Field of the invention

The present invention generally relates to methods and kits for the detection analytes in a biological sample. More specifically it relates to methods and kits which enable the increase in the number of analytes that can be measured concurrently in antibody- based assays.

Background of the invention

Antibody-based immunoassays are the cornerstone in healthcare and scientific research because they can detect and quantify proteins in biological samples and can serve as predictive, diagnostic, or prognostic biomarkers. Immunoassays rely on binding between an antibody and the corresponding biological analyte to form a complex that can be later detected. One standard immunoassay technique termed “sandwich ELISA” involves the use of two antibodies: the first antibody is usually bound to a solid support and serves to capture and to isolate the molecule from the biological sample typically via washing steps. Subsequently, a secondary antibody that has been modified (labeled) to generate a signal is used to bind to the same molecule on a different site (epitope) and produce a signal that can be detected and be used for the quantification of the protein of interest. If the protein of interest is significantly altered between biological states (i.e. normal vs diseased, responder vs non-responder) then the protein is used as biomarker with significant impact in healthcare.

There are three main characteristics of every immunoassay: i) High throughput, ii) Multiplexability, iii) Immunoassay scheme i) High throughout capacity is the number of samples that can be measured in each run and is usually driven but the sample handling format (i.e. 96well plate, 384 well plate, or custom holder for protein array glass slides/membranes) ii) Multiplexability refers to the number of analytes that can be measured simultaneously in the same sample. There are a plethora of multiplexing schemes in which solid supports (spots on glass, microneedles, microparticles etc) and sometime samples obtain a unique identity required for multiplexing. The uniquely identified solid support not only contributes to the barcoding of the assay, but it is also essential for all steps in the assay including washes, analyte isolation, and barcoded detection. These unique identities can be obtained by either spatially distributed 2D arrangements (i.e. Protein arrays, Mesoscale Discovery, Ayoxxa Biosystems etc), microparticles in suspension with unique spectrometric/magnetic/barcoding signature (BD™ Biosciences' Cytometric Bead Array, xMAP ^® technology from Luminex, FireFly ^® , Nanoplex Technologies, Applied Biocode, Biocartis etc) or more recently with nucleic acid-barcoded antibodies used with sequencing detection or proximity ligation (Olink Proteomics AB etc) iii) The immunoassay scheme refers to the scheme required for capturing, washing, labeling, & detection of the analyte. The capturing scheme typically aims to immobilize the protein of interest on the solid supports. On this front, antibodies are the most widely used, however, other affinity molecules including peptides, nanobodies, aptamers, somamers, and affimers have been developed. In another case, capture antibodies can be skipped and biological samples are directly coupled to the solid support (usually known as reverse phase approach). In all cases, a detection scheme is needed to produce a signal which ideally is linearly proportional to the amount of the protein captured on the support. There are several schemes for tagging and detection including direct sample labeling, single-antibody approach, or double- antibody approach (sandwich assay) together with a signal generation scheme such as enzymatic labeled detection (i.e. horseradish peroxidase), amplified chromogenic products, planar wave guide technology (i.e. Zeptosens), standard fluorescent detection with fluorophores, electrochemiluminescent detection (i.e. Meso Scale Discovery), or proximity ligation procedure in which detection signals can be generated by a PCR- type reaction (i.e. Olink Proteomics AB).

The first multiplex technologies in the market were antibody arrays and later multiplex suspension systems allowed better binding kinetics and higher throughput. Suspension systems are based on microparticles in suspension with unique spectrometric/magnetic/barcoding signature such as BD Biosciences' Cytometric Bead Array, xMAP ^® technology from Luminex, FireFly, Nanoplex Technologies, Applied Biocode, Biocartis etc. Many of those multiplex platforms allow high multiplexability: Luminex xMAP ^® technology with the FlexMAP 3D ^® allows 500 simultaneous analytes, Applied BioCode’s Barcoded Magnetic Beads (BMB) technology has 4096 available barcodes, and Abcam’s Firefly ^® particle technology allows 70 analytes. All those systems employ a typical ELISA sandwich assay where antibodies are coupled to the particles, mixed with the test sample, washed to remove unbound proteins, and then mixed with secondary antibodies that are either labeled or biotinylated so a sandwich scheme is formed. Despite the multiplexability potential of the platforms, commercial assays do not go above a few dozen proteins due to the antibody cross-reactivity in the ELISA sandwich scheme. For example, as of today Thermo Fisher Scientific offers a Cytokine Human Magnetic 35-Plex Panel for the Luminex platform (significantly lower than the 500plex that some instruments allows), R&D Systems offers a maximum 45plex assay as“high-performance assays” (out of >450 developed assays), Bio-Rad Laboratories offers a 48plex assay (out of >450 developed assays), and Applied Biocode offers a 20plex assay. None of these multiplex assays can reach the multiplexability of the instrument presumably due to the cross-reactivity of the secondary antibodies. This issue is more pronounced in complex biological samples such as serum and plasma that matrix effects makes cross-reactivity issues a big problem.

