen
(41), choisi parmi les antigènes de surface responsables du pouvoir infectieux, et (41), chosen from the surface antigens responsible for infectivity, and
- un second antigène de surface (42) dudit agent infectieux, ou fragment dudit second antigène- a second surface antigen (42) of said infectious agent, or fragment of said second antigen
(42), choisi parmi les antigènes de surface autres que lesdits antigènes de surface responsables du pouvoir infectieux. (42), chosen from surface antigens other than said surface antigens responsible for infectivity.
[Revendication 6] Système d’analyse rapide selon l’une quelconque des revendications 4 ou 5, caractérisé en ce que la zone de libération (4) comprend une combinaison de deux réactifs de détection (41, 42) : [Claim 6] A rapid analysis system according to any one of claims 4 or 5, characterized in that the release zone (4) comprises a combination of two detection reagents (41, 42):
- un premier antigène de surface (41) de l’agent infectieux, ou fragment dudit premier antigène- a first surface antigen (41) of the infectious agent, or fragment of said first antigen
(41) (antigènes de surface responsables du pouvoir infectieux), et (41) (surface antigens responsible for infectivity), and
- un second antigène de surface (42) de l’agent infectieux, ou fragment dudit second antigène- a second surface antigen (42) of the infectious agent, or fragment of said second antigen
(42) (antigènes de surface autres que ceux responsables du pouvoir infectieux), et en ce que la zone de capture (5) comprend deux lignes (5a, 5b) de réactifs de capture (51, 52) : (42) (surface antigens other than those responsible for infectivity), and in that the capture zone (5) comprises two lines (5a, 5b) of capture reagents (51, 52):
- une première ligne (5a), amont, dont le réactif de capture (51) comprend un premier antigène de surface de l’agent infectieux ou fragment dudit premier antigène (antigènes de surface responsables du pouvoir infectieux), et - a first line (5a), upstream, of which the capture reagent (51) comprises a first surface antigen of the infectious agent or fragment of said first antigen (surface antigens responsible for infectious power), and
- une deuxième ligne (5b), aval, dont le réactif de capture (52) est choisi parmi les anticorps anti-anticorps humain. - a second line (5b), downstream, the capture reagent (52) of which is chosen from anti-human antibody antibodies.
[Revendication 7] Système d’analyse rapide selon l’une quelconque des revendications 4 à[Claim 7] A rapid analysis system according to any one of claims 4 to
6, caractérisé en ce que, pour un agent infectieux SRAS-Cov-V2 (Covid 19) dont la surface comporte des protéines de nucléocapside N P et des protéines Spike S, ladite protéine S comportant une sous-unité S1, une sous-unité S2 et un domaine RBD (Receptor Binding Domain), ledit au moins un réactif de détection (41, 42) et/ou ledit au moins un réactif de capture (51, 52) comprennent : 6, characterized in that, for an infectious agent SARS-Cov-V2 (Covid 19) whose surface comprises NP core proteins and Spike S proteins, said S protein comprising an S1 subunit, an S2 subunit and an RBD domain (Receptor Binding Domain), said at least one detection reagent (41, 42) and / or said at least one capture reagent (51, 52) comprise:
- un premier antigène de surface (41, 51) de l’agent infectieux choisi parmi les protéines S et les fragments de protéine S, de préférence parmi la sous-unité S1, la sous-unité S2 et le domaine RBD (Receptor Binding Domain), et - a first surface antigen (41, 51) of the infectious agent chosen from S proteins and S protein fragments, preferably from S1 subunit, S2 subunit and RBD domain (Receptor Binding Domain ), and
- un second antigène de surface (42) de l’agent infectieux choisi parmi les protéines de nucléocapside N P et les fragments de protéine de nucléocapside N P. - a second surface antigen (42) of the infectious agent chosen from N P core proteins and N P core protein fragments.
