New product and method

Abstract

The present invention relates to a product comprising a reagent composition, the reagent composition comprising a plurality of detection probes suitable for detection of a set of analytes of interest, each detection probe comprising a nucleic acid moiety, the nucleic acid moiety of a detection probe being capable of generating a reporter nucleic acid molecule comprising an analyte identification sequence identifying the respective analyte of interest, wherein the product further comprises a nucleic acid molecule comprising a product identification sequence identifying the product. The invention also relates to a method for identifying a product used in obtaining detection data for a set of analytes of interest in a sample.

Description

[0001] New product and method

[0002] Field of the invention

[0003] The present invention relates to tools for biotechnological research, and in particular products and methods for obtaining detection data for a set of analytes of interest in a sample

[0004] Background

[0005] Modern proteomics methods require the ability to detect a large number of different proteins (or protein complexes) in a small sample volume. To achieve this, multiplex analysis must be performed. Common methods by which multiplex detection of proteins in a sample may be achieved include proximity extension assays (PEA) and proximity ligation assays (PLA). PEA and PLA are described in US 7,306,904; multiplexed PLA is described in Lundberg et al., Molecular & Cellular Proteomics 2011 , 10, DOI 10.1074 / mcp.M110.004978; aspects of PEA is further described in US 8,013,134, US 8,580,504, US 9,902,993, US 2023 / 0159983, US2023 / 032342, US2022 / 0162589; Assarsson et al., PLoS 1 , 2014, 9(4), e95192; Lundberg et al., Nucleic Acids Research, 2011 , Vol. 39, No. 15 e102; and Wik et al., 2021 , Mol Cell Proteomics 20, 100168, all incorporated herein by reference in their entirety.

[0006] PEA and PLA are proximity assays, which rely on the principle of “proximity probing”. In these methods, an analyte is detected by the binding of multiple (i.e. two or more, generally two or three) probes, which when brought into proximity by binding to the analyte (hence "proximity probes") allow a signal to be generated. Typically, at least one of the proximity probes comprises a nucleic acid domain (or moiety) linked to the analyte-binding domain (or moiety) of the probe, and generation of the signal involves an interaction between the nucleic acid moieties and / or a further functional moiety which is carried by the other probe(s). Thus, signal generation is dependent on an interaction between the probes (more particularly between the nucleic acid or other functional moieties / domains carried by them) and hence only occurs when the necessary probes have bound to the analyte, thereby lending improved specificity to the detection system.

[0007] In PEA, nucleic acid moieties linked to the analyte-binding domains of a probe pair hybridise to one another when the probes are in close proximity (i.e. when bound to the same target molecule, or to target molecules which are in close proximity, for example in a complex, interaction, or aggregate, or when two molecules are closely co-located), and are then extended using a nucleic acid polymerase. The extension product forms a reporter nucleic acid, detection of which demonstrates the presence of a particular analyte (the analyte bound by the relevant probe pair) in a sample of interest.

[0008] In PLA, nucleic acid moieties linked to the analyte-binding domains of a probe pair come into proximity when the probes of the probe pair bind their target, and may be ligated together, or alternatively they may together template the ligation of separately added oligonucleotides which are able to hybridise to the nucleic acid domains when they are in proximity. The ligation product is then amplified, acting as a reporter nucleic acid. Multiplex analyte detection using PEA or PLA may be achieved by including a unique barcode sequence in the nucleic acid moiety of each probe. A reporter nucleic acid molecule corresponding to a particular analyte may be identified by the barcode sequences it contains. The methods of the present invention find particular utility in multiplex PEA and PLA methods.

[0009] Panels of proximity assays, as described above, are commercially available from Olink Proteomics AB (Uppsala, Sweden) under the product lines Olink® Target, Olink® Focus, Olink® Flex, Olink® Explore, and Olink® Explore HT. Some of these are fixed panels of from 48 up to 5,400 pre-determined assays (Olink® Target, Olink® Explore and Olink® Explore HT). Others are custom developed (Olink® Focus and Olink® Flex)and may currently contain from 5 to 30 assays, wherein the specific assays are included according to customer specifications to address specific research questions or research needs. Each panel generally includes assays for proteins that have known functions within certain biological or physiological areas, pathways or organs in the body, such as inflammation, organ-specific proteins, cardiovascular, neurology etc.

[0010] Other proteomics methods relying on the use of one or more analyte specific binding moiety, such as antibodies or aptamers, and a nucleic acid moiety carrying an analyte specific identification sequence include nELISA from Nomic Bio, described i.a. in WO2021181161A1 , Successive Proximity Extension Assay Reaction (SPEAR) by Spear Bio, described i.a. in WO2017205719A1 , Mercy Halo by Mercy Bioanalytics, described i.a. in W02020180741 A1 , NULISA by Alamar Biosciences, described i.a. in W02021113290A1 , and SomaScan by Standard Biotools and Illumina, described i.a. in W02023130049A1.

[0011] WO1 987006383 suggests the use of a nucleic acid as a label for an item or substance, e.g. for authenticating valuable goods such as works of art or high fashion clothes, or for tracing goods along distribution lines. Summary

[0012] The present inventors have realized that it is a complex challenge in the prior art as described above to accurately and clearly identify what product or panel has been used to obtain a certain proteomics data set based on the data present in the data set itself. This may rely on information manually entered by a user or operator, which may be subject to human error. Such identifying information may also be stored linked but separate from the obtained data set, and may be inadvertently fully separated from the original data.

[0013] The present inventors have solved this problem by incorporating an oligonucleotide with a predetermined nucleotide sequence (a Product Identification Sequence, or PIS) in the reagents used to run the assay. This Product Identification Sequence Oligonucleotide (“PISO”) is amplified together with the reporter nucleic acid molecules generated in the detection assay and subsequently detected together with the reporter molecules. The presence of an oligonucleotide with a particular PIS in the original reagents will thus be intrinsic to, and visible in, the generated data output and serves to identify the specific product.

[0014] Thus, the present invention in a first aspect relates to a kit of parts comprising at least one reagent composition, the at least one reagent composition comprising a plurality of detection probes suitable for detection of a set of analytes of interest, each detection probe comprising a nucleic acid moiety, the nucleic acid moiety of a detection probe being capable of generating a reporter nucleic acid molecule comprising an analyte identification sequence identifying the respective analyte of interest, wherein the kit further comprises a nucleic acid molecule comprising a product identification sequence identifying the kit.

[0015] In one embodiment, each detection probe comprises an analyte-specific binding domain bound to the nucleic acid moiety.

[0016] In one embodiment, each detection probe comprises a matched set of proximity probes, each proximity probe comprising an analyte-specific binding domain and a nucleic acid moiety, the analyte-specific binding domains of each matched set of proximity probes being capable of binding specifically to the same analyte of interest, and the nucleic acid moieties of a matched set of proximity probes being capable of together forming a reporter nucleic acid molecule comprising an identification sequence identifying the respective analyte of interest. In one embodiment, the nucleic acid molecule comprising the product identification sequence (PISO) is a double stranded DNA molecule.

[0017] In one embodiment, the nucleic acid molecule comprising the product identification sequence (PISO) is included in a reagent composition comprising detection probes. In one embodiment, the PISO is included in a reagent composition comprising detection probes in the form of matched sets of proximity probes. In one embodiment, the PISO is included in a dried reagent composition comprising detection probes in the form of matched sets of proximity probes. In one embodiment, the dried reagent composition is provided in a reaction container comprised in the kit of parts.

[0018] In one embodiment, the PISO is included in a composition not comprising any detection probes. In one embodiment, the PISO is included in a buffered aqueous solution not comprising any detection probes.

