CONTROLS FOR IMPLEMENTING MULTIPLEX ANALYSIS METHODS

Abstract

The present invention relates to controls which may be used to secure the results of multiplex analysis methods. The present invention thus relates to solid supports comprising one or several controls and their use in multiplex analysis methods to detect several analytes potentially present in a sample.

Claims

1. A set of beads appropriate for a multiplex analysis of a sample, comprising at least one control bead and at least two detection beads for an analyte, characterized in that the control bead is selected from the group consisting of a bead for controlling the deposition of a sample, a bead for controlling the deposition of a detection ligand of an analyte and a bead for controlling the deposition of a reporter.

2. The set of beads according to claim 1, comprising at least one bead for controlling the deposition of a sample, at least one bead for controlling the deposition of a detection ligand of an analyte and optionally at least one bead for controlling the deposition of a reporter.

3. A multiplex analysis method for detecting at least n analytes in at least one sample, n being an integer greater than or equal to 2, said method comprising at least steps a), c) and e) or at least steps a), b), c) and d) as follows: a) providing at least one solid support, b) placing, in the presence of the spots of said compartment: l detection ligands of p analytes to be detected, and, if applicable, at least one additive, l being greater than or equal to p, c) placing a sample to be analyzed in the presence of the spots of said compartment, d) placing, in the presence of the spots of said compartment: l′ detection ligands of m analytes to be detected, and, if applicable, at least one detection ligand of said additive(s), l′ being greater than or equal to m, and e) placing, in the presence of the spots of said compartment: at least one detection ligand of a control compound and l″ detection ligands of y analytes to be detected, l″ being greater than or equal to y, l, l′, l″, m, p and y being integers greater than or equal to 0 and the sum m+p+y being greater than or equal to 1; said solid support comprising at least one compartment, said compartment comprising a plurality of spots, each spot being deposited at a separate location within said at least one compartment, said plurality of spots comprising two or more control spots deposited upon said solid support within said at least one compartment and at least two detection spots comprising a ligand specific for an analyte in a sample, and said at least two control spots being selected from the group consisting of a spot for controlling the deposition of a sample, a spot for controlling the deposition of an analyte detection ligand and a spot for controlling the deposition of a reporter, wherein: a) the spot for controlling the deposition of the sample comprises at least one control capture ligand specific for a control compound naturally present in the sample to be analyzed, said control capture ligand being an antibody, antigen, peptide, carbohydrate, lipid, or nucleic acid; b) the spot for controlling the deposition of the detection ligand for the analyte comprises at least one additive capture ligand specific for an additive that is added to a sample and said additive is not naturally present in the sample or derived from any compound present in the sample, said at least one additive capture ligand being an antibody, antigen, peptide, carbohydrate, lipid, or nucleic acid; c) the spot for controlling the deposition of the reporter comprises a direct marker, an indirect marker or a carrier molecule coupled to an indirect marker, wherein the reporter specifically interacts with the indirect marker or the indirect marker coupled to the carrier molecule, optionally, in presence of a substrate, to produce a detectable signal, said direct marker being selected from a radioisotope, a fluorochrome, a lanthanide, a luminescent compound, a transition metal, and colored, fluorescent or luminescent nanoparticles, said indirect marker being an enzyme, biotin, avidin, streptavidin, neutravidin, a hapten, an antigen or an antibody, said carrier being a polypeptide, functionalized polymer, a copolymer, or an antibody, and said reporter being an enzymatic substrate for an enzyme, an enzyme for a luminescent compound, and avidin, streptavidin, or neutravidin coupled to a direct or indirect marker for biotin, and said ligands for analyte detection are antibodies, antigens, peptides, carbohydrates, lipids, or nucleic acids.

4. The multiplex analysis method according to claim 3, comprising a subsequent step f) of placing at least one reporter in the presence of the spots of said compartment.

5. The multiplex analysis method according to claim 4, characterized in that the reporter or at least one of the reporters in step f) is coupled to an indirect marker, and in that said method comprises a subsequent step g) of placing at least one second reporter of said indirect marker coupled to said reporter of step f) in the presence of the spots of said compartment.

6. The multiplex analysis method according to claim 5, characterized in that said second reporter is a substrate.

7. The multiplex analysis method according to claim 6, wherein said substrate is luminol, isoluminol or a derivative thereof.

8. A multiplex analysis method for detecting at least n analytes in at least one sample, n being an integer greater than or equal to 2, said method comprising at least steps a), c) and e) or at least steps a), b), c) and d) as follows: a) providing at least one solid support comprising at least one set of beads according to claim 1, b) placing, in the presence of said at least one set of beads: l detection ligands of p analytes to be detected, and, if applicable, at least one additive, l being greater than or equal to p, c) placing a sample to be analyzed in the presence of said at least one set of beads, d) placing, in the presence of said at least one set of beads: l′ detection ligands of m analytes to be detected, and, if applicable, at least one detection ligand of said additive(s), l′ being greater than or equal to m, and e) placing, in the presence of said at least one set of beads: at least one detection ligand of a control compound and l″ detection ligands of y analytes to be detected, l″ being greater than or equal to y, l, l′, l″, m, p and y being integers greater than or equal to 0 and the sum m+p+y being greater than or equal to 1.

9. The multiplex analysis method according to claim 8, comprising a subsequent step f) of placing at least one reporter in the presence of said at least one set of beads.

10. The multiplex analysis method according to claim 9, characterized in that the reporter or at least one of the reporters in step f) is coupled to an indirect marker, and in that said method comprises a subsequent step g) of placing at least one second reporter of said indirect marker coupled to said reporter of step f) in the presence of the spots of said compartment or of said set of beads.

11. The multiplex analysis method according to claim 10, characterized in that said second reporter is a substrate.

12. The multiplex analysis method according to claim 11, wherein said substrate is luminol, isoluminol or a derivative thereof.

Description

FIGURES

[0380] FIG. 1: Example of a solid support of the microplate type for a secure multiplex analysis. A shows a block diagram of a microplate including 18 wells. B diagrammatically shows the bottom of a well of the microplate. Each well of the microplate comprises 9 spots, 6 spots of which are each intended to detect one or several analytes making it possible to diagnose an infection (respectively A1, A2, A3, A4, A5 and A6) and 3 control spots: SDC (Sample Deposition Control), PVC (analyte detection ligand deposition control) and RVC (Reporter Deposition Control). C diagrammatically shows the three control spots comprising the detection antibodies bonded to the solid phase (PS). Ac CC: Detection antibody of the control compound. Ac Add: Detection antibody of the additive. MS+B: Carrier molecule marked with biotin.

