Control marker for implementing analysis methods on spots

Abstract

The present invention relates to the use of a control marker for implementing analysis methods on spots, in particular in the context of multiplex analyses. The present invention thus relates to solid supports containing said control marker, their preparation method and their use in analysis methods. The present invention makes it possible to verify the presence, location and/or integrity of the spots at the end of the analysis method, and thus to secure the obtained results while guaranteeing that the yielded result indeed results from a present, intact and localized spot.

Claims

1. A method of using at least one resistant control marker in at least one spot intended to detect an analyte to secure a method for detecting at least one analyte in a sample, characterized in that it comprises: checking the quality of said spot, said spot or at least one of said spots comprising at least one resistant control marker and at least one capture ligand of an analyte, after said spot has been placed in the presence of the sample and at least one detection ligand of an analyte to be detected, and the reading of the signal produced by at least one detection marker of a detection ligand of an analyte to be detected on a reading grid defined from the location of the signal produced by said resistant control marker(s) and detected at the end of the analysis method, wherein the at least one resistant control marker is a carbopyronine, a carbopyronine derivative, an oxazine, an oxazine derivative, a benzopyrylium derivative, a phycoerythrin, ##STR00004## ##STR00005## or its amine form.

2. The method of claim 1, wherein the quality of said spot provides for quality control for the detection of said analyte and the quality control of the spot comprises determining the presence, location and/or integrity of the spot.

3. The method of claim 1, said method comprising: a) placing a sample to be analyzed in the presence of the spot(s) of the compartment of a solid support, said spot or at least one of said spots comprising at least one resistant control marker and at least one capture ligand of an analyte, b) placing at least one detection ligand of an analyte in the presence of the spot(s) of said compartment, said detection ligand of an analyte being coupled to a direct detection marker selected from the group consisting of a radioisotope, a fluorophore, a heavy element from the periodic table, a luminescent compound, a transition metal, a chromogenic, colored nanoparticles, fluorescent nanoparticles and luminescent nanoparticles, c) detecting a signal produced by at least one resistant control marker in said compartment, d) defining a reading grid from the location of the signal detected in step c), e) detecting a signal produced by at least one detection marker of a detection ligand of an analyte, and f) reading the signal detected in step e) on the reading grid defined in step d).

4. The method of claim 1, said method comprising: a) placing a sample to be analyzed in the presence of the spot(s) of the compartment of a solid support, said spot or at least one of said spots comprising at least one resistant control marker and at least one capture ligand of an analyte, b) placing at least one detection ligand of an analyte in the presence of the spot(s) of said compartment, said detection ligand of an analyte being coupled to an indirect detection marker selected from the group consisting of an enzyme, a ligand of a ligand-receptor pair, a receptor of a ligand-receptor pair, a hapten, an antigen and an antibody, c) when at least one detection ligand of an analyte is coupled to an indirect detection marker, placing a reporter of said indirect detection marker in the presence of the spot(s) of said compartment, d) when the reporter used in step c) is coupled to an indirect detection marker, placing a reporter of the indirect detection marker coupled to said reporter used in step c) in the presence of the spot(s) of said compartment, e) detecting a signal produced by at least one resistant control marker in said compartment, f) defining a reading grid from the location of the signal detected in step e), g) detecting a signal produced by at least one detection marker of a detection ligand of an analyte, and h) reading the signal detected in step g) on the reading grid defined in step f).

5. The method of claim 4, wherein said ligand-receptor pair is biotin, an analogue of biotin, avidin, streptravidin, neutravidin or digoxigenin and the reporter is an enzyme coupled to avidin, streptravidin, neutravidin or digoxigenin or the reporter is selected from the group consisting of a radioisotope, a fluorophore, a heavy element from the periodic table, a luminescent compound, a transition metal, a chromogenic, colored nanoparticles, fluorescent nanoparticles and luminescent nanoparticles coupled to biotin, an analogue of biotin, avidin, streptravidin, neutravidin or digoxigenin.

6. The method of claim 1, wherein said at least one resistant control marker(s) being selected from a carbopyronine, a carbopyronine derivative, an oxazine, an oxazine derivative, a benzopyrylium derivative, and a phycoerythrin.