[0019] In a second aspect, the present invention relates to a method for identifying a kit of parts used in obtaining detection data for a set of analytes of interest in a sample, wherein the detection data has been obtained through use of a kit of parts adapted for performing a method comprising the steps:

[0020] 51) contacting the sample with a plurality of detection probes suitable for detection of a set of analytes of interest, each detection probe comprising a nucleic acid moiety, the nucleic acid moiety of a detection probe being capable of generating a reporter nucleic acid molecule comprising an analyte identification sequence identifying the respective analyte of interest, and a nucleic acid molecule comprising a product identification sequence identifying the product; and

[0021] 52) generating the reporter nucleic acid molecules; and

[0022] 53) detecting the, optionally amplified, reporter nucleic acid molecules and the nucleic acid molecule comprising the product identification sequence; wherein the kit is identified by the detected product identification sequence.

[0023] In a preferred embodiment, step S2 of the method that the kit of parts is adapted to perform further comprises amplifying any generated reporter nucleic acid molecules and the nucleic acid molecule comprising the product identification sequence. In this embodiment, the detection step (S3) comprises quantifying the amount of amplified nucleic acid molecules comprising the respective identification sequences.

[0024] In one embodiment, the kit of parts used for obtaining the detection data is a kit of parts according to the first aspect.

[0025] Definition of terms and abbreviations

[0026] All terms and abbreviations used in the present specification shall be construed to have the meaning normally given to them in the relevant art, unless another meaning is clearly intended. For the sake of clarity, a few terms and abbreviations are defined below.

[0027] A “kit of parts”, or simply “kit”, is a product. In particular, it is a product comprising multiple parts combined and arranged to be provided as a single entity to a user. The parts of a kit, in the present technological field, are generally one or more reagent compositions comprised in vials or other containers, optionally reaction plates, optionally instructions for use or internet hyperlinks to such instructions, and optionally a box in which the parts are packaged.

[0028] “PISO” is an abbreviation of Product Identification Sequence Oligonucleotide and refers to a nucleic acid molecule comprising a product identification sequence.

[0029] In some embodiments, the present invention makes use of “matched” proximity probes. As generally described herein, proximity probes comprise an analyte-binding domain and a nucleic acid moiety. That two (or more) proximity probes are “matched” indicate that they are capable of simultaneous binding to different parts of the same analyte of interest and that, when the matched proximity probes are bound to the analyte of interest, their respective nucleic acid moieties are capable of interacting, directly (e.g, by hybridization to each other) or indirectly (e.g. by hybridization to a common splint oligonucleotide), to yield, form or otherwise generate a reporter nucleic acid.

[0030] “Readout” is intended to refer to the process of quantifying the amount of reporter molecules with the respective unique identification sequences and correlating these amounts to the amounts of the respective analytes of interest in the analyzed sample. Accordingly, a readout can be seen as a step of detecting the signal in the assay, or more particularly the reporter molecules, in a quantitative manner.

[0031] Multiplexing of biological assays, such as proximity assays, means performing a plurality of assays in parallel and, preferably, in the same reaction container, e.g. a test tube or a well in a microtiter plate. Multiplexing thus has the potential to massively increase throughput of samples and reduce foot-print of the necessary equipment.

[0032] The terms “a” and “an” are defined as one or more unless explicitly stated otherwise herein.

[0033] The present invention makes use of “identification sequences”. An identification sequence may e.g. be a unique sequence (usually termed a “barcode sequence” or simply “barcode”) that is detected in a sequence-specific manner, for example, sequenced for identification in the readout step, or which provides a specific binding (hybridization) site for a probe or primer used in the detection, e.g., a unique primer binding site that can be used for readout using quantitative PCR (qPCR).

[0034] Detailed description

[0035] The present invention relates to a product comprising a reagent composition, the reagent composition comprising a plurality of detection probes suitable for detection of a set of analytes of interest, each detection probe comprising a nucleic acid moiety, the nucleic acid moiety of a detection probe being capable of generating a reporter nucleic acid molecule comprising an analyte identification sequence identifying the respective analyte of interest, wherein the product further comprises a nucleic acid molecule comprising a product identification sequence (“PISO”) identifying the product.

[0036] As the product comprises at least two components, the reagent composition and the PISO, the product can be viewed as a kit of parts as defined above.

[0037] The product identification sequence shall serve to identify the product, i.e. kit, at some level. This is achieved by the product identification sequence being specific to the product. That is, all manufactured units of a certain product, or kit, comprise nucleic acid molecules with the same product identification sequence.

[0038] The number of available unique identification sequences using all four standard nucleotides (A, T / U, G, and C) of length X is 4X. Using identification sequences of e.g. 5-10 nucleotides thus provides a large number of potential identification sequences that can be used both as analyte identification sequences and product identification sequences. This provides the opportunity to assign product identification sequences to a large range of product information, such as product category, product line, product version, manufacturing site, manufacturing lot number, etc. If the product is custom made, the product identification sequence may identify the entity for which the product is produced or developed. In one embodiment, the PISO is a double stranded DNA molecule. In one embodiment, the PISO is a double-stranded DNA molecule having the same general structure as a reporter nucleic acid molecule generated in the detection assay. That is to say, the PISO comprises a barcode sequence which identifies it as a PISO and links it to the product used in the detection assay, and also common primer binding sites, shared with all other reporter nucleic acids generated in response to analyte detection, to enable binding of the primers used in any amplification reaction(s). The PISO may or may not include sequencing adapters and / or a sample index as known in the prior art and explained further below - these are preferably added to the PISO molecule at the same time as they are added to the reporter nucleic acid molecules generated in response to analyte detection, as is known in the art (e.g. in PCR amplification). Generally, the PISO has a length in the range of 20-100 nucleotides, but may be shorter or longer as required in the specific detection assay in which the product specific nucleic acid molecule is intended to be used.

[0039] In one embodiment, each detection probe comprises an analyte-specific binding domain bound to the nucleic acid moiety.

[0040] As is known in the art, the analyte-specific binding domain of a detection probe may be any entity capable of binding specifically to a target analyte (or part thereof), and being coupled to a nucleic acid moiety. That the binding domain is “specific” to a certain analyte means, as is known to the skilled person, that it recognizes the analyte with low cross-reactivity (off- target binding) with other potentially present analytes, within the relevant application and experimental context. A framework for determining specificity for binders have been established by an International Working Group for Antibody Validation (llhlen et al. Nat Methods, 2016 Oct; 13(10), 823-827, incorporated herein by reference).

[0041] Typically, the analyte-specific binding domain may be a protein, for example, an antibody, or an antigen-binding part thereof, including, but not limited to, monoclonal, recombinant monoclonal, and polyclonal antibodies and antigen-binding antibody derivatives and fragments. However, the analyte-specific binding domain may be of any nature, including lectins, soluble cell surface receptors, combinatorially derived proteins from phage display or ribosome display, peptides, carbohydrates, molecularly imprinted polymers (MIPs), nucleic acids, such as an aptamer or a nucleic acid molecule comprising the complementary sequence for a target nucleic acid, or combinations thereof.