[0381] FIG. 2: Diagram of a positive control of the deposition of a sample (SDC) in heterologous format. The control of the deposition of a sample comprises the capture antibody of the control compound fixed to the solid phase (PS). The capture antibody here is specific to the soluble receptor of the transferrin (sTfR). The control compound, here complex 2:2 soluble transferrin receptor (sTfR): transferrin (Tf) is fixed to the capture antibody at the control of the deposition of a sample and to the detection antibody (Ac 2) that is specific to the transferrin. The detection antibody (Ac 2) is marked with biotin (B). The developing is done by adding streptavin coupled to a peroxidase (S-POD), then the substrate of this enzyme (not shown). The capture antibody and the detection antibody can be monoclonal or polyclonal antibodies. In all cases, it involves a heterologous sandwich.

[0382] FIG. 3: Diagram of a positive control of the deposition of a sample (SDC) in homologous format. The control of the deposition of a sample comprises the capture antibody of the control compound fixed to the solid phase (PS). The capture antibody here is specific to the soluble receptor of the transferrin (sTfR). The control compound, here complex 2:2 soluble transferrin receptor (sTfR): transferrin (Tf) is fixed to the capture antibody at the control of the deposition of a sample and to the detection antibody (Ac 2) that is also specific to the soluble transferrin receptor (sTfR). The detection antibody (Ac 2) is marked with biotin (B). The developing is done by adding streptavin coupled to a peroxidase (S-POD), then the substrate of this enzyme (not shown). The capture antibody and the detection antibody are for example monoclonal antibodies. In involves a homologous sandwich.

[0383] FIG. 4: Diagram of a positive control of the deposition of a detection ligand of an analyte (PVC) The method control comprises the capture antibody of the additive fixed to the solid phase (PS). The capture antibody here is specific to digoxigenin (Dig). The additive comprises digoxigenin and BSA, the digoxigenin being complexed to the BSA. The additive is bonded via the digoxigenin to the capture antibody at the control of the deposition of a detection ligand of an analyte and to the detection antibody (Ac 2). The detection antibody (Ac 2) is marked with biotin (B). The developing is done by adding streptavin coupled to a peroxidase (S-POD), then the substrate of this enzyme (not shown). The capture antibody and the detection antibody can both be monoclonal antibodies or both polyclonal antibodies, in which case it involves a homologous sandwich. The capture antibody can be a monoclonal antibody and the detection antibody a polyclonal antibody, or vice versa, in which case it involves a heterologous sandwich.

[0384] FIG. 5: Diagram of a positive control of the deposition of a reporter (RVC). The control of the deposition of a reporter comprises a carrier molecule (MS) coupled to biotin (B) fixed to the solid phase (PH). The developing is done by adding streptavin coupled to a peroxidase (S-POD), then the substrate of this enzyme (not shown).

[0385] FIG. 6: “Spotting” grid situated at the bottom of a well of a microplate and including the spots of 5 analytes to be assayed (A1 to A5) and the three control spots SDC, PVC and RVC.

[0386] FIG. 7: Distribution of the signal of the SDC control beads in simplex format of a population of 94 samples. The Y-axis shows the number of samples and the X-axis shows intervals of Relative Fluorescence Intensity (RFI).

[0387] FIG. 8: Distribution of the signal of the PVC control beads in simplex format of a population of 38 samples. The Y-axis shows the number of samples and the X-axis shows intervals of Relative Fluorescence Intensity (RFI).

EXAMPLES

Example 1: “Liquid Chip” Method

Principle

[0388] The two controls described in this example make it possible to validate (cf. table 1): [0389] for the SDC (“Sample Deposit Control” or “Control of the deposition of a sample”): the deposition of the sample and also step 2 (deposition of the conjugates 2, i.e., the detection ligands deposited in step 2) and step 3) (deposition of the S-PE reporter: streptavidin coupled to Phycoerythrin), and [0390] for the PVC (“Process Verification Control” or “control of the deposition of a detection ligand of an analyte”): the deposition of step 1 (deposition of the conjugates 1, i.e., the detection ligands deposited in step 1) and also step 2 (deposition of the conjugates 2, in dotted lines because not illustrated in this example) and step 3) (deposition of the S-PE reporter).

[0391] Materials

[0392] (i) Analysis System

[0393] The BioPlex 200® analyzer (Bio-Rad, Marnes-la-Coquette, France) is used according to the manufacturer's instructions. This immunoanalysis machine contains a flow cytometer and a Luminex 100™ detector (Luminex Corp., Austin, Tex., United States) and uses heterogeneous sets of superparamagnetic particles. Each homogenous group of particles, polystyrene compounds and methacrylic acid (COOH function), and having a size of 8 μm in diameter, is manufactured with different percentages of fluorochromes (CL1 and CL2) producing a unique identification code assigned to each group of particles and detectable by the laser of the Luminex 100™ detector (Luminex Corp., Austin, Tex., United States). After the immunological reaction, the beads of one set pass one by one through a flow cell, at the center of a liquid sheath, to be simultaneously excited and read by two separate lasers. The measurements are done upon the passage of each bead.

[0394] The laser with a 638 nm red ray excites the identification fluorochromes (CL1 and CL2) encrusted in the surface of each particle and the composite signal is interpreted to identify the analyte detected by the particle. This laser therefore serves, by identifying the particle category, to identify the test in progress.

[0395] The laser with a 532 nm green ray excites the S-PE (streptavidin coupled with Phycoerythrin) reporter and the emitted fluorescence is proportional to the quantity of reporter fixed on the particle. This laser therefore serves to measure the reactivity of the analyte immobilized on said particle.

[0396] The software of the system converts the signal related to the presence of the detection ligand into a relative fluorescence intensity (RFI) value. A ratio may be calculated in order to rate the result qualitatively, as positive or negative.