7. The method of claim 1, wherein said resistant control marker(s) is ##STR00006##

8. The method of claim 1, wherein said resistant control marker(s) is ##STR00007## or its amine form.

9. The method of claim 1, wherein said resistant control marker(s) is ##STR00008##

10. The method of claim 1, wherein said resistant control marker(s) is ##STR00009## or its amine form.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Use of different fluorophores and resulting fluorescence images in different steps of the protocol described in example 1.

(2) FIG. 2: Acquisition of the fluorescence signal of 12 wells comprising 9 spots at the end of an analysis method according to the invention.

(3) FIG. 3: Fluorescence image: Comparison of the theoretical reading grid (solid white circles) relative to the actual position of the spots by fluorescence (dotted white circles) based on the signal produced by the resistant control marker.

(4) FIG. 4: Chemiluminescence image: Improvement of the accuracy of the analysis method by defining the reading grid on the actual position of the spots detected through the resistant control marker (dotted line), versus the theoretical position grid (solid lines).

(5) FIG. 5: Chemiluminescence image: Improvement of the accuracy of the analysis method by verifying the integrity of the spots detected before validating the results.

(6) FIG. 6: Diagram of an optical bench comprising a lighting system, a telecentric objective, a filter wheel, said telecentric objective being coupled on its output lens to the filter wheel, and a camera.

EXAMPLES

Example 1: Example Method for Selecting a Resistant Control Marker

(7) Materials and Method

(8) Within each well of a polystyrene microplate (Greiner, Germany) are deposited, well by well, drops of 500 nl of a fluorophore solution in the buffer traditionally used for spotting of antigens or antibodies. The following fluorophores are used in this example: Atto 633-carboxylic acid (i.e., Atto 633-COOH or Atto 633) (supplier: ATTO-TEC; Germany), Atto 633-amine (i.e., Sérivé amine from Atto 633) (supplier: ATTO-TEC; Germany), Dye 634-carboxylic acid (i.e., Dye 634-COOH or Dye 634) (supplier: Dyomics, Germany), Dye 634-amine (i.e., Sérivé amine from Dye 634) (supplier: Dyomics, Germany), Dye 630-amine (i.e., Sérivé amine from Dye 630) (supplier: Dyomics, Germany), APC (Allophycocyanin) (supplier Febico; Taiwan), B-Phycoerythrin (Febico; Taiwan). The bottom of each well of these microplates has molecule adsorption capacities known in themselves by those skilled in the art. The surface of each well thus obtained is saturated with a saturation solution known in itself by those skilled in the art; the wells are filled with the saturation solution, the saturation solution is removed and the wells are next dried; after a rehydration step of these wells, a substrate solution containing the luminol (ELISTAR ETA C Ultra ELISA (Cyanagen, Italy) (cf. example 2) is then added at a rate of 50 μL/well. The fluorescence images are done at the different steps of the protocol described above (i.e., after the steps for depositing drops, saturation, drying, rehydration, and after adding the substrate solution containing the luminol), by using the Chemidoc™ MP System (Bio-Rad) having the appropriate filters for fluorophores.

(9) Results

(10) FIG. 1 describes the use of different fluorophores and resulting images in different steps of the protocol described above.

(11) The fluorescence obtained with the 4 fluorophores is fully visible at the end of the deposition of the drops on the plate. This fluorescence persists for the Atto 633-amine, the allophycocyanin and the B-Phycoerythrin after elimination of the saturation solution (cf. spots after saturation). It also persists after adding the substrate solution containing the luminol for the Atto 633-amine and for the B-Phycoerythrin, very weakly for the allophycocyanin. Similar results are obtained with the Dye 634-amine and the Dye 630-amine (results not shown). Conversely, no residual fluorescence is visible at the end of the washing step with the Dye 634-carboxylic acid and a fortiori after adding the substrate solution, as well as for the Atto 633-carboxylic acid (results not shown).

(12) Thus, the Atto 633-amine, the Dye 634-amine, the Dye 630-amine and the B-Phycoerythrin are resistant control markers according to the present invention.

(13) Furthermore, as illustrated below in example 2, the Dye 634 coupled to the BSA is also a resistant control marker according to the invention.