[0042] Reagents useful as analyte-specific binding domains are commercially available from a number of manufacturers that offer off-the-shelf reagents or develop new binding reagents for specific analytes and specific needs. Such manufacturers presently include, among others, Thermo Fisher Scientific (Boston, MA, USA), Abeam (Cambridge, United Kingdom), Bio-Techne (Minneapolis, MN, USA), Proteogenix (Schiltigheim, France), Sino Biological (Beijing, China), Agrisera (Vannas, Sweden), Novaptech (Pessac, France), Aptamer Group (York, United Kingdom). Reagents useful as analyte-specific binding domains may also be developed independently of commercial suppliers, according to protocols well-known to the skilled person. Such protocols are e.g. described in “Monoclonal Antibody Production” (National Academy Press, Washington, DC, USA, 1999), Carey-Hanly et al. (ILAR Journal, Volume 37, Issue 3, 1995, Pages 93-118), llgu and Nilsen-Hamilton (Analyst. 2016 March 7; 141(5): 1551-1568). Reagents may also comprise antibody derivates or fragments, such as Fab, Fab', F(ab')2, Fv fragments; diabodies; single-domain antibodies (sdAb, Desmyter et al. (1996) Nat. Structure Biol. 3:803-811), nanobodies, single-chain Fv (scFv, Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883), divalent scFV (di-scFvs), tandem scFvs, triabodies, diabodies, single-chain diabodies (scDb), bi-specific T-cell engagers (BiTEs, Kufer et al. (2004) Trends Biotechnol. 22:238-244), and Dual Affinity Retargeting molecules (DARTs, diabodies additionally stabilized through a C-terminal disulfide bridge). The specificity of analyte-specific reagents with regard to the intended detection assay may be evaluated using the framework proposed by the International Working Group for Antibody Validation, cited above.

[0043] Further, the analyte-specific binding domain may bind to the analyte directly or indirectly. In other words, the detection probe may be a primary reagent which binds directly to the analyte, or a secondary reagent which binds indirectly, by virtue of binding to an intermediate molecule (a primary reagent) which is itself bound directly to the analyte.

[0044] In addition to the analyte-specific binding domain, a detection probe as used in the present invention also comprises a nucleic acid moiety, also referred to herein as an oligonucleotide. The oligonucleotide must be long enough to comprise the necessary functional elements used in the detection assay for which the detection probe is intended to be used. That is, at least a sequence capable of generating an identification sequence in the reporter molecule. This is typically 5-20 nucleotides, such as 5-10, 5-15, 10-15 or 15-20 nucleotides. The oligonucleotide may also contain sequences related to primer sites and / or sequencing adaptors for read-out, as known in the art. Generally, the oligonucleotide has a length in the range of 20-100 nucleotides, but may be shorter or longer as required in the specific detection assay in which the detection probe is intended to be used. Conjugation of a nucleic acid moiety to an antibody can be performed in several ways known to the skilled person, e.g. as reviewed by Dugal-Tessier et al. (J. Clin. Med.2021 , 10, 838). Commercial kits for preparing antibody-oligonucleotide conjugates are also readily available from a number of suppliers. The oligonucleotides may be coupled to the analyte binding domains by any means known in the art, and which may be desired or convenient and may be direct, or indirect, e.g. via a linking group. For example, the domains may be associated with one another by covalent linkage (e.g. chemical cross- linking) or by non-covalent association e.g. via streptavidin-biotin based coupling (biotin being provided on one domain, particularly the oligonucleotide domain, and streptavidin on the other).

[0045] The oligonucleotide and analyte binding domain are joined together either directly through a bond or indirectly through a linking group. Where linking groups are employed, such groups may be chosen to provide for covalent attachment of the nucleic acid moiety and analyte binding domain through the linking group. The linking group, when present, is in many embodiments biologically inert. In representative embodiments, the linking group is generally at least about 50 Daltons, usually at least about 100 Daltons and may be as large as 1000 Daltons or larger, for example, up to 1000000 Daltons if the linking group contains a spacer, but generally will not exceed about 500 Daltons and usually will not exceed about 300 Daltons. Generally, such linkers will comprise a spacer group terminated at either end with a reactive functionality capable of covalently bonding to the nucleic acid domain or analyte binding domain. Spacer groups of interest may include aliphatic and unsaturated hydrocarbon chains, spacers containing heteroatoms such as oxygen (ethers such as polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems that may possibly contain heteroatoms. Spacer groups may also be comprised of ligands that bind to metals such that the presence of a metal ion coordinates two or more ligands to form a complex. Specific spacer elements include: 1 ,4-diaminohexane, xylylenediamine, terephthalic acid, 3,6-dioxaoctanedioic acid, ethylenediamine-N,N- diacetic acid, 1 ,1 '-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid), 4,4'- ethylenedipiperidine.

[0046] Potential reactive functionalities include nucleophilic functional groups (amines, alcohols, thiols, hydrazides), electrophilic functional groups (aldehydes, esters, vinyl ketones, epoxides, isocyanates, maleimides), functional groups capable of cycloaddition reactions, forming disulfide bonds, or binding to metals. Specific examples include primary and secondary amines, hydroxamic acids, N- hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters, glycidyl ethers, vinylsulfones, and maleimides. Specific linker groups that may find use in the subject proximity probes include heterofunctional compounds, such as azidobenzoyl hydrazide, N-[4-(p- azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide, bis-sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N- maleimidobutyryloxysuccinimide ester, N- hydroxy sulfosuccinimidyl-4- azidobenzoate, N-succinimidyl [4-azidophenyl]-1 ,3'- dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl-4-[N- maleimidomethyl]cyclohexane-1 -carboxylate, 3-(2-pyridyldithio)propionic acid N- hydroxysuccinimide ester (SPDP), 4-(Nmaleimidomethyl)-cyclohexane-1 -carboxylic acid N-hydroxysuccinimide ester (SMCC), and the like.

[0047] The nucleic acid domain of the detection probes may be made up of ribonucleotides and / or deoxyribonucleotides as well as synthetic nucleotide residues that are capable of participating in Watson-Crick type or analogous base pair interactions. Thus, the nucleic acid domain may be DNA or RNA or a combination or any modification thereof e.g. PNA or other derivatives containing non-nucleotide backbones.

[0048] In an embodiment, the analyte-specific binding domain is not a nucleic acid which binds to the analyte by hybridization. In another embodiment, the analyte-specific binding domain is not a nucleic acid. In another embodiment, the detection probe is not composed wholly of nucleic acid. In another embodiment, the detection probe is not a gene-specific probe. In another embodiment, the detection probe is not a padlock probe. In another embodiment, the detection probes are not for use in a microscopy-based optical detection method.

[0049] In one embodiment, the detection probes are manufactured by coupling a universal oligonucleotide to the analyte-specific binding domain and subsequently hybridizing a tag oligonucleotide to the universal oligonucleotide, wherein the tag oligonucleotide comprises a sequence capable of generating the identification sequence in a reporter molecule, a sequence complementary to the universal oligonucleotide to facilitate hybridization, and any other functional sequences necessary to perform the detection assay for which the detection probes are intended. Methods for manufacturing such detection probes are described i.a. in U.S. patent 10,781 ,473 .

[0050] In one embodiment, each detection probe comprises a matched set of proximity probes, each proximity probe comprising an analyte-specific binding domain and a nucleic acid moiety, the analyte-specific binding domains of each matched set of proximity probes being capable of binding specifically to the same analyte of interest, and the nucleic acid moieties of a matched set of proximity probes being capable of together forming a reporter nucleic acid molecule comprising an identification sequence identifying the respective analyte of interest. In the most common versions of proximity assays, the matched set comprises two proximity probes wherein the nucleic acid moieties are capable of forming the reporter molecule through hybridization and subsequent extension (PEA) or hybridization to a splint oligonucleotide and subsequent ligation (PLA). However, variant proximity assays using a set of three matched proximity probes (3PLA) have been described, i.a. in US patent 8,268,554. In this assay, the splint oligonucleotide of PLA is provided as the nucleic acid moiety of a third proximity proximity probe comprised in the matched set. Further, versions using four proximity probes (4PLA) have been described by Masood Kamali-Moghaddam and co-workers (Tavoosidana et al., Proc. Natl. Acad. Sci. U.S.A. 108 (21) 8809-8814).

