DIAGNOSTIC MEANS FOR THE DETECTION AND/OR QUANTIFICATION OF A PLURALITY OF ANALYTES PRESENT IN A SAMPLE

Abstract

An immuno-chromatographic diagnosis means for detecting and/or quantifying a plurality of analytes present in an essentially liquid sample, comprising: at least one reaction mixture containing recognition biological molecules and/or competitive ligands labelled with at least one visualisation molecule which is detectable in fluorescence, the reaction mixture being present in a separate container of the recovery system; and at least one recovery system in the form of a solid support to which are bonded competitive ligands and/or recognition biological molecules at distinct and known recovery locations, which are arranged according to a two-dimensional matrix arrangement defined according to a system of coordinates, so as to identify by the localisation of the recovery locations on the support, the analytes present in the sample.

Claims

1. An immuno-chromatographic diagnosis means for respectively, simultaneously and specifically detecting and/or quantifying a plurality of analytes present in an essentially liquid sample comprising: at least one reaction mixture containing recognition biological molecules and/or competitive ligands labelled with at least one visualisation molecule; and at least one recovery system in the form of a solid support to which are bonded competitive ligands and/or recognition biological molecules at distinct and known recovery locations which are arranged according to a two-dimensional matrix arrangement, so as to identify, by the localisation of said recovery locations on said support, said analytes present in said sample, said diagnosis means being characterised in that, a) said two-dimensional matrix arrangement is defined according to a system of coordinates having a first coordinate (X) and a second coordinate (Y) with, for one same coordinate X, several recovery locations each comprising different recognition biological molecules or competitive ligands, arranged along different coordinates Y and, for one same coordinate Y, several recovery locations each comprising different recognition biological molecules or competitive ligands, arranged along different coordinates X; b) for the detection and/or the quantification of a given analyte, a diagnosis couple consisting of a competitive ligand and a recognition biological molecule is present, such that said recognition biological molecule is found in said reaction mixture and said competitive ligand is bonded at at least one recovery location or conversely; c) said at least one visualisation molecule is a molecule which is detectable in fluorescence; and d) said reaction mixture is present in a container, said container being separate from said recovery system.

2. The diagnosis means according to claim 1, wherein said recovery locations are arranged according to a two-dimensional matrix arrangement in the form of points each having a diameter of between 20 m to 2 mm, preferably of between 100 to 500 m, preferably between 250 and 400 m.

3. (canceled)

4. The diagnosis means according to claim 1, wherein said recovery system comprises at least 5 distinct recovery locations that respectively, simultaneously and specifically detect and/or quantify at least 5 distinct analytes present in a sample, and at least one recovery location configured as a control and/or a calibrator location.

5. The diagnosis means according to claim 1, wherein said recovery system is a solid support comprising a membrane or a set of membranes.

6-7. (canceled)

8. The diagnosis means according to claim 1, wherein said at least one visualization molecule is fused to said recognition biological molecules and/or to said competitive ligands via a chemical and/or genetic coupling.

9. The diagnosis means according to claim 8, wherein said chemical and/or genetic coupling is carried out via at least one electrostatic force, at least one peptide bond, at least one reporter gene, or a combination thereof.

10. (canceled)

11. The diagnosis means according to claim 1, wherein said analytes are selected from the group consisting of drug residues, toxins, viruses, bacteria, hormones, heavy metals, adulterants, allergens and the mixtures thereof.

12. The diagnosis means according to claim 11, wherein said analytes are drug residues and are selected from the group consisting of penicillins, cephalosporines, tetracyclines, sulphonamides, aminoglycosides, aminocyclitols, macrolides, quinolones, ionophores, carbadox, nitrofuran antibiotics, phenicols, and the mixtures thereof.

13-14. (canceled)

15. The diagnosis means according to claim 1, wherein the recovery locations are arranged according to a three-dimensional matrix arrangement.

16. (canceled)

17. A method for respectively, simultaneously and specifically detecting and/or quantifying a plurality of analytes present in an essentially liquid sample comprising the following steps: contacting a reaction mixture of a diagnosis means according to claim 1 with the sample to obtain a liquid; incubating at a temperature of between 0 and 70 C., for a duration less than or equal to 15 minutes; soaking an end of a recovery system of the diagnosis means in the liquid; incubating at a temperature of between 0 and 70 C., for a duration less than or equal to 15 minutes; and interpreting qualitatively and/or quantitatively the result on the recovery system using an optical device.

