A sample-specific probe composition and method

Abstract

The present invention relates to a method for detection of at least one analyte of interest in multiple samples simultaneously, wherein said method comprises providing multiple sample- specific probe compositions comprising at least one proximity probe pair, each proximity probe pair comprising: a) a first proximity probe comprising a first analyte binding domain coupled to a first nucleic acid domain, and; b) a second proximity probe comprising a second analyte binding domain coupled to a second nucleic acid domain, wherein the first and / or second nucleic acid domain(s) comprise(s) a sample identification sequence identifying each sample-specific probe composition. The present invention also relates to said sample- specific probe compositions and to a kit comprising said sample-specific probe composition.

Description

[0001] A SAMPLE-SPECIFIC PROBE COMPOSITION AND METHOD

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to the field of detecting the presence of at least one analyte, particularly a protein analyte, in multiple samples simultaneously. More specifically, the present invention relates to dual-recognition immunoassays, particularly PEA (Proximity Extension Assay) and PLA (Proximity Ligation Assay) for detecting and measuring at least one analyte, particularly a protein analyte, in multiple samples simultaneously. The methods and products find particular use in multiplex assays for detecting multiple protein analytes. BACKGROUND OF THE INVENTION

[0004] Modern personalised medicine requires the ability to assess large panels of biomarkers, e.g. in the field of oncology. As personalised medicine becomes ever more widespread, the ability to accurately identify and quantify a large number of biomarkers in a sample is increasing in importance.

[0005] Hence, modern proteomics methods need 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 dual-recognition immunoassays, particularly proximity extension assays (PEA) and proximity ligation assays (PLA). PEA and PLA are described in WO 01 / 61037. PEA is further described in WO 03 / 044231 , WO 2004 / 094456, WO 2005 / 123963, WO 2006 / 137932 and WO 2013 / 113699.

[0006] Multiplexed PLA is also further described in i.a. Lundberg et al. Molecular & Cellular Proteomics 10:10.1074 / mcp.M110.004978, 1-10, 2011 and multiplexed PEA is further described in e.g. Assarsson et al., PLoS 1 , 2014, 9, 4, e95192; Wik et aL, 2021 , Mol Cell 30 Proteomics 20, 100168 and Siegbahn A, et al., PLoS One. 2023 Nov 14; 18(11): e0293465. Briefly, in such assays, analytes (particularly proteins, or parts thereof) are detected through the binding of a pair of binders (usually antibodies, or parts thereof) comprising oligonucleotides (particularly DNA) tails that are attached thereto. A binding event brings the oligonucleotides that are coupled to each member of the pair of binders in proximity (“proximity probes”) and allows them to interact to either extend (PEA) or be ligated (PLA) to generate a reporter nucleic acid molecule. This reporter nucleic acid molecule can then be detected e.g. through qPCR or by sequencing the DNA reporter using Next Generation Sequencing (NGS). The presence of an analyte of interest in a sample is hence indirectly determined through detecting the presence of the reporter nucleic acid molecule producedafter a hybridization event between the oligonucleotides of the respective binders of each pair.

[0007] WO2017068116 relates to a method of manufacturing pairs of proximity probes, wherein each probe of the proximity probe pair comprises a universal oligonucleotide conjugate bound to the analyte binding moiety of the probe. One advantage of using universal oligonucleotide conjugates is that it reduces the number of chemical activation reactions that are required to manufacture a plurality of proximity probe pairs, thereby providing for a greater consistency to be achieved and for the reactions to be performed in large batches. Different tag oligonucleotides comprising unique domains are hybridized to the universal conjugates. As an example, at least one of the probes of each proximity probe pair comprises in its unique domain a sequence that is specific and representative to a particular target analyte. Hence, proximity probes comprising analyte identification sequences are known in the prior art.

[0008] The prior art multiplex analysis protocols for homogenous (i.e. in solution) PEA performed on multiple samples simultaneously build on elaborate fluid handling (for qPCR readout) or the addition of sample index oligonucleotides (for Next Generation Sequencing, NGS, readout, e.g. as described in Wik et al., 2021) to identify the sample origin from the readout. Hence, in methods of the prior art, the sample index oligonucleotide is added to the reporter nucleic acid molecules, see e.g. Fig 1 of Wik et al., 2021. In the protocol of Wik et al., 2021 , the sample index oligonucleotide also comprises a sequence functioning as a PCR primer, thereby incorporating the sample index through a subsequent PCR reaction.

[0009] Various other methods to attach a sample index oligonucleotide also exist in the prior art, e.g. through extension, PCR, ligation and / or recombination.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention aims to solve problems related to scale-up, effectivization and / or automation of dual-recognition immunoassays performed on multiple samples in parallel, while maintaining analytical sensitivity and specificity.

[0012] One example of a problem when analyzing multiple samples for one or more analyte(s) is that multiple reactions are performed in parallel in different reaction spaces resulting in different reaction conditions for different samples. Performing multiple reactions in parallel is also expensive and time-consuming.

[0013] Hence, there would be an advantage in reducing the number of parallel reactions that are performed for each assay, partly for the reason of speeding up the assay, which provides additional advantages, but also for improved precision and robustness due to less variabilityin reaction conditions across separate reactions. However, the probe configurations of the prior art require that samples are reacted separately at least until reporter molecules have been generated.

[0014] The above-mentioned needs and problems have now been addressed by providing improved methods and products for the detection of at least one analyte in multiple samples simultaneously.

[0015] These problems, and others as explained further herein, are addressed by the present invention by providing a sample-specific proximity probe composition comprising at least one proximity probe pair comprising a first and a second proximity probe, wherein said first proximity probe comprises a first analyte binding domain coupled to a first nucleic acid domain and said second proximity probe comprises a second analyte binding domain coupled to a second nucleic acid domain, and wherein the nucleic acid domain of at least one of the proximity probes of the proximity probe pair comprises a sample identification sequence.

[0016] Herein, the sample identification sequence has been introduced into the nucleic acid domain of at least one of the proximity probes of the proximity probe pair prior to combining the probes with the sample to be analyzed, preferably already at manufacture of the proximity probes. In prior art methods, the sample identification sequence is added at a later stage of corresponding methods.