[0051] Where proximity probes are used as the detection probes, each nucleic acid moiety of a proximity probe of the matched set may comprise an identification sequence, or a partial identification sequence. The reporter molecule which is generated may comprise an identification sequence from any or each of the matched proximity probes. In other words, the identification sequence of the reporter molecule may be a combination, or composite, of the identification sequences of the individual nucleic acid moieties of matched proximity probes. The identification sequences of individual matched proximity probes (i.e. members of the same set) may be the same or different. In an embodiment, each identification sequence in matched proximity probes is indicative of, or corresponds to, the analyte of interest. However, it is not required for a particular identification sequence of an individual proximity probe to be indicative of an analyte of interest - it is the identification sequence of the reporter nucleic acid molecule which is indicative of the analyte of interest. As indicated above, the reporter identification sequence may be a combination or composite.

[0052] Alternatively, the identification sequence of the reporter nucleic acid molecule may be derived from the nucleic acid moiety of a single proximity probe (although it will be understood that interaction of the nucleic acid moieties of matched proximity probes will be required for the reporter nucleic acid molecule to form).

[0053] The PISO may be a separate component which is, or comprises, or leads to the generation of, a product identification nucleic acid molecule which is amplified by the same primers as the reporter nucleic acid molecules. It may be present in a pre-determined amount in the product.

[0054] In one embodiment, the PISO is included in the reagent composition also comprising the detection probes. In one embodiment, the PISO is included in a dried reagent composition also comprising the detection probes. In this embodiment, the kit according to the invention may include a device comprising at least one reaction space with a single pre-dispensed dried reagent composition suitable for performing an assay method for detecting at least one analyte in a sample to be added to the reaction space, wherein the reagent composition comprises at least one analyte-specific proximity probe set, at least one PISO, and a drying stabiliser, and wherein the proximity probes in each set comprise a binding domain for binding directly or indirectly to the analyte, and a nucleic acid domain. Such devices, without a PISO, are described in further detail in PCT / EP2024 / 069721 , incorporated by reference herein in its entirety.

[0055] The stabiliser acts to protect the probes and reagents from damage during the drying process.

[0056] Accordingly, the device comprises one or more reaction spaces wherein each reaction space comprises a dried reagent composition disposed thereon or therein (depending on the configuration of the reaction space, the reagent composition may be contained within it, or positioned on it).

[0057] Thus, the reaction space may be an area (i.e. a location or position on a surface) or a reaction container. Preferably, the device is for use in multiplex detection assays. This may involve multiple different proximity probes sets being disposed at the same site, or at different sites. Accordingly, in an embodiment, the device comprises multiple separate reaction spaces, e.g. for different samples. In a further embodiment, the device is a multiwell plate, an array or a microfluidic device.

[0058] In certain embodiments, the multiple separate reaction spaces are not in liquid communication with one another.

[0059] In an embodiment, the, or each, reaction space is configured to receive a volume, e.g. a sample volume, of no more than 10 pl. In other embodiments, larger volumes may be used, e.g. up to 100 pl.

[0060] In an embodiment, the drying stabiliser comprises one or more sugars or sugar alcohols, e.g. trehalose, glucose, sucrose and / or mannitol, and optionally a surfactant, e.g. a detergent such as Tween. The reagent composition is typically prepared in a buffer and the buffer composition is dispensed into or onto the reaction space(s) of the device and dried down. The reagent composition thus typically comprises a buffer. The buffer composition that is prepared for drying may contain further reagents for the probe binding step. It may further comprise one or more further reagents for the detection assay method, e.g. for generation of a reporter nucleic acid molecule, for introduction of a desired sequence element into a reporter molecule (e.g. a barcode or other detection sequence, or a binding site for a primer etc.), for amplification, and / or for detection of a reporter nucleic acid molecule.

[0061] In an embodiment, the reagent composition further comprises a sample index oligonucleotide comprising an ID sequence, for example for identifying a sample, e.g. an ID sequence which is unique to the reaction space or a group of reaction spaces. The sample ID oligonucleotide is used to incorporate the ID sequence into the reporter nucleic acid molecule. For example, the sample index oligonucleotide is a primer, a ligation adaptor, or a ligation and / or extension template.

[0062] In an embodiment, the reagent composition further comprises one or more of the following:

[0063] (i) one or more enzymes, e.g. a DNA polymerase and / or a ligase;

[0064] (ii) dNTPs;

[0065] (iii) one or more primers;

[0066] (iv) a ligation template;

[0067] (v) buffers;

[0068] (vi) salts; and / or

[0069] (vii) blocking agents.

[0070] In another embodiment, the PISO and the detection probes are provided in separate compositions, i.e. the PISO is included in a composition not comprising any detection probes. In one embodiment, the PISO is included in a buffered aqueous solution not comprising any detection probes. In one embodiment, the PISO composition and the composition comprising the detection probes are provided in separate dried compositions, wherein the dried compositions and devices comprising such are substantially as described above.

[0071] In one aspect, the present invention relates to a method for identifying a kit of parts used in obtaining detection data for a set of analytes of interest in a sample, wherein the detection data has been obtained through use of a kit of parts adapted for performing a method comprising the steps:

[0072] 51) contacting the sample with a plurality of detection probes suitable for detection of a set of analytes of interest, each detection probe comprising a nucleic acid moiety, the nucleic acid moiety of a detection probe being capable of generating a reporter nucleic acid molecule comprising an analyte identification sequence identifying the respective analyte of interest, and a nucleic acid molecule comprising a product identification sequence (“PISO”) identifying the product; and

[0073] 52) generating the reporter nucleic acid molecules; and

[0074] 53) detecting any generated, optionally amplified, reporter nucleic acid molecules and nucleic acid molecules comprising the product identification sequence; wherein the kit of parts is identified by the detected product identification sequence.

[0075] In a preferred embodiment, step S2 of the method that the kit of parts is adapted to perform further comprises amplifying any generated reporter nucleic acid molecules and the PISO.

[0076] The method used for obtaining detection data may, as discussed above, be a proximitybased assay or a dual-recognition immunoassay. Proximity-based assays include PLA and PEA as mentioned above. The analytes are thus detected by detecting a reporter nucleic acid molecule generated specifically for each analyte and the product used for generating the reporter nucleic acid molecules is identified by detection of the PISO.

[0077] In one embodiment, each detection probe comprises an analyte-specific binding domain bound to the nucleic acid moiety.

[0078] In one embodiment, each detection probe comprises a matched set of proximity probes, each proximity probe comprising an analyte-specific binding domain and a nucleic acid moiety, the analyte-specific binding domains of each matched set of proximity probes being capable of binding specifically to the same analyte of interest, and the nucleic acid moieties of a matched set of proximity probes being capable of together forming a reporter nucleic acid molecule comprising an identification sequence identifying the respective analyte of interest. A matched set of proximity probes may contain two, three, four or more matched proximity probes. In one embodiment, the nucleic acid molecule comprising the product identification sequence (PISO) is a double stranded DNA molecule.

[0079] In one embodiment, the PISO is a double-stranded DNA molecule having the same general structure as a reporter nucleic acid molecule generated in the detection assay. That is to say, the PISO comprises a barcode sequence which identifies it as a PISO and also identifies the product in which it is present, and also common primer binding sites, shared with all other reporter nucleic acids generated in response to analyte detection, to enable binding of the primers used in any amplification reaction(s). The PISO may or may not include sequencing adapters - these are preferably added to the control DNA molecule at the same time as they are added to the reporter nucleic acid molecules generated in response to analyte detection, as described herein.