Alternative ELISA schemes have been suggested to reduce the problem of cross- reactivity and multiplexability. Sequential multiplex analyte capturing has been suggested by Poetz O. et al, 2010, Mol Cell Proteomics 2010, 9:2474-2481 , where several multiplex analyte panels can be used sequentially on the same sample and overcome cross-reactivity. A nice and detailed overview for“shedding light on the dark side of multiplexing” has been presented in Juncker D. et al, 2014, Curr Opin Chem Biol. 2014 Feb;18:29-37. Among many different techniques, the direct labeling scheme is a well know strategy where samples are labeled and then a single antibody scheme on a solid support is used to capture the labeled protein and generate a signal. The single antibody approach offers high multiplexability and an easy development process since there is no need of antibody pairing. It has been applied extensively in antibody arrays (Raybiotech L-Series Label-Based Antibody Arrays, Sciomics GmbH, etc) and in some multiplex systems but with concerns of performance due to cross-reactivity. Indeed, direct labeling of the whole sample results at the labeling of all proteins that produce significant off-target binding to the capture antibody that results in significant background noise. On this front, a dual capturing scheme have been suggested by Ayoglu B. et al, 2016, Proteomics. Apr; 16(8): 1251-6, Birgersson E. et al, 2017, In: Greening D., Simpson R. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 1619. Humana Press, New York, NY Online ISBN: 978-1-4939-7057-5, and Zhou S. et al, 2010, Anal Biochem. May 15;400(2):213-8, where the labeling takes place only on the bound portion of the captured molecules on a solid surface. In this method, Zhou et al suggests a non-multiplexed plate-based immunoassay method for quantitative detection of proteins using single antibodies and labeling of the bound portion followed by detection. Ayoglou et al. and as follow up Birgersson et al. transferred this dual capture format on Luminex beads where samples are mixed with beads, followed by on-bead-labeling, elution, and then a second capture followed by detection using the Luminex xMAP ^® technology and quantified by the instrument reported MFIs (Median Fluorescence Intensity). They demonstrate that sequential capture/elution has reduced background and noise levels. However, the signals of the multiplex assays with recombinant proteins were low and in most cases they were not reaching more than 1000 MFIs using the Luminex FlexMap3D ^® instrument and the typical streptavidin Phycoerythrin (SAPE) method that many multiplex assays are using for detection. For example, in the main example of Ayoglou et al. in Figure 2A, 2ng/ml spiked CA3 protein resulted in log2MFI=6 (thus MFI=2 ⁶ =64) that is low compared to typical MFI values in sandwich ELISA assays on the same FlexMap3D ^® instrument with recombinant proteins at the same concentration that exceeds 1000 and in some cases 10000 MFIs. To address the issue of sensitivity the authors developed a more sensitive detection method known as rolling circle amplification (RCA) instead of SAPE. The RCA approach resulted in a higher signal but higher Coefficient of Variation (CV).

Many research groups have suggested or shown ( Ayoglu B. et al, 2016, Proteomics. Apr; 16(8): 1251 -6, Tighe P.J. et al, 2015, Proteomics Clinical Applications DOI : 10.1002/prca.201400130, Zheng W. et al., 2012, In: Chiu N.H.L., Christopoulos T.K. (eds) Advances in Immunoassay Technology, DOI: 10.5772/36994, Poetz O. et al, 2010, Mol Cell Proteomics 2010, 9:2474-2481 ) that standard antibody-based multiplex assays follow the conditions of ambient analyte analysis ( Ekins R. P., 1989, J Pharm Biomed Anal, 7, 155-168). Ayoglou et al. presumed that the same assumption holds for direct labeling and thus the measured intensity levels are dependent on target concentration rather than overall available quantities (mass sensing). The number of beads used in their method (1250 beads of 6.5 microns in diameter as a first step and 500 beads for the second step) results in a surface area of 0.17 mm ² for capture and 0.07 mm ² for detection (132 pm ² per bead), thus a surface ratio detection/capture=2.5 and might conform with this ambient assumption. In both cases of Ayoglou et al. and Zhou et al methods, protein sample labeling took place only on the captured portion of the sample which is a much less complex environment from a neat serum or plasma sample and resulted in improved assay performance. Thus, on-particle-labeling offers a great potential for multiplex assays but there is a need in the art for improving the signal and the sensitivity of antibody-based multiplex analyte detection.