[0017] Introducing the sample identification sequence already at such an early stage enables pooling of multiple samples comprising sample-specific probe compositions at an earlier stage than possible with prior art methods. This reduces the number of method steps and the number of parallel reactions as all reactions after pooling may be run in the same reaction space.

[0018] From this also follows a reduction of the number of components that need to be used in the method as the proximity probes are already provided with more functionalities from the very beginning. Further advantages of the invention include increasing sensitivity with solid phase protocols and addressing potential problems with sample-to-sample crosstalk. All of this will result in an improved outcome of the analysis.

[0019] Thus, in a first aspect, the present invention relates to a method for detection of at least one analyte of interest in multiple samples simultaneously, said method comprising the steps of: (i) providing multiple sample-specific probe compositions, each sample-specific probe composition comprising at least one proximity probe pair, each proximity probe pair comprising:a) a first proximity probe comprising a first analyte binding domain coupled to a first nucleic acid domain, and;

[0020] b) a second proximity probe comprising a second analyte binding domain coupled to a second nucleic acid domain,

[0021] wherein the first and second analyte binding domains of said proximity probe pair are capable of simultaneously and specifically binding to a specific analyte of interest present, or suspected of being present, in the samples,

[0022] wherein the first and second nucleic acid domain are capable of directly or indirectly interacting to generate a reporter nucleic acid molecule when said first and second proximity probes are both bound to the same analyte;

[0023] wherein the first and / or second nucleic acid domain(s) comprise(s) a sample identification sequence identifying each sample-specific probe composition,

[0024] (ii) bringing each sample separately into contact with a sample-specific probe composition to form a sample-probe combination, and allowing the proximity probes of said sample-specific probe composition to bind to the analytes in each sample-probe combination;

[0025] (iii) pooling all sample-probe combinations of step (ii);

[0026] (iv) generating reporter nucleic acid molecules from interacting first and second nucleic acid domains; and

[0027] (v) detecting the at least one analyte of interest through detection of the generated reporter nucleic acid molecules of step (iv) comprising the respective sample identification sequences.

[0028] In a second aspect, the present invention relates to a sample-specific probe composition comprising at least one proximity probe pair, each proximity probe pair comprising:

[0029] a) a first proximity probe comprising a first analyte binding domain coupled to a first nucleic acid domain, and;

[0030] b) a second proximity probe comprising a second analyte binding domain coupled to a second nucleic acid domain,

[0031] wherein the first and second analyte binding domains of said proximity probe pair are capable of simultaneously and specifically binding to a specific analyte of interest; wherein the first and second nucleic acid domain(s) are capable of interacting to generate a reporter nucleic acid molecule when said first and second proximity probes are both bound to the analyte of interest; andwherein the first and / or second nucleic acid domain(s) comprise(s) a sample identification sequence identifying the sample-specific probe composition.

[0032] In a third aspect, the present invention relates to a kit comprising multiple sample-specific probe compositions as described herein, optionally further comprising one or more reagents and optionally instructions for use.

[0033] Additional details and advantages of the present invention will be evident as set out further herein.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 shows a number of probe designs useful in the present invention.

[0036] Figure 2 shows a probe design of the prior art (A), a preferred probe design according to the present invention (B), a preferred probe design (C) adapted for a solid-phase protocol, and the probe design of (C) including an anchor oligonucleotide (D) according to the present invention.

[0037] Figure 3 illustrates an embodiment of the method as disclosed herein, using solid-phase capture and toehold-mediated strand displacement for release of probes.

[0038] Figure 4 shows Log2-values of the counts using three different quantitation methods for determining the presence of multiple analytes in multiple samples simultaneously. The quantitation methods are based on sample identifying sequences of either one of the first and second proximity probe (two upper panels) or matched sample identifying sequences of the first and second proximity probe (lower panel), individually plotted against the input concentration of the nine different antigens.

[0039] DEFINITIONS OF TERMS AND ABBREVIATIONS

[0040] 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.

[0041] The singular “a” and “an” shall be construed as including also the plural.

[0042] Compositions “comprising” one or more recited elements may also include other elements not specifically recited. The term "comprising” also encompasses the term “consisting of’. An ’’immunoassay” is a type of assay for a specific analyte to be detected, that utilizes molecules binding specifically and preferentially to the analyte in question and wherein binding events generate or result in a signal that can be detected. While the prefix “immuno-” implies, and originates from, the original use of antibodies as the specific binding molecules,immunoassays may also use other types of specific analyte binding molecules, as further exemplified herein.

[0043] “Dual recognition immunoassay” is an immunoassay wherein two simultaneous and specific binding events are required for a signal to be generated. PEA (Proximity Extension Assay) and PLA (Proximity Ligation Assay) are examples of dual recognition immunoassays. These are assays well-known in the art as further described herein. Dual recognition immunoassays differ from e.g. sandwich ELISAs that generally includes capture of the analyte by a capture antibody and subsequent addition of a second antibody (detection antibody) coupled to a signal-generating moiety. While this involves two binding events, these two events are not both required to generate a signal, as the signal is generated solely by the signal-generating moiety on the detection antibody.

[0044] “Multiplexing” of biological assays, such as proximity assays, means performing a plurality of assays in parallel and, preferably, in the same reaction space, 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 footprint of the necessary equipment. As used herein, the term “multiplex” is used to refer to an assay in which multiple or a plurality of analytes (i.e. at least two) different analytes are assayed at the same time, and more particularly in the same sample.

[0045] The term “plurality” or “multiple” as used in the present invention means more than one (that is to say, two or more), in line with its standard definition.

[0046] The term “analyte” as used herein, in respect of all aspects of the present invention, means any substance (e.g. molecule) or entity it is desired to detect by the method and / or samplespecific probe composition of the invention. The analyte is thus the "target" of a method and / or sample-specific probe composition of the invention, i.e. the substance to be detected or screened for using the method and / or sample-specific probe composition of the invention. The term "detecting" or "detected” and the like is used broadly herein to include any means of determining the presence or absence of an analyte (i.e. determining whether a target analyte is present in a specific sample or not). Accordingly, if a method of the invention is performed and an attempt is made to detect a particular analyte of interest in a sample, but the analyte is not detected because it is not present in the sample, the step of “detecting the analyte” has still been performed, because its presence or absence from the sample has been assessed.