[0080] In proximity based assays, as is known in the prior art (e.g. through WO2021191442), the presence of a particular analyte in the sample results in the production during the detection assay of a nucleic acid molecule with a particular nucleotide sequence, which is known to correspond to the particular analyte. Detection of the particular nucleotide sequence indicates that the analyte to which the sequence corresponds is present in the sample. A “reporter nucleic acid molecule” is thus a nucleic acid molecule whose synthesis during the detection assay indicates the presence in the sample of a particular analyte. The reporter nucleic acid molecule, and the PISO, may be an RNA molecule or a DNA molecule. Preferably it is a DNA molecule.

[0081] The reporter nucleic acid molecule may be generated by any means known in detection assays of the art. For instance, it may be generated by ligation of two (or more) nucleic acids to each other, forming a unique nucleotide sequence indicative of the presence of the analyte in the sample. Alternatively, the reporter nucleic acid molecule may be generated by extension of a provided nucleic acid molecule along a template nucleic acid molecule. Combinations of extensions and ligations may also be used.

[0082] Reporter nucleic acid molecules are thus generated during the multiplex detection assays performed on the sample. To generate a reporter nucleic acid molecule, any detection assay which acts by generation of such nucleic acid molecules may be used. In a particular embodiment, the reporter nucleic acid molecule is generated in the context of a proximity extension assay (PEA). That is to say, a multiplex PEA may be performed in order to detect the analytes in each aliquot, and thus in the sample. In another embodiment, the reporter nucleic acid molecule is generated in the context of a proximity ligation assay (PLA), i.e. a multiplex PLA may be performed in order to detect the analytes in each aliquot. Methods for performing PEAs and PLAs are known in the art, as described above. It is particularly preferred that the detection assay performed is a PEA.

[0083] Following generation of the reporter nucleic acid molecule, it is preferably amplified, together with the PISO, for ease of detection. Amplification of the reporter nucleic acid molecule and PISO is preferably performed by PCR, though any other method of nucleic acid amplification may be utilised, e.g. loop-mediated isothermal amplification (LAMP) or Recombinase Polymerase Amplification (RPA). If the reporter nucleic acid molecules are amplified, it is preferable that also the PISO is amplified in the same reaction, and preferably using the same primers, as applicable.

[0084] As noted above, each reporter nucleic acid molecule is specific for a particular analyte. Thus, a reporter nucleic acid molecule identifies a given analyte, or more particularly, may contain a sequence or domain which functions as an identification (ID) sequence, or tag, by which an analyte may be detected. Correspondingly, the PISO carries the product identification sequence which functions as an identification (ID) sequence, or tag, by which the reagent kit used in the detection may be detected. The ID sequence may be detected for example by serving as a binding site for probes or primers etc., as detailed further below, or more directly by sequencing.

[0085] The analytes in the sample are detected by detection of the specific barcode sequences within the reporter nucleic acid molecules generated during the multiplex detection assay. The reagent kit used in the detection assay is correspondingly identified by detection of the specific barcode sequence within the PISO. The PISO has the same generic composition as the reporter molecules and can thus be included in the read-out / detection of the reporter molecules as described below. This read-out may be achieved in a number of ways. Firstly, specific barcode sequences may be detected by sequencing of all the reporter nucleic acid molecules generated during the multiplex detection assay, including the PISO. By sequencing all reporter nucleic acid molecules generated, all the different reporter nucleic acid molecules generated may be identified by their barcode sequences, and thus all the analytes present in the sample may be identified (based on whether the reporter nucleic acid molecule known to correspond to each target analyte is detected or not). The reagent kit used in the detection assay is identified by correlating the Product Identification Sequence comprised in the PISO to the corresponding reagent kit associated with that specific Product Identification Sequence. Data normalization ensures that the measured changes in assay signal levels reflect actual changes in protein levels and not experimental artifacts. Successful normalization minimizes the variability and will generate more reproducible and precise data. To monitor, control and normalize key steps in the protocol, from immunoreaction to detection, three specifically engineered internal controls may be included in each incubation reaction, as described in Wik et al 2021. The incubation control (Inc Ctrl) is used as a quality control and comprises PEA probes measuring a fixed concentration of non-human green fluorescent protein (GFP). The Inc Ctrl is added in the immunoreaction step. The extension control (Ext Ctrl) is used for normalization of the data and is added at a fixed concentration in the immunoreaction step. It is composed of two paired oligonucleotides coupled to the same antibody molecule, thereby keeping the two oligonucleotides in constant proximity and allowing direct hybridization independent of antigen binding.

[0086] During PCR amplification, all extension products (including the Ext Ctrl) will be amplified at the same rate and the resulting number of amplicons will be relative to the starting concentration of the Ext Ctrl in all samples. In a sample with a high protein concentration, the resulting yield of amplicons will be high relative to the Ext Ctrl and vice versa; in a sample with low protein concentration, the resulting number of amplicons will be low relative to the Ext Ctrl. During data normalization, the number of amplicons from each assay will be normalized against the number of amplicons for the Ext Ctrl to enable comparison of protein levels between samples.

[0087] As is known in the art, different detection modalities for the readout are possible. These include sequencing. Thus, for example, an identification sequence may be a barcode which is sequenced. Any method of sequencing may be used, including sequencing-by-synthesis and sequencing-by-hybridization methods. Thus, depending on the nature of the ID sequence, any suitable method may be used to identify the ID sequence, and this may involve the use of hybridization probes and / or primers. For example, the detection (readout) step may involve amplifying the reporter nucleic acid using one or more primers, at least one of which binds to the ID sequence. Alternatively, the detection method may involve amplifying the reporter and detecting the amplicons by means of specific hybridization probes which bind to the ID sequence (or to a complement thereof). In sequencing-by- hybridization, barcodes can be decoded using labelled hybridization probes, including in combinatorial fashion. Sequencing advantageously allows a high level of multiplexing and is a convenient method of detection. As noted above, any form of sequencing may be used, including any method of sequencing-by-synthesis, for example, pyrosequencing, reversible dye terminator sequencing and ion torrent sequencing. Particularly, high throughput methods of sequencing are used, and especially massively parallel DNA sequencing. Massively parallel DNA sequencing using the reversible dye terminator method may be performed, for instance, using an Illumina® NovaSeq™ system.

[0088] A form of high throughput DNA sequencing is thus preferably used to detect the reporter nucleic acid molecules and PISO. Sequencing by synthesis is the preferred DNA sequencing method. Examples of sequencing by synthesis techniques include pyrosequencing, reversible dye terminator sequencing and ion torrent sequencing, any of which may be utilised in the present method. Preferably the reporter nucleic acids are sequenced using massively parallel DNA sequencing. Massively parallel DNA sequencing may in particular be applied to sequencing by synthesis (e.g. reversible dye terminator sequencing, pyrosequencing or ion torrent sequencing, as mentioned above). Massively parallel DNA sequencing using the reversible dye terminator method is a preferred sequencing method. Massively parallel DNA sequencing using the reversible dye terminator method may be performed, for instance, using an Illumina® NovaSeq™ system.

[0089] As is known in the art, massively parallel DNA sequencing is a technique in which multiple (e.g. thousands or millions or more) DNA strands are sequenced in parallel, i.e. at the same time. Massively parallel DNA sequencing requires target DNA molecules to be immobilised to a solid surface, e.g. to the surface of a flow cell or to a bead. Each immobilised DNA molecule is then individually sequenced. Generally, massively parallel DNA sequencing employing reversible dye terminator sequencing utilises a flow cell as the immobilisation surface, and massively parallel DNA sequencing employing pyrosequencing or ion torrent sequencing utilises a bead as the immobilisation surface.