Summary of the invention

The present invention provides a method and a kit for analyzing a sample for the presence of at least two analytes. The disclosed method concerns improved antibody- based multiplex assays comprising Sequential Microparticle Suspension Systems (SeMiSS) of diverse binding capacities that sequentially come in contact with the test sample and transfer the bound proteins by means of binding and elution.

The first suspension system (SSA) has high binding capacity and plays the role of concentrating and optionally labeling the analytes from the test sample that are bound to its microparticles. Microparticles in SSA are coupled to antibodies recognising at least two analytes. First, SSA microparticles are mixed with the sample in order to immunoprecipitate and concentrate the analytes. Then, microparticles are washed to remove non-bound analytes, and on-bead-analytes are labeled using standard protocols. Then, labeled analytes are eluted from the microparticles and transferred to a second suspension system (SSB). The second suspension system has also antibodies recognizing the same analytes and is used for detection. According to the present invention, the analyte binding capacity of the first suspension system is at least 20 times greater than the analyte binding capacity of the second suspension system. The difference in the binding capacity of the first and the second suspension leads to an increase of the signal and therefore an increase in the sensitivity of the method. Brief description of the drawings

Figure 1 is a schematic illustration of the method comprising Sequential Microparticle Suspension System (SeMiSS) according to the invention. Figure 2 is a schematic illustration of an embodiment of the present invention comprising two Sequential Microparticle Suspension System (SeMiSS).

Figure 3 shows standard curves for PROK1 and IL6 using the Sequential Microparticle Suspension System (SeMiSS) and sandwich ELISA 2plex recombinant proteins in 1 % BSA buffer.

Figure 4 shows the results obtained by Sequential Microparticle Suspension System (SeMiSS) for the detection of naive and spiked-in protein in plasma and BSA buffer. Figure 5 shows the results of the assessment of antibody cross-reactivity using SeMiSS.

Figure 6 shows SeMiSS signal generation by adjusting binding capacity of SSA. Figure 7 shows SeMiSS signal generation by adjusting binding capacity of SSA over SSB.

Figure 8 shows SeMiSS signal amplification by increasing sample volume. Figure 9 shows the results of simultaneous measurement of 96 proteins in plasma using the SeMiSS approach.

Detailed description of the invention

Various terms are generally described or used herein to facilitate understanding of the invention. It will be understood that a corresponding general description or use of these various terms applies to corresponding linguistic or grammatical variations or forms of these various terms. Generally, the term“microparticle” refers to a particle having height, width, length, or diameter below 1 mm on which a plurality of proteins or antibodies can be immobilized either non-covalently or covalently for interaction with analytes present in a sample; and wherein the microparticle may be magnetic or not, and the physical form may assume any physical shape such as a sphere of regular or irregular shape, a sheet of regular or irregular shape, a rod of regular or irregular shape, a bead, a fiber, a matrix, a porous structure, a stick, or the like, merely by way of example, and the material may be selected from the list of sepharose, agarose, latex, dextran, metal, metal oxide, glass, ceramic, quartz, plastic, silicon, polyacrylamide, polystyrene, polyethylene, polypropylene, polymer, a colloid, polycarbonate, polytetrafluoroethylene, silicon oxide, silicon nitride, cellulose acetate membrane, nitrocellulose membrane, nylon membrane and polypropylene membrane, amorphous silicon carbide, castable oxides, polyimides, polymethylmethacrylates, and silicone elastomers and/or the like. The term microparticle comprises also nanoparticles. A microparticle can also have other forms of a closed surface as long as it can be separated from the solution containing or suspected to contain protein molecules by conventional separation methods such as centrifugation, filtration, precipitation, magnetic field, affinity capture and the like.

The term“binding capacity” of a suspension is defined as the maximum mass of analyte that a suspension system can absorb when it comes in contact with the sample comprising the analyte.

The term “surface area” of a suspension is defined as the total surface area of the microparticles comprised in the suspension, i.e. the sum of the surface areas of the microparticles comprised in the suspension.