[0397] (ii) Solid Phase (Beads)

[0398] A set of beads including six separate groups of Luminex™ superparamagnetic particles (Luminex Corp., Austin, Tex., United States) is used. This set of beads comprises a group of beads for controlling the deposition of a sample (SDC beads), a group of beads for controlling the deposition of a detection ligand of an analyte (PVC beads) and 4 groups of beads for detecting an analyte (analytes to be assayed: A1, A2, A3 and A4).

[0399] Each group of particles is coated with a specific capture ligand of a particular test. Each capture ligand is coupled using a hetero-bifunctional reagent.

[0400] The SDC beads are covered with a soluble anti-receptor mouse monoclonal antibody of the Transferrin (Fitzgerald, United States) immobilized at 1 μg/mg of particles.

[0401] The PVC beads are covered with an anti-Digoxigenin sheep polyclonal antibody (Abcam, United States) immobilized at 5 μg/mg of particles.

[0402] The detection beads of the analytes A1, A2, A3 and A4 are covered with one or several specific capture ligands of the analytes to be detected.

[0403] (iii) Detection Ligands

[0404] The detection ligand of the control compound (the SDC control), pAb-anti-Tf-biot, is an anti-Transferrin sheep polyclonal antibody (Bio-Rad, Barnes la Coquette, France) coupled to biotin (Thermo Scientific, France) using a hetero-bifunctional reagent known in itself by those skilled in the art.

[0405] The detection ligand of the additive (of the PVC control), pAb-anti-DIG-biot, is an anti-Digoxigenin sheep polyclonal antibody (Abcam, France) coupled to biotin (Pierce, United States) using a hetero-bifunctional reagent known in itself by those skilled in the art.

[0406] (iv) Additive

[0407] The BSA-DIG additive is Digoxigenin (Sigma, France) grafted on a carrier molecule, in this example Bovine Serum Albumin (Millipore, France), using a hetero-bifunctional reagent known in itself by those skilled in the art.

[0408] (v) Reporter

[0409] The S-PE reporter is streptavidin (Roche, Germany) coupled to the Phycoerythrin (Cyanotech, Hawaii, United States), using a hetero-bifunctional reagent known in itself by those skilled in the art.

[0410] (vi) Diluents

[0411] vi.1. Diluent of the Superparamagnetic Particles

[0412] Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM, EDTA 20 mM, Bovine Serum Albumin at 10%, mouse IgG (Meridian, United States) at 500 μg/mL, Octyl-n-Glucoside at 0.10%, NaN3 at 0.095%.

[0413] vi.2. Diluent of Conjugates 1

[0414] Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM; EDTA 20 mM, Chaps 0.1%, Glycerol 10%, NaN3 at 0.095%.

[0415] vi.3. Diluent of Conjugates 2

[0416] Citrate buffer solution 50 mM, pH 6.7, containing: NaCl 150 mM, EDTA 5.6 mM, Bovine Serum Albumin at 1%, Triton at 2%, Sheep serum at 10%, mouse IgG (Meridian, United States) at 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%.

[0417] vi.4. Diluent of the S-PE Reporter

[0418] Phosphate buffer, pH 7.4 containing: NaCl 150 mM, Tween 20™ (trademark of the company Sigma) at 0.1%, Proclin 300™ (trademark of the company Supelco) at 0.5%, PEG 6000 2.75%, Bovine Serum Albumin 1%, Normal Sheep Serum 1%, NaN3 at 0.095%.

[0419] vi.5. Washing Solution

[0420] 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%.

[0421] (vii) Reaction Dishes

[0422] The immunological reactions take place in the wells of 96-well microplates made from poly propylene having a maximum volume of 355 μL per well.

[0423] (viii) Samples

[0424] The negative samples (serum or plasma) used come from the French blood agency in Lille.

[0425] Methods

[0426] The multiplex format test protocol comprises assaying 4 different analytes, the assay of analytes A1 and A2 being done in one immunological time, the assay of analytes A3 and A4 being done in two immunological times.

[0427] The format of the tests is shown in FIGS. 2 and 4, in which S-PE is used in place of S-POD.

[0428] Step 1:

[0429] 1. In each well of a microplate are successively distributed:

[0430] +100 μL of sample

[0431] +25 μl of diluent of the conjugates 1 containing: [0432] +/−BSA-DIG [0433] +/−the detection ligands of analytes A1 and A2

[0434] +25 μL of immunoreactive superparamagnetic particles (mixture of particles, 1.2 μg per type of beads: SDC, PVC+/−beads of the analytes to be assayed)

[0435] 2. The mixture is incubated for 40 minutes at 37° C. with agitation.

[0436] 3. The following washing steps are carried out: separation of the solid and liquid phases by magnetization and 3 successive washes with at least 300 μl of wash solution. In the last wash, the particles are put back in suspension.

[0437] Step 2:

[0438] 4. Distributed in each reaction well is 90 μl of diluent of conjugates 2 containing:

[0439] +/−the detection ligand of the pAb-anti-Tf-biot control compound

[0440] +/−the detection ligand of the pAb-anti-DIG-biot additive

[0441] +/−the detection ligands of analytes A3 and A4

[0442] 5. The mixture is incubated for 15 minutes at 37° C. with agitation.

[0443] 6. The wash steps (idem point 3) are carried out.

[0444] Step 3:

[0445] 7. 90 μL of the S-PE reporter is distributed in each reaction well.

[0446] 8. The mixture is incubated for 15 minutes at 37° C. with agitation.

[0447] 9. The wash steps (idem point 3) are carried out.

[0448] 10. The particles are put back in suspension in each reaction well while adding 120 μL of washing solution, then the microplate is agitated.

[0449] 11. The suspension of particles in each well is aspirated by the flow cytometer.

[0450] 12. The suspension of particles in each well is read using two laser rays.

[0451] 13. The results of the readings are processed directly by the flow cytometer and recorded in Relative Fluorescence Intensity (RFI) units.

[0452] 14. To interpret the results, for each sample, a ratio is calculated with respect to a threshold value (“cutoff”).

[0453] Calculation of the Ratio

[0454] The SDC ratio of the samples is calculated as follows:

[00001]$\mathrm{Sample}\mathrm{SDC}\mathrm{ratio}=\frac{\mathrm{RFI}\mathrm{signal}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{SDC}\mathrm{threshold}\mathrm{value}}$

[0455] Likewise, the PVC ratio of the samples is calculated as follows:

[00002]$\mathrm{Sample}\mathrm{PVC}\mathrm{ratio}=\frac{\mathrm{RFI}\mathrm{signal}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{PVC}\mathrm{threshold}\mathrm{value}}$

[0456] The samples having ratios (SDC or PVC) above 1 are declared “valid”; those for which the ratios are below 1 are declared “not valid”.