[0090] As is known to the skilled person, immobilisation of DNA molecules to a surface in the context of massively parallel sequencing is generally achieved by the attachment of one or more sequencing adapters to the ends of the molecules. The method of the invention may thus include the addition of one or more adapters for sequencing (sequencing adapters) to the reporter nucleic acid molecules and the PISO.

[0091] Commonly, the sequencing adapters are nucleic acid molecules (in particular DNA molecules). In this instance, short oligonucleotides complementary to the adapter sequences are conjugated to the immobilisation surface (e.g. the surface of the bead or flow cell) to enable annealing of the target DNA molecules to the surface, via the adapter sequences. Alternatively, any other pair of binding partners may be used to conjugate the target DNA molecule to the immobilisation surface, e.g. biotin and avidin / streptavidin. In this case biotin may be used as the sequencing adapter, and avidin or streptavidin conjugated to the immobilisation surface to bind the biotin sequencing adapter, or vice versa.

[0092] Sequencing adapters may thus be short oligonucleotides (preferably DNA), generally 10-30 nucleotides long (e.g. 15-25 or 20-25 nucleotides long). As detailed above, the purpose of a sequencing adapter is to enable annealing of the target DNA molecules to an immobilisation surface, and accordingly the nucleotide sequence of a nucleic acid adaptor is determined by the sequence of its binding partner conjugated to the immobilisation surface. Aside from this, there is no particular constraint on the nucleotide sequence of a nucleic acid sequencing adaptor.

[0093] A sequencing adapter may be added to a reporter nucleic acid molecule of the invention during PCR amplification. In the case of a nucleic acid sequencing adapter this can be achieved by including a sequencing adapter nucleotide within in one or both primers. Alternatively, if the sequencing adaptor is a non-nucleic acid sequencing adaptor (e.g. a protein / peptide or small molecule) an adapter may be conjugated to one or both PCR primers. Alternatively, a sequencing adapter may be attached to a reporter nucleic acid molecule / PISO by directly ligating or conjugating the sequencing adapter to the reporter nucleic acid molecule / PISO. Preferably the one or more sequencing adapters used in the present method are nucleic acid sequencing adapters.

[0094] One or more nucleic acid sequencing adapters may be added to the reporter molecule / PISO in one or more ligation and / or amplification steps. Thus if, for instance, two sequencing adapters are added to the reporter nucleic acid molecule / PISO (one at each end), these may be added in a single step (e.g. by PCR amplification using a pair of primers which both contain a sequencing adapter) or in two steps. The two steps may be performed using the same or different methods, e.g. a first sequencing adapter may be added to the reporter nucleic acid molecule / PISO by ligation and the second by PCR amplification, or vice versa; or a first amplification reaction may be performed to add a first sequencing adapter to the reporter nucleic acid molecule / PISO, followed by a second amplification reaction to add a second sequencing adapter to the reporter nucleic acid molecule / PISO. As noted above, one or more sequencing adapters may be added to the reporter nucleic acid molecule / PISO. By this is meant one or two sequencing adapters - since sequencing adapters are added to the ends of a DNA molecule, the maximum number of sequencing adapters which can be added to a single DNA molecule (e.g. reporter nucleic acid) is two. Thus a single sequencing adapter may be added to one end of a reporter nucleic acid molecule, or two sequencing adapters may be added to a reporter nucleic acid molecule, one to each end. In a particular embodiment the Illumina P5 and P7 adapters are used, i.e. the P5 adapter is added to one end of the reporter nucleic acid molecule / PISO and the P7 adapter is added to the other end.

[0095] Thus in a particular embodiment of the invention, the reporter nucleic acid molecules / PISO are subjected to at least a first (i.e. at least one) PCR amplification, in order to add at least a first (i.e. to add at least one) sequencing adapter to the reporter nucleic acid molecule / PISO. As noted above, a reporter nucleic acid molecule is produced during the detection reaction, in response to the presence of the target analyte to which the reporter nucleic acid molecule corresponds (i.e. the analyte whose presence is indicated by generation of the reporter nucleic acid molecule). As further noted above, the reporter nucleic acid molecule / PISO is preferably amplified in order to enable or improve its detection.

[0096] This amplification may thus be combined with addition of one or more sequencing adapters to the reporter nucleic acid molecule / PISO. This may be achieved by amplification of the reporter nucleic acid molecule / PISO using a primer pair comprising at least one sequencing adapter. In this instance, at least one primer in the primer pair comprises a sequencing adapter upstream of the sequence which binds the reporter nucleic acid molecule / PISO. Thus the sequencing adapter is generally located at the 5’ end of any primer within which it is contained.

[0097] In a particular embodiment, an amplification step is performed using a primer pair comprising one primer which includes a sequencing adapter, such that a single sequencing adapter is added to one end of the reporter nucleic acid molecule / PISO.

[0098] In another embodiment, an amplification step is performed using a primer pair in which both primers comprise a sequencing adapter, such that a sequencing adapter is added to each end of the reporter nucleic acid molecule / PISO in a single amplification step. In another embodiment, two separate amplification reactions are performed to add a sequencing adaptor to each end of the reporter nucleic acid molecule / PISO, wherein each amplification step adds a different sequencing adaptor to a different end of the molecule.

[0099] In another embodiment, an initial amplification step is performed using primers which do not comprise sequencing adapters. The amplified reporter nucleic acid molecules are then subjected to one or more further amplification reactions to add sequencing adapters to each end of the molecule, as described above.

[0100] As detailed above, each reporter nucleic acid molecule generated during the detection assay may comprise a barcode sequence which corresponds to a particular analyte. Thus reporter nucleic acid molecules with different sequences are generated in response to the presence of different analytes in the sample. Nonetheless, for ease of multiplexing it is preferred that all reporter nucleic acid molecules generated in the detection assay, and the PISO, share common primer binding sites, such that the same primer pair can be used for amplification of all different reporter nucleic acid molecules and the PISO.

[0101] If the nucleic acid molecules is subjected to a first PCR amplification in which only a single sequencing adapter is added to the molecule, the amplified nucleic acid molecule (that is to say, the product of the first PCR amplification) may be subjected to a second PCR amplification to add a second sequencing adapter. Thus in this embodiment a first PCR amplification is performed using a primer pair in which one primer comprises a sequencing adapter, thus adding a first sequencing adapter to one end of the nucleic acid molecule. The second PCR amplification is then performed using a different primer pair. The second primer pair comprises one primer which comprises a second sequencing adapter. The second sequencing adapter is different to the first sequencing adapter, i.e. it has a different sequence. The primer comprising the second sequencing adapter binds the nucleic acid molecule at the opposite end to the end comprising the first sequencing adapter, such that the second sequencing adapter is added to the nucleic acid molecule at the opposite end to the first sequencing adapter.

[0102] As necessary for amplification of the product of the first PCR amplification, the second primer of the second primer pair may comprise the sequence of the first sequencing adapter, in order that it can bind to the end of the nucleic acid molecule to which the first sequencing adapter was added during the first PCR amplification. In a particular embodiment, the primer comprising the first sequencing adapter used in the first PCR amplification to add the first sequencing adapter to the nucleic acid molecule, is also used in the second PCR amplification. That is to say, the same primer (comprising the first sequencing adapter) may be used in both the first and second PCR amplifications.

[0103] In embodiments where two sequential PCR amplifications are performed in order to add sequencing adapters to both ends of the reporter nucleic acid molecule, the products of the first PCR may be purified before they are subjected to the second PCR. Standard methods for purification of PCR products are known in the art.