Generally, the term “biological sample” refers to all biological specimens or the derivatives of biological specimens that contain or are suspected to contain biologically relevant molecules (including, but not limited to, proteins, peptides, nucleic acids, steroids or steroid hormones, sugars, lipids, other small molecules). The specimen may contain multiple known or unknown protein(s) or a mixture containing multiple known or unknown protein(s). The specimen may be a biological fluid; a tissue of a plant, fungus, animal or human origin; cell(s) of a bacterium, plant, fungus, animal or human origin; viruses and other micro-organisms; lysates; fractions or other derivatives of the biological specimens described above; or naturally occurring materials (such as water, soil, air) that contain the biological specimens described above.

Generally, the term“biological fluid” refers to all fluids that contain or are suspected to contain biologically relevant molecules (including, but not limited to, proteins, peptides, nucleic acids, steroids or steroid hormones, sugars/carbohydrates, lipids, other small molecules). The biological fluid may be a solution containing multiple known or unknown proteins(s) or a mixture containing multiple known or unknown protein(s). Typical examples of biological fluids include, but are not limited to, bodily fluids such as blood, blood plasma, blood serum, hemolysate, spinal fluid, urine, lymph, synovial fluid, saliva, semen, stool, sputum, cerebral spinal fluid, tear, mucus, amniotic fluid, lacrimal fluid, cyst fluid, sweat gland secretion, bile, milk and the like. Additional examples of “biological fluid” include medium supernatants of culture cells, tissue, bacteria and viruses as well as lysates obtained from cells, tissue, bacteria or viruses. Cells and tissue can be derived from any single-celled or multi-celled organism described above. The herein disclosed method concerns antibody-based multiplex assays comprising a Sequential Microparticle Suspension System (SeMiSS) that sequentially comes in contact with the test sample.

The present invention provides a method for the detection of at least two analytes in a biological sample, wherein the method comprises the steps of

a) exposing the sample to a first suspension comprising microparticles on which antibodies which recognize the at least two analytes are attached under conditions which enable the binding of the analytes to the antibodies,

b) eluting the analytes from the first suspension,

c) exposing the analytes to a second suspension comprising microparticles on which antibodies which recognize the at least two analytes are attached under conditions which enable the binding of the analytes to the antibodies,

wherein the analyte binding capacity of the first suspension is at least 20 times greater than the analyte binding capacity of the second suspension. Preferably, the analyte binding capacity of the first suspension is at least 30 times greater than the analyte binding capacity of the second suspension. More preferably, the analyte binding capacity of the first suspension is at least 100 times greater than the analyte binding capacity of the second suspension. Even more preferably, the analyte binding capacity of the first suspension is at least 200 times greater than the analyte binding capacity of the second suspension.

The binding capacity of a suspension may be determined by bringing it in contact with test sample of known analyte concentration and volume and then measuring with ELISA the drop in the analyte mass before and after the contact with the suspension system.

In one embodiment of the method of the present invention, the first and the second suspension comprise microparticles having the same analyte binding capacity. In this case, the binding capacity of each suspension corresponds to the surface area of the suspension, which in turn corresponds to the number of microparticles in the suspension. Therefore, in this embodiment, the surface area of the first suspension is at least 20 times higher than the surface area of the second suspension. Preferably, the surface area of the first suspension is at least 30 times greater than the surface area of the second suspension. More preferably, the surface area of the first suspension is at least 100 times greater than the surface area of the second suspension. Even more preferably, the surface area of the first suspension is at least 200 times greater than the surface area of the second suspension.

With amounts of antibody coupling protocols typically used in the art, i.e. between 1 ng and 100ng antibody per 1 mm ² the surface area of the first suspension is preferably at least 1 mm ² . More preferably, the surface area of the first suspension is at least 3mm ² . Even more preferably, the surface area of the first suspension is at least 10mm ² . Most preferably, the surface area of the first suspension is at least 20mm ² .

Preferably, the method can be used for the detection of at least 10 analytes. More preferably, the method can be used for the detection of at least 50 analytes.

Preferably, the microparticles comprised in the suspensions are magnetic. Preferably, the macroparticles have a unique spectrometric, magnetic, barcoding or other suitable signature which enables multiplex detection of the analytes.

Preferably, the analytes according to the present invention are proteins.