[0457] The SDC and PVC threshold value has been established according to a statistical study described in the results below.

[0458] Results

[0459] (i) SDC System in Simplex Format

[0460] In the system, the detection beads of analytes A1 to A4 and their detection ligands are not used.

[0461] The study of 94 samples makes it possible to determine the threshold value of the SDC system (cf. FIG. 7). The threshold value of the SDC system is calculated by subtracting 3 times the standard deviation of the signal of the sample population from the average value of the signal of the population.

TABLE-US-00001 TABLE 2 Statistics of the SDC signal for the population of 94 samples and calculation of the threshold value Average (RFI) 334 Standard deviation (σ) (RFI) 44.02 Variation coefficient (CV) in % 13.2% Maximum of the population (RFI) 427 Minimum of the population (RFI) 239 Threshold value = Average − 3 σ 201.5 (RFI)

[0462] The response of the SDC system is measured in the case of a nominal method (Deposited volume of sample=100 μL, Conjugates 2 volume=90 μL, S-PE volume=90 μL) (case no. 1 of table 3). Cases 2, 3, 4 of table 3 are downgraded methods: case 2=absence of sample deposition, case 3=absence of deposition of conjugates 2, case 4=absence of S-PE deposition.

[0463] In this case, the mixture comprising the “conjugates 2” comprises 90 μL of diluent of the conjugates 2 containing the detection ligand of the pAb-anti-Tf-biot control compound.

TABLE-US-00002 TABLE 3 Summary of the SDC ratios obtained during a nominal method (case 1) and downgraded methods (cases 2, 3 and 4) Sample Conjugates Volume Status Volume 2 Volume S-PE SDC VALID/NOT Case (μL) (μL) (μL) RFIs* ratio VALID 1 100 90 90 331 1.6 VALID 2 0 90 90 31 0.2 NOT VALID 3 100 0 90 29 0.1 NOT VALID 4 100 90 0 29 0.1 NOT VALID *RFIs: Relative Fluorescence Intensities

[0464] Cases 2, 3, 4 lead to SDC ratios lower than 1, which makes it possible to invalidate the measurements from these downgraded methods.

[0465] (ii) Impact of the SDC System on Performance in Multiplex Format

[0466] In the SDC system in multiplex format (MPX), the detection beads of analytes A1 to A4 and the corresponding detection ligands are used.

[0467] By comparing the RFI signals obtained between a MPX format without SDC versus MPX with SDC (cf. table 4), the addition of a SDC format does not affect the performance of a multiplex including 4 analytes (i.e., the deviations in % between the RFI signals of a MPX format without SDC versus MPX with SDC are comprised in an interval+/−20%, which is considered statistically acceptable).

TABLE-US-00003 TABLE 4 Comparison in % of the RFI signals obtained between a MPX format without SDC versus a MPX format with SDC Number of samples used for SDC the calculations Analyte 1 Analyte 2 Analyte 3 Analyte 4 Standard 4 positive samples −5.2% deviation in % Analyte 1 between the 3 positive samples −3.0% RFI signal Analyte 2 measured in 5 positive samples −2.2% MPX without Analyte 3 SDC versus 2 positive samples −19.9% MPX with Analyte 4 SDC 32 negative samples   0.1%   1.3% −0.1%  −2.5% for the 4 Analytes

[0468] (iii) PVC in Simplex Format

[0469] As before, in this format, the detection beads of analytes A1 to A4 and the corresponding detection ligands are not used.

[0470] The study of 38 samples makes it possible to determine the threshold value of the PVC system (cf. FIG. 8).

[0471] The threshold value of the PVC system is calculated by subtracting 3 times the standard deviation of the signal of the sample population from the average value of the signal of the population (cf. table 5).

TABLE-US-00004 TABLE 5 Statistics of the PVC signal for the population of 38 samples and calculation of the threshold value Average (RFI) 356 Standard deviation (σ) (RFI) 70.92 Variation coefficient (CV) in % 19.9% Maximum of the population (RFI) 510 Minimum of the population (RFI) 222 Threshold value = Average − 3 σ 143.4 (RFI)

[0472] The response of the PVC system is measured in the case of a nominal method (Deposited volume of sample=100 μL, Conjugates 1 volume=90 μL, S-PE volume=90 μL) (case no. 1 of table 6). Cases 2, 3 of table 6 are downgraded methods: case 2=absence of conjugates 1 deposition, case 3=absence of S-PE deposition.

TABLE-US-00005 TABLE 6 Summary of the PVC ratios obtained during a nominal method (case 1) and downgraded methods (cases 2, 3) Conjugates 1 Volume Volume S-PE Status Case (μL) (μL) RFIs* Ratio VALID/NOT VALID 1 90 90 356 2.5 VALID 2 0 90 93 0.6 NOT VALID 3 90 0 30 0.2 NOT VALID

[0473] Cases 2 and 3 lead to PVC ratios lower than 1, which makes it possible to invalidate the measurements from these downgraded methods.

[0474] (iv) Impact of the Simplex Versus Multiplex Mode on the Performance of the PVC System

[0475] In the PVC system in multiplex format, the detection beads of analytes A1 to A4 and the corresponding detection ligands are used.

[0476] The performance of the PVC system is similar in simplex mode and multiplex mode (cf. table 7).

TABLE-US-00006 TABLE 7 Comparison of the PVC performance in simplex versus multiplex mode 4 Analytes, PVC PVC (multiplex) (simplex) Average RFI of 38 samples (RFI) 356 320 CV (%) 19.9% 20.6% Max (RFI) 510 472 Min (RFI) 222 194 Threshold value = Average − 3 σ 143.4 121.7 (RFI)

[0477] Furthermore, by comparing the RFI signals obtained between a MPX format without SDC or PVC versus a MPX format with SDC and PVC, it appears that the addition of SDC and PVC tests does not affect the performance of a multiplex including 4 analytes to be assayed (i.e., the deviations in % between the RFI signals of a MPX format without SDC or PVC versus a MPX format with SDC and PVC are comprised in an interval+/−20%, which is considered statistically acceptable).