Example 2: Absence of Impact of the Presence of a Resistant Control Fluorophore on the Detection of an Analyte of Interest

(14) Materials and Method

(15) (i) Preparation of the microplate

(16) Within each well of a polystyrene microplate (Greiner, Germany), a spotter robot is used to deposit 50 nL drops of a solution containing the capture ligand(s) as well as a fluorophore selected using the method described in the application in rows.

(17) The capture ligand solution can be: either an antigen or mixture of antigens able to be made up of a recombinant protein associated with one or more synthetic peptides as part of an antibody detection test, or an antibody or mixture of antibodies against the sought marker in the case of an antigen detection test.

(18) These capture ligand solutions contain the selected fluorophore, for example the Atto 633-amine (Atto-tec, Germany) or a Dye 634-BSA complex, obtained using a protocol known by those skilled in the art from Dye 634 (Dyomics, Germany) in NHS-ester (N-Hydroxysuccinimide ester) form coupled with the BSA; these fluorophores are added at the appropriate dose, determined for each one; for the Dye 634-BSA, the indicated dose corresponds to the concentration of the Dye 634-BSA complex. The bottom of each well of these microplates has adsorption capacities for these different proteins known in themselves by those skilled in the art.

(19) The spots thus obtained are saturated with a saturation solution known in itself by those skilled in the art. The plates are next dried.

(20) (ii) Implementation of the Analysis Method

(21) Description of the various elements used during the implementation of the analysis method:

(22) Reporter The Streptavidin-POD (S-POD) reporter is streptavidin (Roche, Germany) coupled with Peroxidase (Roche Germany) according to the method described by Nakane and Kawaoi [J Histochem Cytochem (1974) Vol. 22, No. 12. pp. 1084-1091] known in itself by those skilled in the art.

(23) Wash Solution Tris 10 mM buffer solution, pH 7.4, containing: NaCl 218 mM, Tween 20™ (trademark of the company Sigma) at 0.1%, Proclin 300™ (trademark of the company Supelco) at 0.002%.

(24) Developing Substrate The ELISTAR ETA C Ultra ELISA developing substrate (Cyanagen, Italy) is made up of two solutions: XLSE024L Luminol enhancer solution (A) and XLSE024P Peroxide solution (B).
Description of the different steps carried out:
The test protocol comprises the following steps:

(25) Step 1:

(26) 1. In each well of a microplate (comprising the spots) are distributed:

(27) 40 μl of sample: the sample can for example be a serum or a control sample 40 μl of diluent
2. The mixture is incubated for 40 minutes at 37° C. with agitation.
3. Three successive washes with at least 300 μl of wash solution are done.
4. Next, an incubation step is done in the presence of the detection ligand, then washing under the same conditions as point 3.
5. Then, an incubation step of the reporter, then washing.
6. Lastly, a final developing step, including the addition of 25 μl of each of developing substrate solutions B and A.
7. The mixture is incubated for 1 minute at 37° C. with agitation.
8. The readings are done with a Chemidoc™ reader to measure the fluorescence and a Qview™ reader to measure the chemiluminescence. The results of the readings are processed directly by an image analysis system and recorded in Relative Light Units (RLU); also, Relative Fluorescence Intensity (RFI).

(28) Results

(29) Each datum corresponds to an average of a triplicate; the fluorescence values correspond to the fluorescence signal level after deducting the background noise.

(30) Results obtained with the Atto 633-amine fluorophore added into the specific antibody solution of the marker of interest to be detected.