In an embodiment the invention the method comprises the following steps of: a) preparing a first suspension comprising microparticles coupled with antibodies recognizing at least two analytes; b) preparing a second suspension comprising microparticles coupled with antibodies recognizing the same analytes as the first suspension; c) exposing a biological sample to the first suspension which is capable of immunoprecipitating the analytes to said antibodies under conditions which enable the binding of the analytes to the antibodies; d) washing the first suspension to remove unbound compounds; e) On-particle-labeling the bound analytes with biotin or fluorophore or other signal-generating molecule; f) washing the first suspension to remove labeling reagents; g) eluting labeled analytes from the first suspension; h) exposing labeled analytes to the second suspension under conditions which enable the binding of the analytes to the antibodies; i) performing a multiplex detection on the second suspension.

In another embodiment, the labeling of the analytes is performed after the analytes have been exposed to the second suspension.

The antibodies of the microparticles of the second suspension may be the same or different to the antibodies of the microparticles of the first suspension. When they are different, they must recognize the same analytes as the antibodies of the microparticles of the first suspension.

Optionally, the microparticles of the second suspension are coupled with antibodies recognizing other proteins that have not been captured on the first suspension, as a mean to assess cross-reactivity and assay quality.

The present application provides the unexpected finding that the increase of the analyte binding capacity of the first suspension results in a large increase of the measured signal. This finding is unexpected because it shows that the suggestions of the prior art that antibody-based multiplex assays follow the conditions of ambient analyte analysis and thus the measured intensity levels are dependent on the concentration of the analyte rather than its overall available quantity, do not apply.

Figure 1 is a schematic illustration of the method of the present invention. Suspension System A (SSA) has strong binding capacity and comprises microparticles coupled with antibodies recognizing at least two analytes. Suspension System B (SSB) comprises microparticles coupled to antibodies that recognize the same analytes. A biological sample is mixed with SSA and washed so only the immunoprecipitated analytes are retained and concentrated. The concentrated proteins are then labeled. As illustrated by the number of microparticles, the first microparticle suspension system has at least 20 times larger analyte binding capacity. The final suspension system (SSB) is multiplexed and used for detection. The number of antibodies and the corresponding number of analytes shown in the scheme are only for illustrative purpose. The bead-shaped objects represent microparticles with coupled antibodies. The Y-shaped objects represent antibodies.

FIG. 2 is a schematic illustration of an embodiment of the method of the present invention The following steps are used: a) preparing suspension system A comprising microparticles coupled to antibodies that recognize at least two analytes. Suspension system A has at least 20 times larger binding capacity than Suspension system B, b) preparing suspension system B comprising microparticles coupled to antibodies that recognize the analytes as the suspension system A; c) exposing a biological sample to suspension system A which is capable of immunoprecipitating the analytes to said antibodies; d) washing suspension system A to remove unbound compounds; e) On- particle-labeling the bound analytes with biotin or fluorophore or other signal-generating molecule; f) washing suspension system A to remove labeling reagents; g) eluting labeled analytes from suspension system A; h) transfer labeled analytes to suspension system B; i) use suspension system B for detection using standard protocols. The Y- shaped objects represent antibodies. The bead-shaped objects represent microparticles with coupled antibodies. The irregular-shaped-lines represent protein molecules. Sun- shaped objects represent tags that are bound to labeled proteins. In the method of the present invention, an increase of the binding capacity of the first suspension leads to a big increase of the signal, which can be measured using methods well known to a person skilled in the art. Thus, with the method of the present invention there is no need for the development of specialized detection methods as disclosed in the prior art.

The present invention also provides a kit for the detection of at least two analytes in a biological sample, wherein the kit comprises a first container and a second container, each container comprising microparticles on which antibodies which recognize at least two analytes are attached or may be attached and wherein the kit comprises instructions to perform the method of the present invention as disclosed herein. The kit preferably further comprises a dilution buffer and a washing buffer, used in the washing and elution steps of the method.

Examples

Example 1

This example shows a comparison between the method of the present invention to Sandwich ELISA.