[0047] A “proximity probe” as referred to herein, is a probe for the detection of an analyte in a sample that is generally used in pairs, i.e. in general a first and a second proximity probe isused for detection of an analyte (but it may also comprise more than two probes in combination). A first and a second proximity probe that are used as a pair are herein referred to as a “proximity probe pair”. 'The terms “proximity probing” and “proximity assays” are also used herein.

[0048] The “analyte binding domain” or “analyte binding moiety”, which are terms used interchangeably herein, of a proximity probe of a proximity probe pair, may comprise any entity capable of binding specifically to an analyte of interest (or part thereof) and capable of being coupled to “a nucleic acid domain”. That the analyte binding domain binds “specifically” to or 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. Further, the analyte binding domain may bind to the analyte directly or indirectly. In other words, the proximity probe(s) 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.

[0049] The “nucleic acid domain” or “nucleic acid moiety”, which are terms that may be used interchangeably herein, of a proximity probe of a proximity probe pair, comprises a domain or moiety capable of generating a detectable signal when dual recognition is achieved between the nucleic acid domain of the first and second proximity probe of a proximity probe pair. The nucleic acid domain is coupled to the analyte binding domain of each proximity probe. The nucleic acid domain or moiety is sometimes referred to herein as an “oligonucleotide”.

[0050] Generally, the nucleic acid domain has a length in the range of 20-100 nucleotides but may be shorter or longer as required in the specific assay / method in which the proximity probe is intended to be used.

[0051] A “sample” is a discrete volume of material which is subjected to the method according to the invention.

[0052] The present invention makes use of “identification sequences”. An identification sequence may e.g. be a unique sequence (sometimes termed a “barcode sequence” or simply “barcode” or “index”) that is detected in a sequence-specific manner and used to identify the origin of the molecule in which it is present. It may, for example, be 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). Recognition sites for restriction enzymes may also be used as identification sequences.A “sample identification sequence” is a nucleic acid sequence that serves to identify the sample from which a reporter nucleic acid molecule is derived.

[0053] An “analyte identification sequence” is a nucleic acid sequence that serves to identify a specific analyte, analogous to a sample identification sequence identifying a specific sample. A “sample-specific probe composition” as referred to herein, comprises a proximity probe pair comprising a first proximity probe and a second proximity probe wherein the nucleic acid domain of at least one of the first or the second proximity probe comprises a sample identification sequence. The term “sample-specific probe composition” may also be referred to herein as a “sample-specific proximity probe composition” or in short to as a “probe composition”. When bringing the sample-specific probe composition into contact with a sample, a “sample-probe combination” is obtained. Hence, a “sample-probe combination” comprises the sample-specific proximity probe composition and the sample.

[0054] Herein, the sample-probe combinations are “pooled” before reporter nucleic acid molecules are generated from the interacting first and second nucleic acid domains of the proximity probes. This procedure is also referred to herein as “pooling” of the sample probe combinations. This means that different sample-specific probe compositions combined with their respective samples are mixed to allow for a more efficient analysis of the reaction products by enabling single reaction condition for the steps of the method following the pooling.

[0055] A “reporter nucleic acid molecule” is a nucleic acid molecule whose synthesis and detection indicates the presence of one or more analyte(s) in a specific sample. The reporter nucleic acid molecule may be an RNA molecule or a DNA molecule, preferably a DNA molecule. When both analyte binding domains of the first and second proximity probe of a proximity probe pair simultaneously and specifically bind to a specific analyte of interest in the sampleprobe combination, allowing the first and second nucleic acid domains of the proximity probes to interact, the subsequently generated and detected reporter nucleic acid molecule will indirectly determine the presence of an analyte in a specific sample.

[0056] “Readout” as used herein is intended to refer to the process of detecting the presence of and / or quantifying the amount of reporter nucleic acid molecules comprising the respective sample identification sequence(s) of one of the proximity probes or both, and optionally the respective analyte identification sequence(s), of one of the proximity probes or both, and correlating these amounts to the amounts of the respective at least one analyte of interest originating from a specific sample. Accordingly, a readout can be seen as a step of detecting the signal in the assay, or more particularly the reporter nucleic acid molecules, in a quantitative manner.An “anchor moiety” or “anchor group” as referred to herein, is a physical structure used for capturing a proximity probe or a proximity probe-analyte complex onto a solid phase to purify proximity probe pairs bound to an analyte from unbound probes and other impurities. The anchor moiety is bound or captured directly or indirectly to the solid phase. The moiety on the solid phase capturing the anchor moiety is correspondingly termed “capture moiety”.

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] Hence, as set out previously herein, the first aspect of the present invention relates to a method for detection of at least one analyte of interest in multiple samples simultaneously. More particularly, in a first aspect, the present invention relates to a method for detection of at least one analyte of interest in multiple samples simultaneously, said method comprising the steps of:

[0059] (i) providing multiple sample-specific probe compositions, each sample-specific probe composition comprising at least one proximity probe pair, each proximity probe pair comprising:

[0060] (a) a first proximity probe comprising a first analyte binding domain coupled to a first nucleic acid domain, and;

[0061] (b) a second proximity probe comprising a second analyte binding domain coupled to a second nucleic acid domain,

[0062] wherein the first and second analyte binding domains of said proximity probe pair are capable of simultaneously and specifically binding to a specific analyte of interest present, or suspected of being present, in the samples,

[0063] wherein the first and second nucleic acid domain are capable of directly or indirectly interacting to generate a reporter nucleic acid molecule when said first and second proximity probes are both bound to the same analyte;

[0064] wherein the first and / or second nucleic acid domain(s) comprise(s) a sample identification sequence identifying each sample-specific probe composition,

[0065] (ii) bringing each sample separately into contact with a sample-specific probe composition to form a sample-probe combination, and allowing the proximity probes of said sample-specific probe composition to bind to the analytes in each sample-probe combination;

[0066] (iii) pooling all sample-probe combinations of step (ii);

[0067] (iv) generating reporter nucleic acid molecules from interacting first and second nucleic acid domains; and

[0068] (v) detecting the at least one analyte of interest through detection of the generated reporter nucleic acid molecules of step (iv) comprising the respective sample identification sequences.As mentioned elsewhere herein, said method may be performed using dual recognition assays (also referred to herein as proximity assays), such as proximity extension assays (PEA) or proximity ligation assays (PLA).

[0069] 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, sometimes two or three) proximity probes, which when brought into proximity by binding to the analyte of interest (hence "proximity probes") allow a signal to be generated.