(31) TABLE-US-00001 TABLE 1 Impact of the presence of the Atto 633-amine fluorophore on the detection of the analyte for different fluorophore doses. E1, E2 and E3 are positive samples, containing the antigen to be detected, at different positivity levels. Atto 633-amine fluorophore Comparison of the Chemiluminescence chemiluminescence Composition of the signal signal relative to the Sample deposit solutions RLU absence of fluorophore E1 Antibody without fluorophore 1489.5 reference Antibody + Fluo 0.1 μg/ml 1726  16% Antibody + Fluo 0.35 μg/ml 1703  14% Antibody + Fluo 1.2 μg/ml 1377  −8% Antibody + Fluo 2.5 μg/ml 1438  −3% Antibody + Fluo 5 μg/ml 1210 −19% E2 Antibody without fluorophore 4651 reference Antibody + Fluo 0.1 μg/ml 5033  8% Antibody + Fluo 0.35 μg/ml 4861  5% Antibody + Fluo 1.2 μg/ml 3679 −21% Antibody + Fluo 2.5 μg/ml 4115 −12% Antibody + Fluo 5 μg/ml 3779 −19% E3 Antibody without fluorophore 2188 reference Antibody + Fluo 0.1 μg/ml 2117  −3% Antibody + Fluo 0.35 μg/ml 2264  3% Antibody + Fluo 1.2 μg/ml 1650 −25% Antibody + Fluo 2.5 μg/ml 1479 −32% Antibody + Fluo 5 μg/ml 1226 −44%

(32) The results shown in table 1 show the absence of impact of the presence of the Atto 633-amine fluorophore on the detection of the analyte for fluorophore doses of 0.35 μg/ml or less. The calculation of the detection limit of the analyte shows the absence of impact of the presence of the fluorophore up to a dose of 1.2 μg/ml. The presence of the capture ligands only very slightly modifies the fluorescence (cf. table 2), which remains very significant and fully detectable at the end of the analysis.

(33) TABLE-US-00002 TABLE 2 Impact of the presence of capture ligands on the fluorescence signal (case of the Atto 633-amine fluorophore). E1 and E3 are positive samples, containing the antigen to be detected, at different positivity levels. N1 is a negative sample, not containing the antigen to be detected. ‘Fluo’ stands for Fluorophore. Atto 633-NH2 fluorophore Comparison of the Fluorescence Chemiluminescence fluorescence signal Composition of the signal signal relative to the absence Sample deposit solutions RLU RLU of markers of interest E1 Fluorophore alone 0.1 μg/ml 26843 54 −21% Antibody + Fluo 0.1 μg/ml 21158 1726 E3 Fluorophore alone 0.1 μg/ml 29039 56 −31% Antibody + Fluo 0.1 μg/ml 20021 2117 N1 Fluorophore alone 0.1 μg/ml 24999 57 −29% Antibody + Fluo 0.1 μg/ml 17641 49
Results obtained with the Dye 634-BSA fluorophore added into the specific antibody solution of the marker of interest to be detected:

(34) The results shown in table 3 show the absence of impact of the presence of the Atto 634-BSA on the detection of the analyte for fluorophore doses of 6 μg/ml or less, this dose being compatible with the detection of the fluorescence at the end of analysis and the implementation of the data processing method as described in the present application.

(35) TABLE-US-00003 TABLE 3 Impact of the presence of the Atto Dye 634-BSA fluorophore on the detection of the analyte for different fluorophore doses. E1, E2 and E3 are positive samples, containing the antigen to be detected, at different positivity levels. ‘Fluo’ stands for Fluorophore. Dye 634-BSA fluorophore Comparison of the Chemiluminescence chemiluminescence Composition of the signal signal relative to the Sample deposit solutions RLU absence of fluorophore E1 Antibody without fluorophore 1856 reference Antibody + Fluo 3 μg/ml 1716  −8% Antibody + Fluo 6 μg/ml 1782  −4% Antibody + Fluo 12 μg/ml 1562 −16% Antibody + Fluo 25 μg/ml 1379 −26% Antibody + Fluo 50 μg/ml 1297 −30% E2 Antibody without fluorophore 5576 reference Antibody + Fluo 3 μg/ml 5471  −2% Antibody + Fluo 6 μg/ml 4908 −12% Antibody + Fluo 12 μg/ml 4133 −26% Antibody + Fluo 25 μg/ml 4231 −24% Antibody + Fluo 50 μg/ml 4629 −17% E3 Antibody without fluorophore 2183 reference Antibody + Fluo 3 μg/ml 2397  10% Antibody + Fluo 6 μg/ml 2184  0% Antibody + Fluo 12 μg/ml 1714 −21% Antibody + Fluo 25 μg/ml 1445 −34% Antibody + Fluo 50 μg/ml 1347 −38%
Results obtained with the Atto 633-amine fluorophore added into the antibody solution corresponding to the antibodies to be detected:

(36) The results shown in table 4 show the absence of impact of the presence of the Atto 633-amine fluorophore on the detection of the analyte for the fluorophore dose of 0.1 μg/ml or less. The presence of capture ligands (cf. table 5) modifies the fluorescence, but the signal remains very significant, fully detectable at the end of analysis and usable to implement the data processing method as described in the present application.