The first microparticle suspension system comprises two antibodies IL6 and PROK1 at 10 million magnetic beads per well. Each coupling step is using 15pg antibody to 600 million magnetic beads of size 1 micrometer in diameter. The coupling takes place via standard epoxy coupling techniques but other methods including carboxyl and tosyl techniques can be used. The first suspension system (SSA) has high binding capacity and plays the role of immunoprecipitation of the protein sample that is bound to its microparticles. The suspension system B comprising two plex magnetic Luminex beads coupled to the IL6 and PROK1 antibodies using standard Luminex coupling techniques as described at the Luminex Cookbook. IL6 and PROK1 standard are serially diluted in 1 % BSA in PBS starting at 100ng/ml concentration. Recombinant samples are then exposed to suspension system A overnight and then washed with PBS containing BSA. During washing unbound proteins are removed. On-particle-labeling comprising biotinylation of the SSA bound proteins using standard protocols from Pierce. SSA is then washed to remove labeling regents. The bound proteins that are still attached to the SSA are then eluted using standard elution buffer of low pH 3.0. Eluted proteins are neutralized with standard protocols found in Pierce. The neutralized and labeled proteins are then transferred to suspension system B comprising Luminex beads. Detection is performed using a FlexMAP 3D ^® Luminex instruments and results are shown in Figure 3. For comparison purposes, a standard sandwich ELISA of IL6 and PROK1 using the same capture antibodies is shown in the same graph. Interestingly, it is possible to achieve similar assay performance using the sandwich ELISA and SeMiSS methods when recombinant proteins are used.

Figure 3 shows a signal of -9,500 MFIs at 2 ng/ml PROK1 (interpolation at 2ng/ml data point in sandwich PROK1 ELISA) compared to -64 MFIs obtained by the method of Ayoglu B. et al, 2016, Proteomics. Apr; 16(8): 1251 -6 (Figure 1A, C3A at 2 ng/ml of that publication) and 70,528 MFIs at 100 ng/ml (100 ng/ml data point in sandwich PROK1 ELISA) compared to -250 MFIs (Ayoglou Figure 1A, C3A at 100 ng/ml of that publication). Even though the instrument settings used in Ayoglou et al. are not known and there are differences due to the Low/High PMT settings, background subtraction, antibody affinity, and other reasons, the MFIs differences are between the method of the present invention and the prior art method are very high.

Example 2

This example shows the use of the method of the present invention for protein detection in plasma. To evaluate the specificity of the method, the first suspension system (SSA) comprises 2plex beads (PROK1 and IL20). The second suspension system (SSB) comprises an 1 1 -plex (the said 2plex and extra 9plex).

The first microparticle suspension system comprises two antibodies IL20 and PROK1 at 10 million magnetic beads per well. Each coupling step is using 15pg antibody to 600 million magnetic beads of size 1 micrometer in diameter. The coupling takes place via standard carboxyl coupling techniques but other methods including epoxy and tosyl techniques can be used. The first suspension system (SSA) has high binding capacity and plays the role of immunoprecipitation of the protein sample that is bound to its microparticles. The suspension system B comprising of an 1 1 plex magnetic Luminex beads coupled to TNFA, CSF3, IL17F, IL1A, FST, CXCL10, CNTF, CCL2, DEFB1 antibodies using standard Luminex coupling techniques as described at the Luminex Cookbook. IL20 and PROK1 standards are prepared in 1 % BSA in PBS at 10ng/ml, 1 ng/ml and 0 ng/ml concentration. IL20 and PROK1 standards were also spiked in at 1 :10 diluted human plasma and 1 :100 diluted human plasma in final concentrations at 10ng/ml, 1 ng/ml and 0 ng/ml. Recombinant protein and plasma samples are then exposed to suspension system A overnight and then washed with PBS containing BSA. During washing unbound proteins are removed. On-particle-labeling comprising biotinylation of the SSA bound proteins using standard protocols from Pierce. SSA is then washed to remove labeling regents. The bound proteins that are still attached to the SSA are then elluted using standard elution buffer of low pH 3.0. Eluted proteins are neutralized with standard protocols found in Pierce. The neutralized and labeled proteins are then transferred to suspension system B comprised of Luminex beads. Detection is performed using a FlexMAP 3D ^® Luminex instruments and results are shown in Figure 4. High signal was detected in PROK1 and IL20 but not in the 9plex in all sample types. Low signal is detected in the 9plex due to filtering-out at SSA and thus cannot be detected in SSB.

Example 3

This example shows the use of the method of the present invention to evaluate antibody cross reactivity.