[0070] Each proximity probe of a proximity probe pair comprises a nucleic acid domain (or moiety) coupled to an analyte binding domain (or moiety) of the probe, and generation of the signal involves an interaction between the nucleic acid domain of the first proximity probe and the nucleic acid domain of the second proximity probe of the proximity probe pair.

[0071] Thus, signal generation in the form of the generation of a reporter nucleic acid molecule is dependent on an interaction between the respective nucleic acid domains of the respective proximity probes of each proximity probe pair, more particularly between the nucleic acid or other functional moieties / domains carried by them. This only occurs when the necessary proximity probes of the same proximity probe pair have bound to the analyte of interest, thereby lending improved specificity to the detection system.

[0072] In PEA, nucleic acid domains linked to the analyte binding domains of the first and second proximity probe of a proximity probe pair hybridise to one another when the proximity probes are in close proximity (i.e. when bound to a target) and are then extended using a nucleic acid polymerase. The extension product forms a reporter nucleic acid molecule, the detection of which demonstrates the presence of at least one analyte of interest, i.e. wherein the analyte is the analyte bound by each proximity probe of the relevant proximity probe pair. In PLA, nucleic acid domains linked to the analyte binding domains of a proximity probe pair come into proximity when the probes of the proximity probe pair bind their target analyte, and may at that stage 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. Such oligonucleotides are also referred to as splint oligonucleotides. The ligation product is then amplified, acting as a reporter nucleic acid molecule.

[0073] While a number of dual-recognition immunoassays may be used in the present invention, as discussed herein, presently preferred dual-recognition immunoassays are PEA and PLA. PEA and PLA are also described in US 7,306,904 and further information regarding PLA may be found in Lundberg et al., Molecular & Cellular Proteomics 10:10.1074 / mcp.M110.004978,1-10, 2011. PEA is further described in WO 03 / 044231 (US2005003361 A1), WO 2004 / 094456 (US2005009050A1), WO 2005 / 123963 (US2009162840A1), WO 2006 / 137932 (US2008131883A1), WO 2013 / 113699 (US2015044674A1), WO 2021 / 191442 (US2023159983A1), WO 2021 / 191448 (US2023159983A1), WO 2021 / 191449 (US2023159983A1), W02021 / 191450 (US2023107654A1), and WO 2022 / 112300 (US2022162589A1); Lundberg et al. Nucleic Acids Research, 2011, Vol. 39, No. 15 e102; Wik et al., 2021 , Mol Cell Proteomics 20, 100168, and Siegbahn A, et al., PLoS One. 2023 Nov 14;18(11): e0293465, all incorporated herein by reference in their entirety.

[0074] In addition, a methodology to develop and run a panel detecting 94 unique target proteins is provided in Assarsson et al. PLoS One, 2014, 9(4), e95192. Similarly, the development of a multiplexed PLA assay is described in Lundberg et al., Molecular & Cellular Proteomics 10:10.1074 / mcp.M110.004978, 1-10, 2011, all incorporated herein by reference in their entirety.

[0075] To detect at least one analyte of interest in multiple samples simultaneously, multiple samplespecific probe compositions are provided. Each sample-specific probe composition comprises at least one proximity probe pair, each proximity probe pair comprising a) a first proximity probe comprising a first analyte binding domain coupled to a first nucleic acid domain, and; b) a second proximity probe comprising a second analyte binding domain coupled to a second nucleic acid domain. The nucleic acid domain of at least one proximity probe of each proximity probe pair comprises a specific sample identification sequence identifying each sample-specific probe composition. Hence, in combination, multiple samplespecific probe compositions provide multiple different sample-specific identification sequences which make it possible to detect at least one analyte in multiple samples simultaneously in the same reaction.

[0076] In a preferred embodiment, each sample-specific probe composition contains one and only one sample identification sequence. That is, the sample identification sequence is the same for all probes within a certain sample specific probe composition. It is however possible, if desired, to have a group of two or more sample identification sequences being indicative of the same sample-specific probe composition. Likewise, it is possible to have a single sample identification sequence being indicative of multiple samples, e.g. multiple samples from the same patient.

[0077] According to the method of the present invention, a sample-specific probe composition is added to each individual sample in different reaction spaces wherein the first and second proximity probes are allowed to bind to their respective analytes present in the respective sample to provide a sample-probe combination.In this regard, the first and second analyte binding domains of a proximity probe pair are capable of simultaneously and specifically binding to a specific analyte of interest present, or suspected of being present, in the samples and the first and second nucleic acid domain are capable of directly or indirectly interacting to generate a reporter nucleic acid molecule when said first and second proximity probes are both bound to the same analyte.

[0078] After this event, all sample-probe combinations are pooled, as described in more detail elsewhere herein, and reporter nucleic acid molecules are generated from interacting first and second nucleic acid domains, such as by an extension or ligation reaction (e.g. PEA or PLAas described elsewhere herein). The generated reporter nucleic acid molecules contain at least one sample identification sequence from at least one proximity probe, and optionally one or two analyte identification sequence(s) from one or two proximity probes of the same proximity probe pair.

[0079] Introducing a sample identification sequence into the nucleic acid domain of at least one of the proximity probes of the sample-specific probe composition prior to bringing the proximity probes into contact with a sample facilitates an improved method of detecting at least one analyte of interest in multiple samples simultaneously. This is further explained in the following.

[0080] In the prior art, aliquots of the same proximity probe composition are added to all individual samples. Thereafter, a separate sample identification sequence, unique for each sample, is also added and one or two PCR reactions are performed before pooling of the samples followed by sequencing of the reporter nucleic acid molecules. Hence, in methods of the prior art, pooling of the sample and the proximity probes is performed at a later stage as compared to a method of the present invention.

[0081] Accordingly, a proximity probe composition of the present invention provides several advantages to probe compositions of the prior art. One advantage of the current invention is thus that the extension / ligation reactions and the PCR amplifications are performed for all sample probe combinations in the same reaction space and thus under identical reaction conditions. Thereby, the risk for differences in the results arising from different reaction conditions for the different samples is reduced. Also, such a procedure will simplify sample handling and reduce the amount of individual sample handling.