(37) TABLE-US-00004 TABLE 4 Impact of the presence of the Atto 633-amine fluorophore on the detection of the analyte for different fluorophore doses. Samples S1 and S2 are positive samples, containing antibodies reacting selectively and respectively relative to 2 types of antigens used in mixture: recombinant protein or synthetic peptide. ‘Fluo’ stands for Fluorophore. Atto 633-amine fluorophore Comparison of the Chemiluminescence chemiluminescence Composition of the signal signal relative to the Sample deposit solutions RLU absence of fluorophore S2 Antigens without fluorophore 410 reference Antibody + Fluo 0.1 μg/ml 355 −13% Antigens + Fluo 0.35 μg/ml 351 −14% Antigens + Fluo 1.2 μg/ml 366 −11% Antigens + Fluo 2.5 μg/ml 330 −20% Antigens + Fluo 5 μg/ml 247 −40% S1 Antigens without fluorophore 1638 reference Antibody + Fluo 0.1 μg/ml 1547 −6% Antigens + Fluo 0.35 μg/ml 1215 −26% Antigens + Fluo 1.2 μg/ml 1261 −23% Antigens + Fluo 2.5 μg/ml 1253 −24% Antigens + Fluo 5 μg/ml 1194 −27%

(38) TABLE-US-00005 TABLE 5 Impact of the presence of capture ligands on the fluorescence signal (case of the Atto 633-amine fluorophore). Samples S1 and S2 are positive samples, containing antibodies reacting selectively and respectively relative to 2 types of antigens used: recombinant protein or synthetic peptide. N2 is a negative sample, not containing antibodies recognizing the antigen to be detected. ‘Fluo’ stands for Fluorophore. Atto 633-amine fluorophore Comparison of the fluorescence Chemiluminescence fluorescence signal Composition of the signal signal relative to the absence Sample deposit solutions RLU RLU of markers of interest N2 Fluorophore alone 0.1 μg/ml 70881 70 −47% Antigens + Fluo 0.1 μg/ml 37781 67 S2 Fluorophore alone 0.1 μg/ml 72852 93 −45% Antigens + Fluo 0.1 μg/ml 39877 355 S1 Fluorophore alone 0.1 μg/ml 73721 117 −47% Antigens + Fluo 0.1 μg/ml 39096 1547
Results obtained with the Dye 634-BSA fluorophore added into the antibody solution corresponding to the antibodies to be detected:

(39) TABLE-US-00006 TABLE 6 Impact of the presence of the Atto Dye 634-BSA fluorophore on the detection of the analyte for different fluorophore doses. The sample S1 is a sample containing antibodies reacting with respect to the recombinant protein used. ‘Fluo’ stands for Fluorophore. Dye 634-BSA fluorophore Comparison of the Chemiluminescence chemiluminescence Composition of the signal signal relative to the Sample deposit solutions RLU absence of fluorophore S1 Antigens without fluorophore 1066 reference Antigens + Fluo 3 μg/ml 1218 14% Antigens + Fluo 6 μg/ml 1003 −6% Antigens + Fluo 12 μg/ml 1277 20% Antigens + Fluo 25 μg/ml 1291 21% Antigens + Fluo 50 μg/ml 1247 17%

(40) The results shown in table 6 show the absence of impact of the presence of the Dye 634-BSA fluorophore on the detection of the analyte irrespective of the fluorophore dose. The fluorescence signal obtained at the end of analysis is fully detectable and usable to implement the data processing method as described in the present application.

Example 3: Use of a Resistant Control Marker to Secure a Method for Detecting at Least One Analyte in a Sample

(41) Materials and Method

(42) (i) Preparation of the Microplate

(43) Within each well of a polystyrene microplate (Greiner, Germany), a spotter robot is used to deposit 50 nL drops of a solution containing the capture ligand(s) as well as a fluorophore selected using the method described in the application in rows.