[Revendication 8] Système d’analyse rapide selon l’une quelconque des revendications 1 à[Claim 8] A rapid analysis system according to any one of claims 1 to
7, caractérisé en ce que l’agent infectieux est choisi parmi les virus, notamment les virus responsables de pneumopathies, avantageusement les Coronaviridae, de préférence encore Orthocoronavirinae ou coronavirus. 7, characterized in that the infectious agent is chosen from viruses, in particular viruses responsible for pneumopathies, advantageously the Coronaviridae, more preferably Orthocoronavirinae or coronavirus.
[Revendication 9] Système d’analyse rapide selon l’une quelconque des revendications 1 à[Claim 9] A rapid analysis system according to any one of claims 1 to
8, caractérisé en ce que ledit système d’analyse rapide (1) comprend encore avantageusement : 8, characterized in that said rapid analysis system (1) also advantageously comprises:
- au moins un dispositif de prélèvement (8), adapté au prélèvement dudit échantillon biologique chez un individu, par exemple un échantillon de sang capillaire, et - at least one sampling device (8), suitable for taking said biological sample from an individual, for example a capillary blood sample, and
- une solution tampon (9), destinée à être mélangée avec l’échantillon biologique, pour la mise en œuvre d’une technique chromatographique. - a buffer solution (9), intended to be mixed with the biological sample, for the implementation of a chromatographic technique.
[Revendication 10] Procédé d’analyse rapide d’un échantillon biologique par la mise en œuvre d’un système d’analyse rapide (1) selon l’une quelconque des revendications 1 à 9, pour discriminer les anticorps sérologiques neutralisants et les anticorps sérologiques non- neutralisants qui sont spécifiques d’un agent infectieux, caractérisé en ce que ledit procédé comprend : [Claim 10] A method of rapid analysis of a biological sample by implementing a rapid analysis system (1) according to any one of claims 1 to 9, to discriminate between neutralizing serological antibodies and antibodies non-neutralizing serological tests which are specific for an infectious agent, characterized in that said method comprises:
- une étape de dépôt d’un mélange solution tampon (9) / échantillon biologique sur la zone de dépôt (3) de ladite au moins une bandelette chromatographique (2), pour la mise en œuvre d’une technique chromatographique, et - une étape de lecture de ladite au moins une bandelette chromatographique (2) après migration de la solution tampon (9), au sein de la zone de capture (5), pour discriminer lesdites anticorps sérologiques neutralisants et les anticorps sérologiques non-neutralisants spécifiques d’un agent infectieux. - a step of depositing a buffer solution (9) / biological sample mixture on the deposit zone (3) of said at least one chromatographic strip (2), for the implementation of a chromatographic technique, and - a step of reading said at least one chromatographic strip (2) after migration of the buffer solution (9), within the capture zone (5), to discriminate said neutralizing serological antibodies and non-neutralizing serological antibodies specific for an infectious agent.
[Revendication 11] Procédé d’analyse rapide selon la revendication 10, caractérisé en ce que l’échantillon biologique est un échantillon de sang capillaire ou un échantillon de sérum / plasma. [Claim 11] A rapid analysis method according to claim 10, characterized in that the biological sample is a capillary blood sample or a serum / plasma sample.
[Revendication 12] Procédé d’analyse rapide selon l’une quelconque des revendications 10 ou 11, en combinaison avec la revendication 2, caractérisé en ce que l’étape de lecture comprend : - une phase de détermination de l’intensité de chaque ligne (5a, 5b), puis [Claim 12] A rapid analysis method according to any one of claims 10 or 11, in combination with claim 2, characterized in that the reading step comprises: - a phase of determining the intensity of each line (5a, 5b), then
- une phase de détermination du ratio d’intensité entre lesdites lignes (5a, 5b). - a phase of determining the intensity ratio between said lines (5a, 5b).
PCT/EP2021/066103 2020-06-16 2021-06-15 System for rapid analysis of a biological sample for distinguishing specific serological antibodies of an infectious agent present in the biological sample WO2021255021A1 (en)

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