[0082] One or more analyte(s) of interest is detected by an improved probe composition of the present invention. An analyte may be any biomolecule or chemical compound, for example a peptide or protein, a nucleic acid molecule, or a small molecule, including organic and inorganic molecules.An analyte can be any substance or entity for which a specific binding partner (analytebinding domain) can be developed. All that is required is that the analyte is capable of simultaneously binding at least two binding partners (more particularly, the analyte-binding domains of at least two proximity probes as disclosed herein). As is well known, proximity probe-based assays have found particular utility in the detection of proteins or polypeptides. Analytes of particular interest thus include proteinaceous molecules such as peptides, polypeptides, proteins or prions or any molecule which includes a protein or polypeptide component, etc., or fragments thereof. In a particularly preferred embodiment of the invention, the analyte is a wholly or partially proteinaceous molecule, most particularly a protein. Thus, preferably, the analyte is or comprises a protein or a polypeptide.

[0083] The analyte may be a single molecule or a complex that contains two or more molecular subunits, which may or may not be covalently bound to one another, and which may be the same or different. Thus, in addition to cells or microorganisms, such a complex analyte may also be a protein complex, or a biomolecular complex comprising a protein and one or more other types of biomolecule. Such a complex may thus be a homo- or hetero-multimer.

[0084] Aggregates of molecules e.g. proteins may also be target analytes, for example aggregates of the same protein or different proteins.

[0085] The analyte may also be a complex between proteins or peptides and nucleic acid molecules such as DNAor RNA. Of particular interest may be the interactions between proteins and nucleic acids, e.g. regulatory factors, such as transcription factors, and DNAor RNA. Thus, the analyte may be a protein-nucleic acid complex (e.g. a protein-DNA complex or a protein-RNA complex).

[0086] The analyte may also be a small molecule or a lipid.

[0087] Detecting an analyte may include any form of measurement of the concentration or abundance of the analyte in the sample. Either the absolute or the relative concentration of a target analyte may be determined. To determine the relative concentration of an analyte, the concentration of the target analyte may be compared to the concentration of one or more other target analyte(s) in the sample or in other samples. A relative concentration of an analyte may also be accomplished by inclusion of known concentration(s) of one or more control analytes and / or referencing the detected level of the target analyte with known control analytes (e.g. through generation of a standard curve). Methods by which quantification can be achieved in the method of the invention are discussed further below.

[0088] The method of the present invention is used for detecting one or more analytes in multiple samples. The analytes may be of the same type (e.g. all the analytes may be proteins, or protein complexes), or of different types (e.g. some analytes may be proteins, others proteincomplexes, others lipids, others protein-DNAor protein-RNA complexes, etc., or any combination of such types of analytes).

[0089] As noted above, a target analyte may be a single entity, in particular an individual protein. If so, both proximity probes in the proximity probe pair bind the analyte (e.g. protein), but at different epitopes. The epitopes are non-overlapping, so that the binding of one proximity probe in the proximity probe pair to its epitope does not interfere with or block binding of the other proximity probe in the proximity probe pair to its epitope. Alternatively, as noted above the target analyte may be a complex, e.g. a protein complex, in which case one proximity probe in the proximity probe pair binds one member of the complex and the other probe in the pair binds the other member of the complex at sites different to the interaction sites of the different parts of the complex (e.g.. a site different the sites in the respective proteins of a protein complex where the proteins interact with each other to form the complex).

[0090] The first and second proximity probes of a proximity probe pair work together by binding to the same analyte in close proximity to be able to generate a detection signal through interaction between the first and second proximity probe. Generation of a signal from the interacting first and second proximity probe means that a specific analyte of interest has been detected in the sample.

[0091] Any sample of interest may be assayed according to the invention. The sample may contain an analyte(s) of interest or may be suspected to contain an analyte(s) of interest and the method of the present invention enables determining the presence and / or concentration of a specific analyte in a specific sample.

[0092] The sample may for example be a 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.

[0093] A biological or clinical sample may e.g. contain a viral or cellular material, including prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles. Such biological material may thus comprise any type of mammalian and / or nonmammalian animal cell, plant cells, algae including blue-green algae, fungi, bacteria, protozoa etc. Environmental samples, e.g. soil and water samples, or food samples may also be analysed according to the invention. The samples may be freshly prepared or they may be prior-treated in any convenient way e.g. for storage. Further non-limiting examples of samples are food and allied products.

[0094] In particular, the sample may be a clinical sample, for instance whole blood and blood-derived products such as plasma, serum, buffy coat and blood cells, urine, faeces,cerebrospinal fluid or any other body fluid (e.g. respiratory secretions, saliva, milk etc.), tissues and biopsies. It is particularly preferred that the sample is a plasma or serum sample. Thus, the method or products of the invention may be used in the detection of biomarkers, for instance, or to assay a sample for pathogen-derived analytes.

[0095] The sample may in particular be derived from a human, though the method and products of the invention may equally be applied to samples derived from non-human animals (i.e. veterinary samples), plants, fungi, bacteria or cell cultures. The sample may be pre-treated in any convenient or desired way to prepare it for use in the method of the invention, for example by cell lysis or removal, etc.

[0096] By the present method any number of samples may be analysed simultaneously. Typically, from about 2 to about 200 samples may be analysed simultaneously, such as from about 10 to about 200 samples, such as from about 20 to about 150 samples, such as from about 80 to about 120 samples, such as about 90 to about 100 samples.

[0097] An analyte of interest is detected by the binding thereto of a first and a second proximity probe of a sample-specific proximity probe pair. A proximity probe of a proximity probe pair of the present invention comprises an analyte binding domain and a nucleic acid domain, wherein the nucleic acid domain is coupled to the analyte binding domain. The analyte binding and nucleic acid domains, and how they may be attached to each other to form proximity probes are generally known in the art, and also further explained elsewhere herein. At least one of the proximity probes of a proximity probe pair comprises the sample identification sequence in the nucleic acid domain.

[0098] The exact position of the sample identification sequence within the nucleic acid domain is not critical. The prime requisite is that the sample identification sequence(s), and any analyte identification sequence, are incorporated into the reporter molecule and can be identified using the read-out method. As an example, if the reporter molecules are to be sequenced using a sequencing technology requiring sequencing adapters at the ends of the nucleic acid sequence, the respective identification sequences should be located between those sequencing adapters. A sample identification sequence may be of any length but is preferably relatively short, e.g. 3-12, 4-10 or 4-8 nucleotides.