18. A diagnosis kit for respectively, simultaneously and specifically detecting and/or quantifying analytes present in a sample comprising a diagnosis means according to claim 1, wherein the kit further comprises a device for optically reading a removable solid support, comprising: a placement to receive said solid support; an optical unit to analyse said solid support and comprising: a first light source to emit according to an emission intensity and in a first wavelength range, a first light beam to said placement; an imaging system comprising an optical detector to provide an image of a visualisation zone, said visualisation zone comprising at least one portion of said placement; a filter to filter a defined wavelength range defined, and positioned between the placement and said imaging system; a communication means to obtain an item of information relative to a solid support; a selection means to: select from a list of predefined analytes corresponding to said recovery locations bonded on the solid support, a selection of analytes to be detected and/or to be quantified for said sample from one same solid support; an image processing means of said image to: determine, from the information relating to said solid support to be read, a finite number of subassemblies of said image, each subassembly corresponding to an analyte; provide data relating to light intensities coming from said subassemblies; a determination means to: calculate, for each subassembly corresponding to an analyte selected in said selection of analytes, a subassembly intensity; determine, based on said subassembly intensity, analyte information from said sample for each subassembly corresponding to an analyte selected in said selection of analytes; transmission means configured to transmit said analyte information from said sample analysed for each subassembly corresponding to an analyte selected in said selection of analytes.

19. (canceled)

20. The diagnosis kit according to claim 18, wherein the optical device further comprises: a means making it possible to read a selection profile; and wherein said selection means are configured to carry out said selection of analytes based on said selection profile.

21-24. (canceled)

25. A diagnosis kit for respectively, simultaneously and specifically detecting and/or quantifying analytes present in a sample comprising a diagnosis means according to claim 1, wherein the kit further comprises a device for optically reading a removable solid support, comprising: a placement to receive said solid support; an optical unit to analyse said solid support and comprising: a first light source to emit according to an emission intensity and in a first wavelength range, a first light beam to said placement; a light intensity sensor to measure the emission intensity emitted by said first light source; a feedback means to modulate said emission intensity of said first light source according to the emission intensity measured by said light intensity sensor such that said first light source emits a target intensity; an imaging system comprising an optical detector to provide an image of a visualisation zone, said visualisation zone comprising at least one portion of said placement; a filter to filter a defined wavelength range, and positioned between the placement and said imaging system; an image processing means of said image to: determine a finite number of subassemblies of said image, provide data relating to light intensities coming from said subassemblies; a determination means to: calculate, for each subassembly, a subassembly intensity, and a transmission means to transmit an item of information relating to said subassembly intensity for each subassembly.

26-27. (canceled)

28. A method of using the diagnosis means according to claim 1, for respectively, simultaneously and specifically detecting and/or quantifying analytes present in a sample, wherein the method detects and/or quantifies at least 5 analytes belonging to separate classes, forming part of families of analytes, which are different or not.

29. A method of using the diagnosis kit according to claim 18, for respectively, simultaneously and specifically detecting and/or quantifying classes of analytes present in a sample, wherein the method detects and/or quantifies at least 5 analytes belonging to separate classes, forming part of families of analytes, which are different or not.

Description

DESCRIPTION OF THE FIGURES

[0193] FIG. 1a is a schematic view of a diagnosis means according to document EP1712914.

[0194] FIG. 1b is a schematic view of a diagnosis means according to the document by Taranova et al.

[0195] FIG. 2 is a schematic view of a diagnosis means according to the invention.

[0196] FIG. 3 is a schematic view illustrating in detail, a recovery system according to the invention.

[0197] In the figures, identical or similar elements have the same references.

[0198] FIG. 1a represents a diagnosis means 1 according to document EP1712914 and illustrates the positioning of the recovery elements 4.sub.1, 4.sub.2, 4.sub.3 and 5 on a recovery system 3 in the form of a nitrocellulose solid support in the case of the simultaneous dosage of -lactams 4.sub.1, tetracyclines 4.sub.2 and sulphadimethoxine 4.sub.3, a bonded control zone 5 also being provided, with respect to a migration direction M. According to this document, the reaction mixture 2 is provided in a separate container with which a sample E to be tested in put into contact.