Example 2: Spotting Method

[0478] Materials and Methods

[0479] The three controls described in this example make it possible to validate (cf. table 8): [0480] for the SDC (“Sample Deposit Control” or “control of the deposition of a sample”): the deposition of the sample and also step 2 (deposition of the conjugates 2), step 3 (deposition of the S-POD reporter) and step 4 (deposition of the Luminol substrate). [0481] for the PVC (“Process Verification Control” or “control of the deposition of a detection ligand of an analyte”): the depositions of step 1 (deposition of the additive and deposition of the conjugates 1), step 3 (deposition of the S-POD reporter) and step 4 (deposition of the Luminol substrate), and [0482] for the RVC (“Revelation Verification Control” or “control of the deposition of a reporter”): the revelation step, by controlling both step 3 (deposition of the S-POD reporter) and step 4 (deposition of the Luminol substrate).

[0483] Materials

[0484] (i) Analysis System

[0485] The technology used for this system is an innovative nanospotting on biochip multiplex technology (cf. definition below), with developing by chemiluminescence owing to a reporter marked by the enzyme of the horseradish peroxidase and developed by a substrate of the luminol type.

[0486] The term “biochip” is a collection of miniaturized test sites (or “micro-array”) arranged on a solid support that makes it possible to perform many tests at the same time in order to obtain a faster rhythm.

[0487] Within each well of a microplate (Greiner, Germany), a spotter robot is used to deposit 50 nL drops of a protein solution containing proteins or antibodies specific to the analyte to be assayed (A1, A2, A3, A4, A5, SDC, RVC and PVC) (cf. FIG. 6). The bottom of each well of these microplates has protein and peptide adsorption capacities known in themselves by those skilled in the art. The spots thus obtained are saturated with a saturation solution known in itself by those skilled in the art.

[0488] It is next possible to perform a traditional immunological reaction in 1 or 2 immunological times within these biochips.

[0489] After the immunological reaction, the addition of the developing substrate causes a light emission. Indeed, the oxidation of the luminol by enzymatic catalysis leads to a light emission proportional to the quantity of Streptavidin-peroxidase reporter fixed by the spot. The acquisition of the signal is done by a scientific camera. The resulting image is then analyzed in order to determine the intensity of the luminescence produced by each geographical zone of the bottom of the well corresponding to each spot (addressing information).

[0490] The software of the system converts the signal measured by spot into a value in Relative Luminescence Units (RLU). A ratio may be calculated in order to rate the result qualitatively, as positive or negative, as outlined below.

[0491] (ii) Solid phase (spots)

[0492] The different control spots are: [0493] the SDC spot, comprising a soluble anti-receptor mouse monoclonal antibody of the Transferrin (Fitzgerald, United States) immobilized at 50 μg/mL, [0494] the PVC spot, comprising a soluble anti-Digoxigenin mouse monoclonal antibody (Covalab, France) immobilized at 25 μg/mL, and [0495] the RVC spot comprising an anti-KLH (Keyhole Limpet Hemocyanin) mouse monoclonal antibody (Genway, United States) coupled to biotin (Thermo Scientific, France) using a hetero-bifunctional reagent known in itself by those skilled in the art and immobilized at 1 μg/mL.

[0496] (iii) Detection Ligands

[0497] The detection ligand of the control compound (relative to the SDC control), pAb-anti-Tf-biot, is an anti-Transferrin sheep polyclonal antibody (Bio-Rad, Barnes la Coquette, France) coupled to biotin (Thermo Scientific, France) using a hetero-bifunctional reagent known in itself by those skilled in the art.

[0498] The detection ligand of the additive (relative to the PVC control), mAb-anti-DIG-biot, is an anti-Digoxigenin mouse monoclonal antibody coupled to biotin (Covalab, France).

[0499] (iv) Additive

[0500] The BSA-DIG additive is Digoxigenin (Sigma, France) grafted on a carrier molecule, in this example Bovine Serum Albumin (Millipore, France). The coupling is done using a hetero-bifunctional reagent known in itself by those skilled in the art.

[0501] (v) Reporter

[0502] The 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.

[0503] (vi) Diluents

a) Diluent of the Additive

[0504] Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM, EDTA 20 mM, mouse IgG (Meridian, United States) at 500 μg/mL, Cow's milk (100% skim) at 15%, Sheep serum at 10%, NaN3 at 0.095%.

b) Diluent of Conjugates 1

[0505] Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM; EDTA 20 mM, Chaps 0.1%, Glycerol 10%, NaN3 at 0.095%.

c) Diluent of Conjugates 2

[0506] 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/m L, Proclin 300™ (trademark of the company Supelco) at 0.5%, cow's milk (100% skim) at 15%, Glycerol 10%. NaN3 at 0.095%.

d) Diluent of the S-POD Reporter

[0507] Citrate buffer solution 50 mM, pH 6.7, containing: NaCl 2053 mM, Tween 20™ (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%.

e) Wash Solution

[0508] 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%.

f) Developing Substrate

[0509] 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).

[0510] (vii) Reaction Dishes

The immunological reactions take place in 96-well microplates made from polystyrene having a maximum volume of 392 μL per well.

[0511] (viii) Samples

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

[0512] Methods

[0513] The test protocol comprises the following steps.

[0514] Step 1:

1. In each well of a microplate (comprising the spots) are successively distributed:
+20 μl of diluent of the additive containing the BSA-DIG additive
+20 μl of diluent of the conjugates 1 comprising: [0515] +the detection ligand of the mAb-anti-DIG-biot additive, and [0516] +the detection ligands of analytes 1, 2 and 3 to be assayed
+40 μl of sample
2. The mixture is incubated for 40 minutes at 37° C. with agitation.
3. Three successive washes with at least 400 μl of wash solution are done.

[0517] Step 2:

4. Distributed in each reaction well is 50 μl of diluent of conjugates 2 containing: [0518] +the detection ligand of the pAb-anti-Tf-biot control compound [0519] +the detection ligands of analytes 4 and 5 to be assayed.
5. The mixture is incubated for 15 minutes at 37° C. with agitation.
6. The wash steps (idem point 3) are carried out.