[0099] The nucleic acid domain of the proximity probe must be long enough to comprise the necessary elements. That is, at least a sequence capable of generating a sample identification sequence in the reporter nucleic acid molecule. The nucleic acid domain may also contain sequences related to primer sites and / or sequencing adaptors for read-out, as is well known in the art and as further described herein.Generally, the nucleic acid domain 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.

[0100] In order for the reporter nucleic acid molecule to form, the nucleic acid domains of the proximity probes in each pair are typically designed to hybridise to one another, or to one or more common oligonucleotide molecules, to which the nucleic acid domains of both proximity probes of a pair may hybridise.

[0101] Accordingly, the nucleic acid domains of each proximity probe must be at least partially single-stranded for the reporter nucleic acid molecule to form. Sometimes the nucleic acid domains of the proximity probes are wholly single-stranded. However, the nucleic acid domains may also be at least partially double stranded.

[0102] When a nucleic acid domain is at least partially double stranded, the sample identification sequence may be positioned on either one of the strands of the at least partially double stranded nucleic acid domain. Thus, in a proximity probe pair, the sample identification sequence may form part of the nucleic acid strand of the proximity probe that hybridises with the nucleic acid domain of the other proximity probe of the proximity probe pair or with an oligonucleotide splint that hybridizes with the nucleic acid domain of the other proximity probe. The sample identification sequence may also form part of the other strand of a double stranded nucleic acid domain that does not hybridise with the nucleic acid domain of the other proximity probe or to a splint oligonucleotide.

[0103] It is presently preferred that both of the first and second nucleic acid domains of each proximity probe pair of a sample-specific probe composition comprises a sample identification sequence. This is also referred to herein as dual index or dual-indexing.

[0104] Dual-indexing is particularly preferred as using a sample identification sequence on both proximity probes of a proximity probe pair assists in discarding signals from mismatched probes, i.e. signals based on reporter nucleic acid molecules that comprise sample identification sequences from different sample-specific probe compositions due to that the probes originate from different sample-specific probe compositions. It was also shown that using the dual-indexing feature, it was possible to detect lower concentrations of analyte (LLOD, example 1).

[0105] When a sample-specific probe composition comprises sample identification sequences on both proximity probes of a proximity probe pair, only signals, i.e. reporter nucleic acid molecules, comprising two sample identification sequences assigned to the same specificsample are counted as a true signal and are assigned to the respective sample. This means that irregularities in the analysis due to sample-to-sample cross talk will be reduced.

[0106] Furthermore, when a sample-specific probe composition comprises sample identification sequences on both proximity probes of a proximity probe pair, the sample identification sequences of the proximity probes of the individually matched proximity probe pairs may have the same nucleic acid sequence, or it may be different as long as each proximity probe of the pair can be assigned to an original specific sample.

[0107] When it is intended to detect a plurality of analytes of interest, the first and / or second nucleic acid domain of each proximity probe pair comprise an analyte identification sequence identifying the specific analyte of interest. Sometimes, both of the first and second nucleic acid domains of each proximity probe pair comprise an analyte identification sequence identifying the specific analyte of interest.

[0108] A plurality of analytes may comprise at least 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, 4000, 5000, 5500, 6000, 7000 or more analytes, but is not limited to such a number.

[0109] Hence, a reporter nucleic acid molecule generated from an interacting first and second proximity probe of a proximity probe pair may comprise one or both of sample identification sequences from one or both of the first and second proximity probe of a proximity probe pair and optionally one or both of analyte identification sequences from one or both of the first and second nucleic acid domains of the first and second proximity probe of a proximity probe pair.

[0110] Particularly there is provided herein a proximity probe pair wherein both of the first and second proximity probe of each proximity probe pair comprise a sample identification sequence identifying each sample-specific probe composition and an analyte identification sequence identifying the specific analyte of interest. Thus, this means that both proximity probes of such a proximity probe pair carry one sample identification sequence and one analyte identification sequence, respectively. This may be referred to herein as a proximity probe pair with a quadruple indexing.

[0111] Herein, each sample-probe combination may be diluted, when the sample-probe combinations are pooled in step (iii). As an example, each sample-probe combination is diluted at least 1:10, such as at least 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80 or 1:90, preferably at least 1:100, when the sample-probe combinations are pooled in step (iii) of a method of the invention.Dilutions of the sample may be made with any suitable diluent, which may depend on the type of sample being assayed. For instance, the diluent may be water or saline solution, or a buffer solution, in particular a buffer solution comprising a biologically-compatible buffer compound (i.e. a buffer compatible with the detection assay used, for instance a buffer compatible with a PEA or PLA). Examples of suitable buffer compounds include HEPES, Tris (i.e. Tris(hydroxymethyl)aminomethane), disodium phosphate, etc. Suitable buffers for use as diluent include PBS (phosphate-buffered saline), TBS (Tris-buffered saline), HBS (HEPES-buffered saline), etc. The buffer (or other diluent) used must be made up in a purified solvent (e.g. water) such that it does not contain contaminant analytes. The diluent should thus be sterile, and if water is used as diluent or the base of the diluent, the water used is preferably ultrapure (e.g. Milli-Q water).

[0112] Pooling of the sample-probe combinations occurs before the step of generating the reporter nucleic acid molecules.

[0113] Pooling of the sample-probe combinations in step (iii) may be performed sequentially or simultaneously, optionally into a dilution buffer. When the sample-probe combinations are pooled simultaneously, this means that, as compared to when the pooling is sequential, all sample-probe combinations are pooled into one reaction space in principle at the same time. When the pooling is sequential, two or more but not all sample-probe combinations are pooled at the same time. Sequential pooling includes starting off with one or more sampleprobe combinations and adding one or more sample-probe combinations to the first sampleprobe combination until all sample-probe combinations have been pooled into the same reaction space.

[0114] The sample-probe combinations may be pooled without the presence of a dilution buffer, wherein the dilution of each sample-probe combination is then a result of the pooling of the multiple sample-probe combinations perse, i.e. each sample-probe combination is diluted by being mixed with the other sample-probe combinations. The sample-probe combinations may also be pooled into a dilution buffer, which means that each sample-probe combination will be further diluted in addition to the dilution of each sample-probe combination occurring when pooling the respective sample probe combinations.