The first particle suspension system comprises two antibodies IL20 and PROK1 at 10 million magnetic beads per well. Each coupling step is using 15pg antibody to 600 million magnetic beads of size 1 micrometer in diameter. The coupling takes place via standard carboxyl coupling technique. The first suspension system (SSA) has high binding capacity and plays the role of immunoprecipitation of the protein sample that is bound to its microparticles. The suspension system B comprises 12plex magnetic Luminex beads coupled to TNFA, CSF3, IL17F, IL1A, FST, CXCL10, CNTF, CCL2, DEFB1 , and IL12A (last column in Figure 5) antibodies using standard Luminex coupling techniques as described at the Luminex Cookbook. Three samples are prepared: 1 :10 diluted human plasma, 1 :100 diluted human plasma, and 1 % BSA in PBS. Samples are then exposed to suspension system A overnight and then washed with PBS containing BSA. During washing unbound proteins are removed. On-particle- labeling comprising biotinylation of the SSA bound proteins using standard protocols from Pierce. SSA is then washed to remove labeling regents. The bound proteins to SSA, theoretically only the PROK1 and IL20, are then eluted using standard elution buffer of low pH 3.0. Eluted proteins are neutralized with standard protocols found in Pierce. The neutralized and labeled proteins are then transferred to suspension system B comprising 12 plex Luminex beads. SSB comprises 10 off-target assays that should not recognize the pass-through eluate. Detection is performed using a FlexMAP 3D ^® Luminex instrument and theoretically only PROK1 and IL20 beads should detect a signal. Results are shown in Figure 5 and strong signal is detected in the off-target bead IL12A. This cross-reactivity event implied that the SSB antibody IL12A recognize the PROK1/IL20 eluate or that the SSA antibodies recognize and immunoprecipitate IL12A protein that is later pass through to the eluate or that all PROK1/IL20-SSA antibodies and IL12A-SSB antibodies recognize some other protein.

Example 4

This example shows the importance of the binding capacity in the process of the present invention, by adjusting the binding capacity of SSA.

The first particle suspension system comprises one antibody CSF3 coupled using 15pg antibody to 600 million magnetic beads of size 1 micrometer in diameter. The surface area of each bead is calculated at 3.14pm ² . Different surface areas were tested by adjusting the number of beads starting from 83.33 millions and using 3 fold dilutions down to 141 1 beads (Figure 6): (a)83.33 Million beads or 261.1 mm ² (b) 27.7 Million beads or 87 mm ² , (c) 9.25 Million beads or 29 mm ² , (d) 3.09 Million beads or 9.69 mm ² , (e) 1.02 Millions beads or 3.23 mm ² , (f) 342.9 thousands beads or 1.08 mm ² (g) 1 14.3 thousands beads or 0.359mm ² (h) 38.1 thousands beads or 0.120mm ² (i) 12.7 thousands beads or 0.040 mm ² (j) 4233 beads or 0.013mm ² (k) 141 1 beads or 0.004mm ² . The suspension system B comprising 500 Luminex beads coupled to CSF3 antibody using standard Luminex coupling techniques as described at the Luminex Cookbook. The corresponding ratio of surface area of suspension system A to Suspension System B is (a) 5314 , (b)1771 , (c) 590, (d) 196, (e) 65, (f) 22, (g) 7.3, (h) 2.4, (i) 0.8, G) 0.3, (k) 0.09. CSF3 samples at 10 ng/ml were then exposed to different bead numbers (a)-to-(k) overnight and then washed with PBS containing BSA. During washing unbound proteins were removed. Subsequently beads were labeled with standard biotinylation process from Pierce. SSA were then washed to remove labeling reagents. The bound proteins to SSA were then eluted using standard elution buffer of low pH 3.0. Eluted proteins were neutralized with standard protocols found in Pierce. The neutralized and labeled proteins were then transferred to suspension system B comprising 500 Luminex CSF3 beads. Detection was performed using a FlexMAP 3D ^® Luminex instrument. Results are shown in Figure 6. The lower limit of strong signal is shown with the black arrow and defined as 5 times higher MFI than basal MFI. Strong signals are detected only in cases (a), (b), (c), (d), (e), and (f). Thus, to achieve strong signals we need a surface area A that is at least 20 times larger than surface area B ([SSA surface]/[SSB surface]>20), or we need a surface area A that of at least 1 mm ² (square millimeters) to detect a strong signal.

The 0.17 mm ² area used in the publication Ayoglu B. et al, 2016, Proteomics. Apr; 16(8): 1251 -6 (estimated using the 1250 magnetic Luminex beads of 6.5pm in diameter) results in low binding capacity compared to the surface area of at least 1 mm ² (points (a) to (f) in Figure 6). For example, when 29 mm ² is used (9.2Million beads) a signal of 15028 MFI is obtained, significantly higher than 450 MFIs that was measured with 0.17 mm ² surface area. Thus, it is postulated that the ambient assumptions should not be followed and large surface areas should be used in the first system in combination with a smaller area in the second system.