[0520] Step 3:

7. 50 μL of the S-POD reporter is distributed in each reaction well.
8. The mixture is incubated for 15 minutes at 37° C. with agitation.

[0521] Step 4:

9. 25 μL of developing solution “B” is distributed in each reaction well.
10. 25 μL of developing solution “A” is distributed in each reaction well.
10. The mixture is incubated for 1 minute at 37° C. with agitation.
11. The acquisition of the luminescence signal is done for 180 seconds.
12. The results of the readings are processed directly by an image analysis system and recorded in Relative Light Units (RLU).
13. To interpret the results, for each sample, a ratio is calculated with respect to a threshold value (or “cutoff”).

[0522] Calculation of the Ratio

[0523] Two analysis modes are illustrated below:

[0524] Analysis Mode 1: A Single Threshold Value

The SDC ratio of the samples is calculated as follows:

[00003]$\mathrm{Sample}\mathrm{SDC}\mathrm{ratio}=\frac{\mathrm{RLU}\mathrm{signal}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{SDC}\mathrm{threshold}\mathrm{value}}$

Likewise, the PVC ratio of the samples is calculated:

[00004]$\mathrm{Sample}\mathrm{PVC}\mathrm{ratio}=\frac{\mathrm{RLU}\mathrm{signal}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{PVC}\mathrm{threshold}\mathrm{value}}$

Similarly, the RVC ratio of the samples is calculated:

[00005]$\mathrm{Sample}\mathrm{RVC}\mathrm{ratio}=\frac{\mathrm{RLU}\mathrm{signal}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{RVC}\mathrm{threshold}\mathrm{value}}$

[0525] The samples having ratios (SDC or PVC or RVC) above 1 are declared “valid”; those for which the ratios are below 1 are declared “not valid”. The SDC, PVC and RVC threshold value has been established according to a statistical study described in the results chapter below.

[0526] Analysis Mode 2: Two Threshold Values

Two SDC ratios of the samples are calculated as follows:

[00006]$\mathrm{Sample}\mathrm{SDC}\mathrm{ratio}\left(\mathrm{threshold}-\right)=\frac{\mathrm{RLU}\mathrm{signal}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{SDC}\left(\mathrm{threshold}-\right)\mathrm{value}}$$\mathrm{Sample}\mathrm{SDC}\mathrm{ratio}\left(\mathrm{threshold}+\right)=\frac{\mathrm{RLU}\mathrm{signal}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{SDC}\left(\mathrm{threshold}+\right)\mathrm{value}}$

Likewise, the PVC ratios of the samples are calculated:

[00007]$\mathrm{Sample}\mathrm{PVC}\mathrm{ratio}\left(\mathrm{threshold}-\right)=\frac{\mathrm{RLU}\mathrm{signal}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{PVC}\left(\mathrm{threshold}-\right)\mathrm{value}}$$\mathrm{Sample}\mathrm{PVC}\mathrm{ratio}\left(\mathrm{threshold}+\right)=\frac{\mathrm{RLU}\mathrm{signal}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{PVC}\left(\mathrm{threshold}+\right)\mathrm{value}}$

Similarly, the RVC ratios of the samples are calculated:

[00008]$\mathrm{Sample}\mathrm{RVC}\mathrm{ratio}\left(\mathrm{threshold}-\right)=\frac{\mathrm{RLU}\mathrm{signal}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{RVC}\left(\mathrm{threshold}-\right)\mathrm{value}}$$\mathrm{Sample}\mathrm{RVC}\mathrm{ratio}\left(\mathrm{threshold}+\right)=\frac{\mathrm{RLU}\mathrm{signal}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{RVC}\left(\mathrm{threshold}+\right)\mathrm{value}}$

[0527] The samples having ratios (threshold−) (SDC or PVC or RVC) above 1 are declared “valid”; those for which the ratios are below 1 are declared “not valid”.

[0528] The samples having ratios (threshold+) (SDC or PVC or RVC) below 1 are declared “valid”; those for which the ratios are above 1 are declared “not valid”.

[0529] The SDC, PVC and RVC threshold value has been established according to a statistical study described in the results chapter below.

[0530] Results

[0531] The multiplex described in this example includes 5 analytes to be assayed, 3 analytes whereof the detection ligands are added in step 1 (A1, A2 and A3), 2 analytes whereof the detection ligands are added in step 2 (A4 and A5) and the three SDC, PVC and RVC controls.

[0532] Table 9 groups together the scenarios that may be encountered upon isolated or cumulative invalidation of the SDC, PVC and RVC controls.

[0533] The responses of the SDC, PVC and RVC controls are measured in the case of a nominal method (Additive Volume=20 μL, Conjugates 1 Volume=20 μL, Deposited Sample Volume=40 μL, Conjugates 2 Volume=50 μL, S-POD Volume=50 μL, Luminol Volume=25 μL solution B+25 μL solution A) for 22 samples.

[0534] The study of these 22 samples makes it possible to characterize the SPC, PVC, RVC response in terms of average spots, standard deviation, minimum and maximum value (cf. table 10a and 10b). The threshold values of the SDC, PVC and RVC controls are also calculated.

TABLE-US-00007 TABLE 9 Interpretation of all of the SDC, PVC and RVC controls and identification of the deficient step in the method. SDC and PVC and SDC PVC RVC RVC Status Status Status Status VALID/ VALID/ VALID/ VALID/ NOT NOT NOT NOT Deficient step at the source of the VALID VALID VALID VALID invalidation of the method NOT VALID VALID NOT Sample deposition or deposition of VALID VALID detection ligands in step 2 VALID NOT VALID NOT Additive deposition or deposition of VALID VALID detection ligands in step 1 NOT NOT VALID NOT Sample deposition or deposition of VALID VALID VALID detection ligands in step 2 and Additive deposition or deposition of detection ligands in step 1 NOT NOT NOT NOT S-POD or Luminol deposition VALID VALID VALID VALID Furthermore, in this scenario, the sample, additive, detection ligands of step 1, detection ligands of step 2 depositions cannot be validated or invalidated. *VALID *VALID *NOT *Cases *A not valid RVC signal involves an VALID unable to invalidation of the SDC and PVC *NOT *VALID *NOT arise VALID VALID *VALID *NOT *NOT VALID VALID