[0104] As noted above, the Illumina P5 and P7 sequencing adapters are a preferred pair of sequencing adapters for use in the present invention. In a particular embodiment, the P5 sequencing adapter is added to the nucleic acid molecule in the first PCR amplification and the P7 sequencing adapter is added to the nucleic acid molecule in the second PCR amplification. In another embodiment, the P7 sequencing adapter is added to the nucleic acid molecule in the first PCR amplification and the P5 sequencing adapter is added to the nucleic acid molecule in the second PCR amplification.

[0105] In another embodiment, the ID sequences are primer binding sites, e.g. for a PCR primer, although other amplification methods may be used.

[0106] Suitable methods for detecting the reporter nucleic acid molecule include PCR-based methods. For instance, quantitative PCR utilising “TaqMan” probes may be performed. In this instance, the reporter nucleic acid molecules (or at least a section of each reporter nucleic acid molecule comprising the barcode sequence) and the PISO are amplified, and a probe complementary to each barcode sequence is provided, with each different probe being conjugated to a different, distinguishable fluorophore. The presence or absence of each barcode (and thus reporter nucleic acid molecule, and thus analyte, or PISO) can then be determined based on whether the particular barcode is amplified. However, it is apparent that PCR-based methods such as described above are only suitable for analysis of relatively small numbers of different sequences at the same time, although combinatorial methods using probes for decoding barcode sequences are known and may be used to extend multiplexing capacity to a degree. Nucleic acid sequencing does not have any real limit on the number of sequences which can be identified in any one go, enabling higher levels of multiplex reaction than detection using PCR, hence sequencing is the preferred method for reporter nucleic acid molecule detection.

[0107] Amplification methods based on PCR are convenient and conveniently the readout may involve quantitative PCR (qPCR) or real-time PCR. The amplicons may be detected using any convenient protocol, including the use of dyes and stains, or labels, e.g. intercalating dyes, or labelled probes which bind to the amplicons. These include molecular beacons and such like, e.g. probes with FRET labels etc.

[0108] For instance, when readout is performed by qPCR, it is preferable to be able to provide a limited set of qPCR primers that work for all panels of assays, regardless of the assay content of the various panels. It is also preferable to keep the number of qPCR primers low to reduce cost, risk of mismatched binding and other biological artefacts. With the present invention, it is possible to select a set of detection probes where all detection probes generate reporter molecules with unique identification sequences, while at the same time all those identification sequences also correspond to a limited set of qPCR primer binding sites, so that a corresponding limited set of qPCR primers can be used for read-out of the panel. In this way, the same set of qPCR primers can be used for readout of any panel compiled from the library according to the invention.

[0109] Thus, in one embodiment, the unique identification sequences can be made to correspond to a set of qPCR primers that can be used for readout of any panel compiled from the library according to the invention.

[0110] In one embodiment, the identification sequences are binding sites for qPCR primers.

[0111] In one embodiment, the identification sequences are barcode sequences.

[0112] In still further embodiments, the ID sequences may be restriction sites (i.e. a nucleotide sequence recognized by a restriction enzyme). In this embodiment, the nucleic acid domain of a proximity probe may comprise a different restriction site (such that it is recognized and cleaved by a different restriction enzyme). Different combinations of restriction enzymes may thus be applied to differentiate different reporter nucleic acids.

[0113] The sample may be any sample of interest - that is to say, any sample which contains or may contain analytes of interest. It may be any biological or clinical sample, e.g. any cell or tissue sample of or from an organism, or any body fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates etc. Environmental samples, e.g. soil and water samples, or food samples are also included. The samples may be freshly prepared or they may be prior-treated in any convenient way e.g. for storage.

[0114] The analyte of interest may be any analyte it is desired to detect. In an embodiment, the analyte is or comprises a protein. However, it may be any biological or chemical entity it desired to detect. As indicated above, proximity probes are used in the art to detect a wide variety of analytes, and these may include interactions and complexes etc. Thus, the proximity probes of a set (a proximity probe pair or a larger set of matched proximity probes) may each bind to the same target molecule (but at different sites, so that the individual proximity probes may each bind to their respective target binding sites at the same time (i.e. simultaneously), or to different molecules (e.g. where the target analyte is an interaction, and each proximity probe binds to a different member of the interaction, or where a post- translational modification of a given protein is being detected). Indeed, the target analyte may be the co-localization of two molecules in close proximity.

[0115] The invention is described herein in relation to the two main aspects, one relating to a product (kit of parts) and one relating to a method. These aspects are inter-related as the product is adapted for use in a method as disclosed herein, and the method preferably makes use of a product as disclosed herein. A feature primarily described in relation to one of these aspects is thus generally applicable to the other aspect as well, unless expressly indicated otherwise.

[0116] All prior publications cited in the present specification are incorporated herein by reference in their entirety.

[0117] The invention is further explained in the following Examples. The Examples are purely illustrative and shall not be construed as limiting the scope of the invention, which is that of the appended claims.

[0118] Examples

[0119] Example 1 - Exemplary Experimental Protocol (PEA)

[0120] This exemplary protocol uses up to four separate panels each comprising 384 probe pairs, that is assays for 384 different analytes. Further, each such 384-assay panel is split into four different sets, or blocks, wherein the assays, i.e. probe pairs, are allocated to a block based on the predicted abundance of the respective analyte in the sample under investigation. Assays for analytes of expected to be present in similar concentrations are allocated to the same block. This concept is further detailed in WO2021191442.

[0121] Step 1 - Sample Preparation and Incubation 16 Aliquots from each of 48 to 96 plasma samples are incubated with each of up to 16 proximity probe pools (four abundance blocks for each of four 384-probe pair panels) in 96- well or 384-well incubation plates.

[0122] - Samples may be pre-diluted 1 :10, 1 :100, 1:1 ,000, 1 :10,000 and 1 :100,000 for those probe pools containing assays that require it.

[0123] - Dilution and dispensing of plasma samples into incubation solution can be performed manually, or by pipetting robot e.g. LabTech’s Mosquito® HTS. Incubation solution is dispensed into the wells of the plate.

[0124] - 1 pl of sample is added to 3 pl of incubation mix at the bottom of each well, the plate is sealed with adhesive film, spun at 400 x g for 1 minute at room temperature and incubated overnight at 4°C.

[0125] - If using the above-mentioned pipetting robot, volumes may be decreased to 0.2 pl sample and 0.6 pl incubation mix (5x reduction).

[0126] The tables below give exemplary reagent formulations. Other components may be included, for example other blocking agents in the probe solutions.

[0127] Table 1 - Sample Diluent and Negative Control Solution

[0128] Table 2 - Incubation Mix

[0129] Table 3 - Panel specific incubation Solution The PISO is a dsDNA oligonucleotide comprising a panel barcode unique to the panel.

[0130] Table 4 - Forward Probe Solution

[0131] Table 5 - Reverse Probe Solution

[0132] Step 2 - Proximity Extension and PCR1 Amplification

[0133] Extension and amplification are performed using Pwo DNA polymerase. PCR1 is performed using common primers for amplification of all extension products. The incubation plate (from step 1) is brought to room temperature and centrifuged at 400 x g for 1 minute. The extension mix (comprising ultrapure water, DMSO, Pwo DNA polymerase and PCR1 solution) is added to the plate, and the plate is then sealed, briefly vortexed and centrifuged at 400 x g for 1 minute, then placed in a thermal cycler for the PEA reaction and preamplification (50 °C 20 min, 95 °C 5 min, (95 °C 30s, 54 °C 1 min, 60 °C 1 min) x25 cycles, 10°C hold). Preferably, a dispensing robot may be used to dispense the extension mix into the plate, e.g. the Thermo Scientific™ Multidrop™ Combi Reagent Dispenser. The forward common primer comprises the Illumina P5 sequencing adapter sequence.