[0115] Diluting the sample-probe combinations is advantageous as the dilution effect may contribute to reducing sample-to-sample crosstalk which interferes with the outcome of the analysis. Without being bound by theory, diluting the sample-probe combinations in a dilution buffer provides a greater number of proximity-probe-analyte complexes which will reduce the probability that two probes spontaneously detach from an analyte from one sample and re-bind to the same analyte species from another sample, i.e. jumping of proximity probes between analytes originating from different samples.

[0116] A fast pooling of the sample-probe combinations, i.e. reducing the time elapsed between the pooling step (iii) and the reporter nucleic acid molecule generation of step (iv) will thus also add advantages. Fast pooling may also reduce the number of sample-to-sample crosstalk interactions that may occur before the generation of the reporter nucleic acid molecules as there will be less time for interaction between the proximity probe analyte-complexes originating from different samples that may cause the probes to jump between samplespecific analytes.

[0117] Hence to achieve this purpose, step (iv) of a method may be initiated within 60 minutes after initiating pooling step (iii), such as after no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, or 45 minutes after initiating pooling step (iii).

[0118] Non-limiting examples of how pooling may be effected include separated aspiration (e.g. air gap, oil etc.) of multiple samples and pooling via fast dispensation, incubations (or deposition) of immune reactions on a flat surface and either “scraping” (i.e. window cleaning scraper) or electrowetting for fast pooling, or by centrifugation of samples through a funnel directly onto a solid phase capture reaction (spin column).

[0119] Step (iv) of the method may be immediately preceded by a step of capturing the first or second proximity probe of the sample-specific probe compositions comprised in the pooled sample-probe combinations on a solid phase and subjecting the captured proximity probes to one or more wash steps. The proximity probes may subsequently be released from the solid phase prior to step (iv).

[0120] The first and / or the second proximity probe of a proximity probe pair may be captured on, and directly bound to, the solid phase. When one of the first or the second proximity probe is captured on the solid phase, the other proximity probe of the same proximity probe pair may be described as indirectly captured to the solid phase as the probe is interacting with the other proximity probe of the proximity probe pair through its concorruent binding to the same analyte molecule, while not being directly bound to the solid phase. A proximity probe-analyte complex comprising one of the proximity probes of a proximity probe pair may also be captured on a solid phase. Examples of attachment means for capturing a proximity probe or a proximity probe-analyte complex on a solid phase are described elsewhere herein.

[0121] Capturing on a solid phase to remove impurities and unbound proximity probes is a way to increase sensitivity and is therefore, in certain situations, an advantageous addition to proximity assays. However, using solid phase capture in individual reaction spaces for eachsample-probe combination can come with a complex and / or unrobust or imprecise workflow as performing capture, several wash steps and release steps for each sample are a large source for variation. Hence, the early pooling of the sample-probe combinations also adds further advantages to the solid phase protocol of the method, simplifying solid phase workflow by reducing the number of reactions.

[0122] Solid phase protocols that may be applied to a method of the invention are known in the art. One example of a suitable protocol comprises streptavidin coated magnetic beads (e.g. Dynabeads™ MyOne™ Streptavidin T1) that are used for the isolation and handling of biotinylated nucleic acids, antibodies, and other biotinylated ligands.

[0123] An anchor moiety, as explained elsewhere herein, may be added to at least one of the proximity probes for performing solid phase capture. Incorporation of the anchor moiety into one or both of the proximity probes in a proximity probe pair may be done in a number of ways, some of which are illustrated in the figures of the present disclosure.

[0124] Herein, the step of capturing the first or second proximity probe of all sample-specific probe compositions comprised in the pooled sample-probe combinations may be initiated within 60 minutes after initiating pooling step (iii), such as after no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, or 45 minutes after initiating pooling step (iii). Similarly to reducing the time elapsed between the pooling step and the reporter nucleic acid molecule generation (i.e. when no solid phase capture is performed), it is advantageous to perform the capture on the solid phase as fast as possible e.g. to reduce sample-to-sample crosstalk.

[0125] A method comprising a combination of at least one proximity probe of a proximity probe pair comprising a sample identification sequence, particularly wherein both the first and the second proximity probe of a proximity probe pair comprises a sample identification sequence (dual-indexing), an early (before reporter nucleic acid molecule generation) and fast (i.e., reducing the time to the subsequent generation of reporter nucleic acid molecules or to the capture on a solid phase) pooling and reaction, is particularly preferred and enables proximity probe assays to become faster, cheaper and more robust.

[0126] In example 1 , figure 4, It was also shown that the dual index design expands the dynamic range for the method when dilution is limited, and no solid phase capture is used for purification. Hence in this situation, dual-indexing seems to at least compensate for these conditions. However, it was also concluded that a preferred set of conditions include dualindexing, dilution of the sample-probe combinations at pooling, a fast pooling and solid phase capture for purification. Such a combination facilitates maintaining low background signals resulting in a method with a broad dynamic range and high sensitivity.Adding one or both of the analyte identification sequences to one or both of the proximity probes of the proximity probe pair will also add advantages as it will enable analysis of multiple analytes in the respective samples. In such a setting, the proximity probes may comprise quadruple indices, namely both analyte identification sequence and sample identification sequences on both the first and the second proximity probe of a proximity probe pair.

[0127] Following generation of the reporter nucleic acid molecule of step (v), the reporter nucleic acid molecule is preferably amplified for ease of detection. Amplification of the reporter nucleic acid molecule may be performed by PCR, or by an isothermal amplification reaction, such as loop-mediated isothermal amplification (LAMP) or Recombinase Polymerase Amplification (RPA) but is not limited thereto.

[0128] Herein, step iv) of generating the reporter nucleic acid molecules comprises hybridization between the first and second nucleic acid domain of each proximity probe through paired hybridization sequences present in said first and second nucleic acid domain of said probes or hybridization through a common splint oligonucleotide, said common splint oligonucleotide comprising hybridization sequences complementary to each of the paired hybridization sequences of the first and second nucleic acid domain of said probes, wherein said hybridization is followed by an extension or a ligation reaction, respectively.

[0129] A PEA (Proximity Extension Assay) and PLA (Proximity Ligation Assay), as defined and explained in detail elsewhere herein, include an extension ora ligation reaction, respectively. Hence, these are preferred ways of performing a method as disclosed herein.