(44) The capture ligand solution can be:

(45) either an antigen or mixture of antigens able to be made up of a recombinant protein associated with one or more synthetic peptides as part of an antibody detection test, or an antibody or mixture of antibodies against the sought marker in the case of an antigen detection test.

(46) These capture ligand solutions contain the selected Atto 633-amine fluorophore (Atto-tec, Germany) at the appropriate dose comprised between 0.1 to 0.5 μg/mL. The bottom of each well of these microplates has adsorption capacities for these different proteins known in themselves by those skilled in the art.

(47) The spots thus obtained are saturated with a saturation solution known in itself by those skilled in the art. The plates are next dried.

(48) (ii) Implementation of the Analysis Method

(49) Description of the various elements used during the implementation of the analysis method:

(50) I. Reporter

(51) The Streptavidin-POD (S-POD) reporter is streptavidin (Roche, Germany) coupled with Peroxidase (Roche Germany) according to the method described by P. Nakane and A. Kawaoi [J Histochem Cytochem (1974) Vol. 22, No. 12. pp. 1084-1091] known in itself by those skilled in the art.

(52) II. Diluents

(53) a) Diluent Step 1

(54) Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM, EDTA 20 mM, mouse IgG (Meridian) at 500 μg/mL, Cow's milk (100% skim) at 15%, Sheep serum at 10%, NaN3 at 0.095%.

(55) b) Diluent of Conjugates 1

(56) Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM; EDTA 20 mM, Chaps 0.1%, Glycerol 10%, NaN3 at 0.095%.

(57) c) Diluent of Conjugates 2

(58) Citrate buffer solution 50 mM, pH 6.7, containing: NaCl 150 mM, EDTA 5.6 mM, Triton at 2%, Sheep serum at 10%, mouse IgG 500 μg/mL, Proclin 300™ (trademark of the company Supelco) at 0.5%, cow's milk (100% skim) at 15%, Glycerol 10%. NaN3 at 0.095%.

(59) d) Diluent of the Streptavidin-POD Reporter

(60) Citrate buffer solution 50 mM, pH 6.7, containing: NaCl 2053 mM, Tween20™ (trademark of the company Sigma) at 0.5%, Proclin 300™ (trademark of the company Supelco) at 0.5%, cow's milk (100% skim) at 7%, Glycerol 20%.

(61) e) Wash Solution

(62) Tris 10 mM buffer solution, pH 7.4, containing: NaCl 218 mM, Tween20™ (trademark of the company Sigma) at 0.1%, Proclin 300™ (trademark of the company Supelco) at 0.002%.

(63) f) Developing Substrate

(64) The ELISTAR ETA C Ultra ELISA developing substrate (Cyanagen, Italy) is made up of two solutions: XLSE024L Luminol enhancer solution (A) and XLSE024P Peroxide solution (B).

(65) III. Reaction Dishes

(66) The immunological reactions take place in 96-well microplates (Greiner, Germany) made from polystyrene having a maximum volume of 392 μL per well.

(67) IV. Samples

(68) The negative samples (serum or plasma) used come from the French blood agency in Lille.

(69) V. Optical Bench

(70) The optical bench used is made up of the following elements:

(71) a lighting system emitting red light centered on the wavelength of 620 nm and assembled such that it illuminates the lower face of the microplate homogenously, a telecentric objective produced to make it possible to image the entire surface of the microplate, a filter wheel inserted between the output lens of the telecentric objective and the camera, a camera having the ability to produce images with exposure times comprised between 0.001 second and 250 seconds, a support chassis that supports and positions all of the elements, including the microplate.
The optical bench is studied and assembled such that it takes the images from the lower face of the microplate. The development of the objective is done such that the inner face of the microplate wells is clear.
The filter wheel is able to have two different filters: a filter centered on the wavelength of 680 nm making it possible to allow only the signal corresponding to the light emitted by the fluorophore to pass, a filter making it possible to allow all of the wavelengths comprised between 400 nm and 700 nm to pass.
Description of the different steps carried out:
The test protocol comprises the following steps:

(72) Step 1:

(73) 1. In each well of a microplate (comprising the spots) are successively distributed:

(74) +20 μl of diluent step 1

(75) +20 μl of diluent of conjugates 1 comprising the detection ligands of the analytes to be assayed from the first step.