[0199] FIG. 1b represents a diagnosis means 1 according to the document by Taranova et al. and illustrates the positioning of the recovery elements 4.sub.1a, 4.sub.1b, 4.sub.1c, 4.sub.1d, 4.sub.1e, 4.sub.1f, 4.sub.1g, 4.sub.1h, 4.sub.2a, 4.sub.2b, 4.sub.2c, 4.sub.2d, 4.sub.2e, 4.sub.2f, 4.sub.2g, 4.sub.2h, 4.sub.3a, 4.sub.3b, 4.sub.3c, 4.sub.3d, 4.sub.3e, 4.sub.3f, 4.sub.3g, 4.sub.3h, 4.sub.4a, 4.sub.4b, 4.sub.4cc, 4.sub.4d, 4.sub.4e, 4.sub.4f, 4.sub.4g, 4.sub.4h, on a recovery system 3 in the form of a nitrocellulose solid support in the case of the simultaneous dosage of amphetamines (4.sub.1a, 4.sub.1b, 4.sub.1c, 4.sub.1d, 4.sub.1e, 4.sub.1f, 4.sub.1g, 4.sub.1h), of benzoylecgonine (4.sub.2a, 4.sub.2b, 4.sub.2c, 4.sub.2d, 4.sub.2e, 4.sub.2f, 4.sub.2g, 4.sub.2h), of methamphetamines (4.sub.3a, 4.sub.3b, 4.sub.3c, 4.sub.3d, 4.sub.3e, 4.sub.3f, 4.sub.3g, 4.sub.3h) and of morphine (4.sub.4a, 4.sub.4b, 4.sub.4c, 4.sub.4d, 4.sub.4e, 4.sub.4f, 4.sub.4g, 4.sub.4h). The recovery elements 4 are bonded in the form of points according to a two-dimensional matrix arrangement.

[0200] According to Taranova et al., the reaction mixture 2 is present on said recovery system 3, in a lyophilised form, upstream of said recovery elements 4 bonded on said recovery system 3 with respect to a migration direction M of a liquid comprising the sample E to be tested on the reaction mixture 2. According to this document, the recovery elements arranged on one same row, namely having the same coordinate Y, are specific of the same analyte.

[0201] FIG. 2 represents a diagnosis means 1 according to the invention and illustrates the positioning of the recovery elements 4 and 5 on a recovery system 3 in the form of a solid support with respect to a migration direction M, the recovery elements 4 and 5 being bonded in the form of points according to a two-dimensional matrix arrangement. According to the invention, the reaction mixture 2 is provided in a separate container with which a sample E to be tested is put into contact to obtain a liquid, before soaking the recovery system 3 in the liquid obtained.

[0202] FIG. 3 illustrates in detail, the recovery system 3 according to the invention on which the recovery locations 4 and 5 are arranged according to a two-dimensional matrix arrangement in the form of points having a defined diameter, each of the points being separated by a minimum distance. The two-dimensional matrix arrangement is defined according to a system of coordinates (X; Y) which has a first coordinate X defined on a longitudinal axis (A.sub.L) of a length (L) of said recovery system 3 and a second coordinate Y defined on a longitudinal axis (A.sub.l) of a width (I) of said recovery system 3. According to a preferred embodiment, the recovery system 3 comprises at least 12 separate recovery locations (4.sub.1-4.sub.12) intended to respectively, simultaneously and specifically detect and/or quantify at least 12 analytes of separate classes present in a sample E and at least three recovery locations 5 intended for a control of the detection threshold or being used as a calibrator. Furthermore, each of the recovery locations (4.sub.1-4.sub.12 and 5.sub.1-5.sub.3) is arranged in two samples (4.sub.1A; 4.sub.1B-4.sub.12A; 4.sub.12B).

[0203] It is understood that the present invention is in no way limited to the embodiments described above and that modifications can be applied to them without moving away from the scope of the appended claims.

Embodiments according to the Invention Examples

EXAMPLES

Example 1

Example of Composition of a Buffer for the Reaction Mixture and Example of a Method for Preparing the Reaction Mixture

[0204]

TABLE-US-00001 TABLE 1 Salts and additives Final concentration (nM) TRIS 20-25 HEPES 3-10 NaCl 4-8 MgCl2 0-2 Sugar 50-100 BSA 0-1 Glycerol 10-30 Tween 0-1

[0205] To this buffer are added recognition molecules and/or competitive ligands. After incubating the mixture for one night at 4 C., this is lyophilised. During the carrying out of the test, 250 l of sample to be tested will be added to the reaction mixture thus obtained.

Example 2

Example of Coupling Recognition Molecules to Fluorophore Rhodamine B

[0206] Beta and Tetra receptors and DNA oligonucleotides are obtained according to the method described in EP1712914A1.

[0207] Monoclonal antibodies are purified on the protein A or protein G column according to the species and of the isotype. The antibodies are then stored at 20 C. in the phosphate buffer 10 mM NaCl 140 mM pH7.4.

[0208] The rhodamine B used has a N-hydroxysuccinimidyl(NHS)-esters residue which has the particularity of reacting with the amine groups of proteins with a basic pH.