Example 5

This example shows the importance of the binding capacity in the process of the present invention, by adjusting the binding capacity of the second suspension (SSB).

The strength of the signal was evaluated by adjusting the binding capacity of SSB and consequently the binding capacity of SSA over SSB. TNFa antibody coupling was performed by using 15pg antibody to 600 million magnetic beads of size 1 micrometer in diameter. Now the SSA area was fixed to 3.49 mm ² (1 .1 Million beads) and the eluate from 10ng/ml TNFa was measured at different surface areas of SSB starting from 20000 5.6pm diameter beads (or 2.66 mm ² ) down to 312 beads (or 0.04 mm ² ). The results are shown in Figure 7. The lower the surface area of SSB the higher the signal. For a ratio of 20 fold SSA / SSB and above the signals reach saturation and should be used for detection.

Example 6

This examples shows the evaluation of the signal strength by adjusting sample volume. The surface area of SSA has been fixed to 87 mm ² (-27.7 Million beads) and the eluate has been measured at fixed surface area of SSB of 500 beads (or 0.07 mm ² ). Recombinant CSF3 at 100 ng/ml, 10 ng/ml, and 1 ng/ml concentration was measured. During the first incubation step of the sample with the SSA, two volumes were used (250pl and 20pl). The results are shown in Figure 8. Using 12.5 times more samples volume (250mI/20mI) an average 8.3 fold increase in signal was achieved. Since for the same sample concentration the signal depends strongly on the sample volume, the ambient analyte assumption does not apply. This is an extra advantage of the method of the present invention not observed in standard multiplex antibody-based assays where ambient analysis is assumed.

Example 7

This example shows the simultaneous measurement of 96 proteins in plasma using the method of the present invention. SSA contained 3 Million beads per antibody whereas SSB comprised 600 96plex Luminex beads (96 antibodies). The sample volume was 200mI and plasma was diluted in 1 :100 in PBS/BSA. The diluent was used as blank and measured. The first particle suspension system comprised 96 antibodies for the following proteins: MMP9 NPHS1 COL3A1 ANXA2 CFH MMP1 TGFbl CXCL12/SDF1 CCL19/MIP3b IL20 IL33 CTGF IL6 IL8 CCL1 1 TNF10 TNR1 1 NRG1 PDGFB IL1A IL1 B CXCL10 CXCL1 1 FGF2 ICAM1 IFNG KIM1 KIM1 TFF3 PEDF GROA ALDOA ANXA5 ANXA1 HNRNPA1 AZGP1 C3 AMBP TTR KNG1 ISG15 RRM1 IFIT3 KRT18 CD81 CASP1 CD8A CD14 DDX3X ANXA2 ANXA2 monoclonal TRIM28 CCR1 TGM2 CCL2 SLA MUC1 PLK1 SMAD3 CDK5 ID2 TXNRD1 PRDX1 KRT8 GZMB NPHS1 SNCA CD46 ELAVL1 AURKB MMP1 CSNK2B PRKRA FYN TLR6 EEF2 CFB DNMT3A TIMP3 CASP8 PLAUR CD2 CASP3 PIN1 CXCR4 SNAI 1 TNFRSF10D TACR1 ARPC3 TYR CHEK2 MMP7 CTGF TNF15 IL21. The following steps were followed a) generating suspension system A comprising 3 Million microparticles of 1 pm in diameter coupled to antibodies. For coupling we used 15pg antibody to 600 million magnetic beads of size 1 micrometer in diameter b) generating suspension system B comprising 600 magnetic microparticles 6.5 pm in diameter. Coupling was done following standard COOH protocols found in Luminex CookBook version 3; c) exposing 1 :100 plasma sample to suspension system A which is capable of immunoprecipitating the bound target proteins to said 96 antibodies; d) washing suspension system A to remove unbound proteins; e) On-particle-labeling the bound protein with biotin using standard protocols from Pierce; f) washing suspension system A to remove labeling reagents; g) eluting labeled proteins from suspension system A at low pH and neutralizing; h) transfer labeled proteins to suspension system B; i) use multiplex suspension system B for multiplex detection. The results are shown in Figure 9. and they illustrate that the method can be used for the simultaneous detection of 96 analytes in a biological fluid.