(76) +40 μl of sample

(77) 2. The mixture is incubated for 40 minutes at 37° C. with agitation.

(78) 3. Three successive washes with at least 400 μl of wash solution are done.

(79) Step 2:

(80) 4. Distributed in each reaction well is 50 μl of diluent of conjugates 2 containing the detection ligands of the analytes to be assayed from the second step.

(81) 5. The mixture is incubated for 15 minutes at 37° C. with agitation.

(82) 6. The wash steps (idem point 3) are carried out.

(83) Step 3:

(84) 7. 50 μL of the S-POD reporter is distributed in each reaction well.

(85) 8. The mixture is incubated for 15 minutes at 37° C. with agitation.

(86) Step 4:

(87) 9. 25 μL of developing solution “B” is distributed in each reaction well.

(88) 10. 25 μL of developing solution “A” is distributed in each reaction well.

(89) 10. The mixture is incubated for 1 minute at 37° C. with agitation.

(90) 11. The acquisition of the fluorescence signal is done for 10 seconds.

(91) 12. The acquisition of the luminescence signal is done for 180 seconds.

(92) Results

(93) A) Persistence of the resistant control marker after an analysis method.

(94) The fluorescence signal of the resistant control marker of each of the 9 spots present in the 12 wells is clearly identifiable and fully measurable at the end of the analysis method in FIG. 2.

(95) B) Importance of redefining the reading grid at the end of the analysis method: comparison of the regions of interest obtained by fluorescence by defining the reading grid relative to the theoretical positions versus by defining the reading grid from the signal emitted by fluorescence by the resistant control marker.

(96) In FIG. 3, the solid white circles show the theoretical position of the spots, the dotted white circles showing the detected actual position. The spots are clearly shifted relative to their expected theoretical position.

(97) This image demonstrates the relevance of the method, which always makes it possible to target the position of the detected actual spot through the resistant control marker, which does not cause errors in the reading of the signal emitted by the detection marker of the detection ligand of the analyte.

(98) C) Importance of redefining the reading grid at the end of the analysis method: comparison of the regions of interest obtained by chemiluminescence by defining the reading grid relative to the theoretical positions versus by defining the reading grid from the signal emitted by fluorescence by the resistant control marker.

(99) The location of the actual spots (dotted lines) was obtained based on signals produced by the resistant control marker and applied on the acquisition image of the signals of the detection marker of a ligand of the analyte by chemiluminescence shown here in FIG. 4. The theoretical positioning of the spots is indicated in solid lines and clearly shows a shift relative to the actual position.

(100) The comparison between the median values in chemiluminescence of the pixels situated under the expected theoretical positions (solid lines) and the actual detected positions (dotted lines) by fluorescence is shown in table 7.

(101) TABLE-US-00007 TABLE 7 Signal measured from the analysis of the theoretical presence region of a spot versus signal measured from the analysis of the actual position region of that same spot Spot 1 Spot 3 Spot 5 spot 9 (top left) (top right) (middle) (bottom right) Theoretical 319 301 58 3387 position Actual 689 693 138 5233 position

(102) The analysis of the region containing a spot producing the signal provides significantly higher results than the theoretical presence region of that same spot. The quantification of the signal and the accuracy of the results obtained are therefore improved by basing oneself on the detected actual positioning grid owing to the resistant control marker.

(103) D) Example of deteriorated spot after an analysis method and that could have yielded a false result without verification of the integrity of the spots.

(104) The location of the actual spots (dotted lines) was obtained based on signals produced by the resistant control marker and applied on the acquisition image of the signals of the detection marker of a ligand of the analyte by chemiluminescence shown here (cf. FIG. 5). The theoretical positioning of the spots is indicated in solid lines.

(105) In the image (cf. FIG. 5), the spot located at the center of the well is deformed. The broken white line surrounding it shows that the software detected the actual shape of the spot, which makes it possible to analyze its integrity before validating the results yielded. In this case, the circularity perimeter of the actual shape of the spot makes it possible to eliminate this spot and not yield a false analysis value.