[0209] The recognition molecules (antibodies and/or receptors) are dialysed for one night in a carbonate buffer 50 mM pH 8.5. The fluorophore is dissolved in DMF at 5 mg/ml.

[0210] The recognition molecule and the fluorophore (the visualisation molecule) are brought together in a molar ratio of around 1/4 for one hour away from light.

[0211] Finally, the chemical reaction is stopped during the complex dialysis with a phosphate buffer 10 mM pH 7.4.

[0212] Other types of chemical bonds can be achieved, with fluorochromes having a maleimide or carboxyl group.

[0213] Other types of fluorophores can be used, such as FITC, Alexa, DyLight, etc.

[0214] The coupling of the recognition molecules can also be carried out with colorimetric nanoparticles (gold, latex, carbon nanoparticles, etc.), as much by covalent coupling, as by electrostatic adsorption.

Example 3

Example of Composition of the Reaction Mixture and Example of Recovery Elements Bonded on the Recovery System

[0215]

TABLE-US-00002 TABLE 2 Molecules of Ligands bonded the reaction on the recovery Analytes Channels mixture system Class Family detected CTL1 Control Control antigen 1 control control / antibody 1 BETA Beta receptor lactams lactams antibiotics 27 CEFA Anti-cefalexin cefalexin lactams antibiotics 2 monoclonal antibody TETRA Tetra receptor DNA tetracyclines antibiotics 10 oligonucleotides SULFA Anti- sulphonamides sulphonamides antibiotics 20 sulphonamide antibody SDX Anti- sulphadoxine sulphonamides antibiotics 1 sulphadoxine antibody QUINO Anti- fluoroquinolones fluoroquinolones antibacterial 20 fluoroquinolone agents antibodies CAP Anti- chloramphenicol phenicols antibiotics 1 chloramphenicol antibody MELA Anti-melamine melamine melamine adulterant 4 antibody AFLA Anti- aflatoxineM1 mycotoxins toxins 2 aflaxotineM1 antibody CTL2 Control Control antigen 2 control control / antibody 2 COLI Anti-colistin colistin polymyxins antibiotics 1 antibody NEO Anti-neomycin neomycin aminoglycosides antibiotics 2 antibody GEN1 Anti-gentamicin gentamicin aminoglycosides antibiotics 2 antibody STR Anti- streptomycin aminoglycosides antibiotics 2 streptomycin antibody TYLO Anti-tylosin tylosin macrolides antibiotics 2 antibody LINCO Anti- lincosamides sulphonamides antibiotics 3 lincosamide antibody SPIRA Anti-spyramicin spyramicin macrolides antibiotics 2 antibody ERY Anti- erythromycin macrolides antibiotics 3 erythromycin antibody CTL1 Control Control antigen 3 control control / antibody 3 TOTAL 104 analytes detected and distinguished via 17 channels

Example 4

Example of Carrying Out the Test and Results Obtained

[0216] A milk sample is put into contact with the reaction mixture (comprising the buffer and the recognition molecules and/or the competitive ligands in lyophilised form) for 3 minutes at 30 C. Then, the upstream end of the migration direction of the recovery system is immersed in the solution (comprising the sample and the reaction mixture). After an incubation of 10 minutes at 30 C., the reading of the results is carried out using an optical device.

[0217] The results are outlined in table 3.

TABLE-US-00003 TABLE 3 Concentrations targeted by the Signal test Concentration (arbitrary Instrumental Channels (ppb; g/kg) (ppb; g/kg) unit) interpretation BETA 4 2 1.04 negative 4 0.68 positive CEFA 2 1 1.09 negative 2 0.64 positive TETRA 50 30 1.10 negative 50 0.71 positive SULFA 100 50 1.08 negative 100 0.71 positive SDX 1.00 50 1.08 negative 100 0.69 positive QUINO 20 10 1.29 negative 20 0.69 positive CAP 0.3 0.2 1.01 negative 0.3 0.84 positive MELA 15 10 1.11 negative 15 0.86 positive AFLA 0.3 0.1 1.05 negative 0.3 0.93 positive COLI 25 20 1.14 negative 25 0.72 positive NEO 1200 900 1.04 negative 1200 0.69 positive GEN 80 60 1.03 negative 80 0.68 positive STR 200 150 1.05 negative 200 0.68 positive TYLO 40 30 1.14 negative 40 0.80 positive LINCO 80 60 1.08 negative 80 0.66 positive SPIRA 50 30 1.05 negative 50 0.79 positive ERY 20 10 1.23 negative 20 0.73 positive