[0535] Analysis Mode 1: A Single Threshold Value

TABLE-US-00008 TABLE 10a Response of the SDC, PVC and RVC controls (average response, standard deviation, minimum and maximum value, threshold values) SDC PVC RVC Average response (RLUs) 998 2623 3200 Standard deviation (σ) (RLUs) 124 154 214 Variation coefficient (CV) in % 12.4% 5.9% 6.7% Maximum of the population (RLUs) 1246 2997 3535 Minimum of the population (RLUs) 798 2407 2773 Threshold value = Average − 3 σ 627 2162 2559 (RLUs)

[0536] Analysis Mode 2: Two Threshold Values

TABLE-US-00009 TABLE 10b Responses of the SDC, PVC and RVC controls (average response, standard deviation, minimum and maximum value, threshold values) SDC PVC RVC Average response (RLUs) 998 2623 3200 Standard deviation (σ) (RLUs) 124 154 214 Variation coefficient (CV) in % 12.4% 5.9% 6.7% Maximum of the population (RLUs) 1246 2997 3535 Minimum of the population (RLUs) 798 2407 2773 Value (threshold−) = Average − 3 σ 627 2162 2559 (RLUs) Value (threshold+) = Average + 3 σ 1368 3084 3841 (RLUs)

[0537] By combining the responses of the SDC, PVC, RVC controls and the calculated threshold values (cf. table 10a and 10b), the SDC, PVC and RVC ratios are calculated for each sample (cf. table 11a and 11b).

[0538] Analysis Mode 1: A Single Threshold Value

TABLE-US-00010 TABLE 11a Responses of the SDC, PVC and RVC controls in RLUs and ratios of a population of 22 samples. Status SDC PVC RVC SDC AND Status Status Status PVC AND SDC PVC RVC RVC Spec. SDC SDC VALID/NOT PVC PVC VALID/NOT RVC RVC VALID/NOT VALID/NOT No. RLUs ratio VALID RLUs Ratio VALID RLUs Ratio VALID VALID 1 974 1.55 VALID 2997 1.39 VALID 3368 1.32 VALID VALID 2 970 1.55 VALID 2407 1.11 VALID 3437 1.34 VALID VALID 3 863 1.38 VALID 2489 1.15 VALID 2773 1.08 VALID VALID 4 1072 1.71 VALID 2724 1.26 VALID 3216 1.26 VALID VALID 5 1246 1.99 VALID 2830 1.31 VALID 3340 1.31 VALID VALID 6 1219 1.94 VALID 2617 1.21 VALID 3165 1.24 VALID VALID 7 1002 1.60 VALID 2743 1.27 VALID 3535 1.38 VALID VALID 8 1172 1.87 VALID 2670 1.24 VALID 3150 1.23 VALID VALID 9 835 1.33 VALID 2686 1.24 VALID 3082 1.20 VALID VALID 10 932 1.49 VALID 2575 1.19 VALID 2806 1.10 VALID VALID 11 1061 1.69 VALID 2641 1.22 VALID 3274 1.28 VALID VALID 12 929 1.48 VALID 2617 1.21 VALID 3200 1.25 VALID VALID 13 1098 1.75 VALID 2495 1.15 VALID 3445 1.35 VALID VALID 14 996 1.59 VALID 2551 1.18 VALID 3382 1.32 VALID VALID 15 876 1.40 VALID 2732 1.26 VALID 3459 1.35 VALID VALID 16 896 1.43 VALID 2623 1.21 VALID 3310 1.29 VALID VALID 17 897 1.43 VALID 2429 1.12 VALID 3219 1.26 VALID VALID 18 798 1.27 VALID 2611 1.21 VALID 2977 1.16 VALID VALID 19 906 1.44 VALID 2440 1.13 VALID 2993 1.17 VALID VALID 20 1007 1.61 VALID 2412 1.12 VALID 2894 1.13 VALID VALID 21 1093 1.74 VALID 2868 1.33 VALID 3294 1.29 VALID VALID 22 1108 1.77 VALID 2547 1.18 VALID 3083 1.20 VALID VALID

[0539] Analysis Mode 2: Two Threshold Values

TABLE-US-00011 TABLE 11b Responses of the SDC, PVC and RVC controls in RLUs and (threshold+) ratios, (threshold−) ratios of a population of 22 samples. SDC PVC RVC SDC SDC PVC PVC RVC RVC ratio ratio Ratio Ratio Ratio Ratio Spec. SDC (thres- (thres- PVC (thres- (thres- RVC (thres- (threshold No. RLUs hold−) hold+) RLUs hold−) hold+) RLUs hold−) +) 1 974 1.55 0.71 2997 1.39 0.97 3368 1.32 0.88 2 970 1.55 0.71 2407 1.11 0.78 3437 1.34 0.89 3 863 1.38 0.63 2489 1.15 0.81 2773 1.08 0.72 4 1072 1.71 0.78 2724 1.26 0.88 3216 1.26 0.84 5 1246 1.99 0.91 2830 1.31 0.92 3340 1.31 0.87 6 1219 1.94 0.89 2617 1.21 0.85 3165 1.24 0.82 7 1002 1.60 0.73 2743 1.27 0.89 3535 1.38 0.92 8 1172 1.87 0.86 2670 1.24 0.87 3150 1.23 0.82 9 835 1.33 0.61 2686 1.24 0.87 3082 1.20 0.80 10 932 1.49 0.68 2575 1.19 0.83 2806 1.10 0.73 11 1061 1.69 0.78 2641 1.22 0.86 3274 1.28 0.85 12 929 1.48 0.68 2617 1.21 0.85 3200 1.25 0.83 13 1098 1.75 0.80 2495 1.15 0.81 3445 1.35 0.90 14 996 1.59 0.73 2551 1.18 0.83 3382 1.32 0.88 15 876 1.40 0.64 2732 1.26 0.89 3459 1.35 0.90 16 896 1.43 0.65 2623 1.21 0.85 3310 1.29 0.86 17 897 1.43 0.66 2429 1.12 0.79 3219 1.26 0.84 18 798 1.27 0.58 2611 1.21 0.85 2977 1.16 0.78 19 906 1.44 0.66 2440 1.13 0.79 2993 1.17 0.78 20 1007 1.61 0.74 2412 1.12 0.78 2894 1.13 0.75 21 1093 1.74 0.80 2868 1.33 0.93 3294 1.29 0.86 22 1108 1.77 0.81 2547 1.18 0.83 3083 1.20 0.80

[0540] The status of the interpretations from the (threshold+) and (threshold−) ratios of the SDC; PVC, RVC controls is “Valid” for all 22 samples. The status of the interpretations from the SDC, PVC, RVC controls is “Valid” for all 22 samples.