[0134] Table 6 - PCR1 Reaction Mix Table 7 - PCR1 Solution

[0135] Step 3 - Pooling Abundance Blocks PCR1 products from each of the four abundance blocks from a 384-probe pair panel are pooled together. This results in up to four PCR1 pools per sample, one for each 384-probe pair panel.

[0136] Different volumes can be taken from each block to even out the relative levels of assays between the blocks. Pooling of PCR1 products can be performed manually, or by pipetting robot.

[0137] Step 4 - PCR2 Indexing

[0138] A primer plate containing 48 to 96 reverse primers is provided (generally one primer in each well of a 96-well plate). Each reverse primer comprises the “Illumina P7” sequencing adapter sequence and a sample index barcode. A unique barcode sequence is used for PCR1 products from each different sample. Preferably each of the up to four PCR1 pools comprising the same plasma sample (one for each 384-probe pair panel) receive the same sample index, for easy identification and data processing. A forward common primer comprising the “Illumina P5” sequencing adapter sequence (the same forward primer as used in PCR1) is provided in the PCR2 solution.

[0139] Each PCR1 pool is contacted with PCR2 solution containing the forward common primer, a single reverse (sample index) primer from the primer plate, and a DNA polymerase (Taq or Pwo DNA polymerase). Amplification is performed by PCR until primer depletion (95°C 3 min, (95°C 30 s, 68°C 1 min) x 10 cycles, 10°C hold). The theoretical end concentration of pooled PCR1 product is 1 pM (all primers used). PCR1 amplicons are diluted 1 :20 dilution for PCR2, giving a starting concentration of 50 nM in each PCR2 reaction. The concentration of each PCR2 primer is 500 nM. PCR2 primer depletion should therefore occur after 3.3 cycles (10-fold amplification).

[0140] Table 8 - PCR2 Reaction Mix

[0141] Table 9 - PCR2 Solution

[0142] Table 10 - Sample Index Primer Solution

[0143] Step 5 - End Pool All 48 to 96 indexed sample pools belonging to the same 384-probe pair panel are pooled together, adding the same volume from each sample. This yields up to four final pools (or libraries), one for each 384-probe pair panel.

[0144] Step 6 - Purification and Quantification (Optional)

[0145] The libraries are purified separately using magnetic beads, and purified libraries’ total DNA concentration is determined using qPCR with a DNA standard curve. AMPure XP beads (Beckman Coulter, USA), which preferentially bind longer DNA fragments, may be used in accordance with the manufacturer’s protocol. The AMPure XP beads bind the long PCR products but do not bind short primers, thus enabling purification of the PCR product from any remaining primers.

[0146] Depletion of the PCR2 primers means that this purification step may not be necessary.

[0147] Step 7 - Quality Control (Optional)

[0148] A small aliquot of each (purified) library is analysed on an Agilent Bioanalyser (Agilent, USA), in accordance with the manufacturer’s instructions, to confirm successful DNA amplification.

[0149] Step 8 - Sequencing

[0150] Libraries are sequenced using an Illumina platform (e.g. the NoveSeq platform). Each of the up to four libraries (from each 384-probe pair panel) is run in a separate “lane” of a flow cell. Depending on the size and model of flow cell and sequencer used, the up to four libraries may be sequenced in parallel or sequentially (one after the other) in different flow cells.

[0151] Step 9 - Data Output

[0152] Barcode (from each reporter nucleic acid molecule), sample index (from the sample index primers), and product identification (from PISO) sequences are identified in the data, counted, summed and aligned / labeled according to a known barcode-assay-sample key. “Matching barcodes” represent interactions between two paired PEA probes. The count is relative to the number of interactions in the PEA. Counts for each assay and sample must be normalised using the internal reference controls to be able to compare between samples. Each of the four abundance blocks has its own internal reference control. The panel used to run the assay is identified according to a panelbarcode key. The PISO and the corresponding product identification sequence (barcode) may differ between the panels, but is the same for all abundance blocks within a panel.

[0153] Each 384-probe pair panel is separated based on the lane it is read out in. Each panel comprises the same 96 sample indexes and the same 384 barcode combinations and internal reference controls.

Claims

CLAIMS1 . A kit of parts comprising a reagent composition, the reagent composition comprising a plurality of detection probes suitable for detection of a set of analytes of interest, each detection probe comprising a nucleic acid moiety, the nucleic acid moiety of a detection probe being capable of generating a reporter nucleic acid molecule comprising an analyte identification sequence identifying the respective analyte of interest, characterized in that the kit further comprising a nucleic acid molecule comprising a product identification sequence identifying the kit.

2. The kit of parts according to claim 1 , wherein each detection probe comprises an analyte-specific binding domain bound to the nucleic acid moiety.

3. The kit of parts according to claim 2, wherein each detection probe comprises a matched set of proximity probes, each proximity probe comprising an analyte-specific binding domain and a nucleic acid moiety, the analyte-specific binding domains of each matched set of proximity probes being capable of binding specifically to the same analyte of interest, and the nucleic acid moieties of a matched set of proximity probes being capable of together forming a reporter nucleic acid molecule comprising an identification sequence identifying the respective analyte of interest.

4. The kit of parts according to any one of claim 1-3, wherein the nucleic acid molecule comprising the product identification sequence is a double stranded DNA molecule.

5. The kit of parts according to any one of claim 1-4, wherein the nucleic acid molecule comprising the product identification sequence is included in the reagent composition comprising the plurality of detection probes.

6. The kit of parts according to claim 5, wherein the reagent composition comprising the plurality of detection probes and the nucleic acid molecule comprising the product identification sequence is provided in dried form in a reaction container comprised in the kit of parts.

7. The kit of parts according to any one of claims 1-4, wherein the nucleic acid molecule comprising the product identification sequence and the plurality of detection probes are included in separate reagent compositions.

8. A method for identifying a kit of parts used in obtaining detection data for a set of analytes of interest in a sample, wherein the detection data has been obtained through use of a kit of parts adapted for performing a method comprising the steps:51)contacting the sample with a plurality of detection probes suitable for detection of a set of analytes of interest, each detection probe comprising a nucleic acid moiety, the nucleic acid moiety of a detection probe being capable of generating a reporter nucleic acid molecule comprising an analyte identification sequence identifying the respective analyte of interest, and a nucleic acid molecule comprising a product identification sequence identifying the kit; and52)generating the reporter nucleic acid molecules; and53) detecting the reporter nucleic acid molecules and the nucleic acid molecule comprising the product identification sequence; wherein the kit is identified by the detected product identification sequence.

9. The method according to claim 8, wherein the method for obtaining detection data further comprises, between steps S2 and S3, a step of amplifying any generated reporter nucleic acid molecules and the nucleic acid molecule comprising the product identification sequence; and wherein step S3 comprises detecting the amplified nucleic acid molecules.

10. The method according to any one of claims 8 to 9, wherein the detection step (S3) comprises quantifying the amount of nucleic acid molecules comprising the respective identification sequences.

11. The method according to any one of claims 8 to 10, wherein each detection probe comprises a matched set of proximity probes, each proximity probe comprising an analyte-specific binding domain and a nucleic acid moiety, the analyte-specific binding domains of each matched set of proximity probes being capable of binding specifically to the same analyte of interest, and the nucleic acid moieties of a matched set of proximity probes being capable of together forming a reporter nucleic acid molecule comprising an identification sequence identifying the respective analyte of interest.

12. The method according to any one of claims 8 to 11 , wherein the kit of parts used for obtaining the detection data is a kit of parts according to any one of claims 1-7.

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