[0541] A case study of methods done in downgraded mode is illustrated in tables 12 and 13a and 13b. Six cases are presented: [0542] Case 1=absence of sample deposition. [0543] Case 2=absence of additive deposition. [0544] Case 3=absence of conjugates 1 deposition. [0545] Case 4=absence of conjugates 2 deposition. [0546] Case 5=absence of S-POD deposition. [0547] Case 6=absence of Luminol deposition.

[0548] Analysis Mode 1: A Single Threshold Value

[0549] Cases 1 and 4 lead to SDC ratios lower than 1, which makes it possible to invalidate the measurements from these downgraded methods.

[0550] Cases 2 and 3 lead to PVC ratios lower than 1, which makes it possible to invalidate the measurements from these downgraded methods.

[0551] Cases 5 and 6 lead to SDC, PVC and RVC ratios lower than 1, which makes it possible to invalidate the measurements from these downgraded methods. The RVC signal makes it possible to specifically invalidate the developing step (S-POD deposition and Luminol deposition). However, the invalidation of the procedure by a RVC ratio lower than 1 implies an absence of the systematic signal of the SDC and PVC controls (indicated with “*” in table 9). In this case, it is not possible to determine whether the depositions of the sample and the conjugates 2 (depositions controlled by SDC) or the depositions of additive and the conjugates 1 (depositions controlled by PVC) have taken place correctly.

[0552] Analysis Mode 2: Two Threshold Values

[0553] Cases 1 and 4 lead to SVC (threshold−) ratios lower than 1, which makes it possible to invalidate the measurements from these downgraded methods. In addition to case 1, the PVC (threshold+) ratio is greater than 1, which reflects an indirect impact of the absence of sample on the PVC control.

[0554] Cases 2 and 3 lead to PVC (threshold−) ratios lower than 1, which makes it possible to invalidate the measurements from these downgraded methods.

[0555] Cases 5 and 6 lead to SDC, PVC and RVC (threshold−) ratios lower than 1, which makes it possible to invalidate the measurements from these downgraded methods. The RVC signal makes it possible to specifically invalidate the developing step (S-POD deposition and Luminol deposition). However, the invalidation of the procedure by a RVC (threshold−) ratio lower than 1 implies an absence of the systematic signal of the SDC and PVC controls (indicated with “*” in table 9). In this case, it is not possible to determine whether the depositions of the sample and the conjugates 2 (depositions controlled by SDC) or the depositions of additive and the conjugates 1 (depositions controlled by PVC) have taken place correctly.

TABLE-US-00012 TABLE 12 Detail of the 6 downgraded method cases Step 1 Step 2 Step 3 Step 4 Sample Additive Conjugates Conjugates S-POD Volume Volume Volume 1 Volume 2 Volume Volume Luminol Case (μL) (μL) (μL) (μL) (μL) (μL) 1 0 20 20 50 50 50 2 40 0 20 50 50 50 3 40 20 0 50 50 50 4 40 20 20 0 50 50 5 40 20 20 50 0 50 6 40 20 20 50 50 0

[0556] Analysis Mode 1: A Single Threshold Value

TABLE-US-00013 TABLE 13a Responses of the SDC, PVC and RVC controls in RLU and ratio of a population of 22 samples SDC AND SDC PVC RVC PVC AND SDC PVC RVC RVC VALID/ VALID/ VALID/ VALID/ NOT NOT NOT NOT SDC SDC VALID PVC PVC VALID RVC RVC VALID VALID Case RLUs ratio (NV) RLUs Ratio (NV) RLUs Ratio (NV) (NV) 1 37 0.06 NV 3939 1.82 VALID 3458 1.35 VALID NV 2 991 1.58 VALID 72 0.03 NV 3569 1.39 VALID NV 3 983 1.57 VALID 59 0.03 NV 3046 1.19 VALID NV 4 18 0.03 NV 2727 1.26 VALID 3655 1.43 VALID NV 5 5 0.01 NV 4 0.00 NV 5 0.00 NV NV 6 6 0.01 NV 7 0.00 NV 5 0.00 NV NV

[0557] Analysis Mode 2: Two Threshold Values

TABLE-US-00014 TABLE 13b Responses of the SDC, PVC and RVC controls in RLU and ratio of a population of 22 samples SDC PVC RVC SDC PVC RVC SDC SDC VALID/ PVC PVC VALID/ RVC RVC VALID/ SDC AND PVC ratio ratio NOT Ratio Ratio NOT Ratio Ratio NOT AND RVC SDC (thresh- (thresh- VALID PVC (thresh- (thresh- VALID RVC (thresh- (thresh- VALID VALID/NOT Case RLUs old−) old+) (NV) RLUs old−) old+) (NV) RLUs old−) old+) (NV) VALID (NV) 1 37 0.06 0.03 NV 3939 1.82 1.28 3458 1.35 0.90 VALID NV (direct measurement),  (indirect meusurement of the effect of the absence of sample on PVC) 2 991 1.58 0.72 VALID 72 0.03 0.02 NV 3569 1.39 0.93 VALID NV 3 983 1.57 0.72 VALID 59 0.03 0.02 NV 3046 1.19 0.79 VALID NV 4 18 0.03 0.01 NV 2727 1.26 0.88 VALID 3655 1.43 0.95 VALID NV 5 5 0.01 0.00 NV 4 0.00 0.00 NV 5 0.00 0.00 NV NV 6 6 0.01 0.00 NV 7 0.00 0.00 NV 5 0.00 0.00 NV NV

[0558] Furthermore, by comparing the RLU signals obtained between a MPX format without SDC or PVC versus a MPX format with SDC and PVC, it appears that the addition of SDC and PVC tests does not affect the performance of a multiplex including 4 analytes to be assayed (i.e., the deviations in % between the RLU signals of a MPX format without SDC or PVC versus a MPX format with SDC and PVC are comprised in an interval+/−20%, which is considered statistically acceptable).