[0130] In a PLA, the nucleic acid domains which are coupled to the analyte binding domains of the respective proximity probes of a proximity probe pair do not have regions of complementarity and are therefore unable to form a duplex directly. Instead, a third nucleic acid molecule is provided that has a region of homology with the nucleic acid domain of each proximity probe. This third nucleic acid molecule acts as a “molecular bridge" or a "splint" between the nucleic acid domains. Herein this is referred to a common splint oligonucleotide and it bridges the gap between the nucleic acid domains of the proximity probes, allowing them to interact with each other indirectly.

[0131] Detection of the at least one analyte of interest through detection of the generated reporter nucleic acid molecule may be performed in various ways, such as by sequencing.

[0132] By sequencing all reporter nucleic acid molecules generated, all the different reporter nucleic acid molecules generated may be identified by their sample, and optionally analyte,identification sequences. Nucleic acid sequencing is the preferred method of reporter nucleic acid detection / analysis.

[0133] Preferably, a form of high throughput DNA sequencing is used to detect the reporter nucleic acid molecules. 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.

[0134] Preferably the reporter nucleic acid molecules are sequenced using massively parallel DNA sequencing. Massive parallel sequencing or massively parallel sequencing are any of several high-throughput approaches to DNA sequencing that uses the concept of massively parallel processing. This is also called next-generation sequencing (NGS) or second-generation sequencing. The use of NGS in PEA is further described in Wik et al, 2021.

[0135] 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.

[0136] 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.

[0137] 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 methods herein may thus include the addition of one or more adapters for sequencing (sequencing adapters) to the reporter nucleic acid molecules.

[0138] Herein, the Illumina P5 and P7 sequencing adapters are a preferred pair of sequencing adapters. Sometimes, the P5 sequencing adapter is added to the reporter nucleic acid molecule in a first PCR amplification and the P7 sequencing adapter is added to the reporter nucleic acid molecule in a first or second second PCR amplification. Sometimes, the P7sequencing adapter is added to the reporter nucleic acid molecule in the first PCR amplification and the P5 sequencing adapter is added to the reporter nucleic acid molecule in the second PCR amplification. Preferably, the P5 and P7 sequences are present in the nucleic acid domains already from the start of the method.

[0139] Further details of sequencing as a manner of detecting and analysing the generated reporter nucleic acid molecules and as applicable to the present disclosure may be found in US20230159983, incorporated herein by reference.

[0140] Other 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 is amplified, and a probe complementary to each sequence is provided, with each different probe being conjugated to a different, distinguishable fluorophore. The presence or absence of each sequence can then be determined based on whether the particular sequence is amplified. However, it is apparent that PCR-based methods such as described above may only be suitable for analysis of relatively small numbers of different sequences at the same time, although combinatorial methods using probes for decoding identifying sequences are known and may be used to extend multiplexing capacity to a degree. Nucleic acid sequencing enables higher levels of multiplex reaction than detection using PCR, hence sequencing is the preferred method for reporter nucleic acid molecule detection.

[0141] In a second aspect of the present invention, there is provided a sample-specific probe composition comprising at least one proximity probe pair, each proximity probe pair comprising:

[0142] a) a first proximity probe comprising a first analyte binding domain coupled to a first nucleic acid domain, and;

[0143] b) a second proximity probe comprising a second analyte binding domain coupled to a second nucleic acid domain,

[0144] wherein the first and second analyte binding domains of said proximity probe pair are capable of simultaneously and specifically binding to a specific analyte of interest; wherein the first and second nucleic acid domain(s) are capable of interacting to generate a reporter nucleic acid molecule when said first and second proximity probes are both bound to the analyte of interest; and

[0145] wherein the first and / or second nucleic acid domain(s) comprise(s) a sample identification sequence identifying the sample-specific probe composition.Details, features and advantages of a sample-specific probe composition in relation to the first aspect of the present invention have been described elsewhere herein and are equally applicable to the second aspect of the invention.

[0146] Hence, as described elsewhere herein, both the first and second nucleic acid domains of a sample-specific probe composition may comprise a sample identification sequence.

[0147] Furthermore, the first and / or second nucleic acid domain(s) of each proximity probe pair of a sample-specific probe composition may further comprise an analyte identification sequence identifying the specific analyte of interest. Alternatively, one or both of the first and second nucleic acid domain(s) of each proximity probe pair comprises an analyte identification sequence identifying the specific analyte of interest. Examples of suitable first and / or second analyte-binding domain(s) of each proximity probe are described elsewhere herein.

[0148] Furthermore, at least one member of each proximity probe pair, i.e. the first and / or the second proximity probe of a proximity probe pair may comprise an anchor moiety for capturing the proximity probe or a proximity probe-analyte complex on a solid phase carrying a corresponding capture moiety. The anchor moiety may be incorporated in the proximity probe by coupling it to the analyte binding moiety, optionally through a linker or spacer to avoid steric hindrance and facilitate that the anchor moiety is available to bind to the capture moiety. The anchor moiety may also be incorporated in the proximity probe by coupling it to the nucleic acid domain, either directly to the nucleic acid domain coupled to the analyte binding domain or to a nucleic acid strand hybridized to the nucleic acid domain.

[0149] If two capture steps are desired, with an intermediate release from the solid-phase, it is possible to include two different anchor moietys adapted for use with different capture moietys, to use two different capture-release mechanisms in the method. Various configurations are shown in Figure 1 and 2, and further explained below. Examples of suitable anchor and capture moieties include but are not limited to biotin and streptavidin / avidin / neutravidin (using e.g. Dynabeads from Thermo Fisher Scientific) and complementary nucleic acid molecules, e.g. a poly-A strand as anchor moiety for capture on an oligo-dT bead. Other examples of binding pairs that may make up an anchor-capture pair include, but are not limited to, an antigen and an antibody against the antigen (including its fragments and derivatives), a ligand and its receptor, lectin and carbohydrates (such as fucose and fucose binding peptides as described in US8178319, incorporated herein by reference).

[0150] Furthermore, in a third aspect of the present invention, there is provided a kit comprising multiple sample-specific probe compositions as described elsewhere herein, optionally further comprising one or more reagents and optionally instructions for use to perform themethod of the present invention. The sample-specific probe compositions and / or the one or more reagents of a kit may be provided in dried form, generally as described in WO2025012398, but is not limited thereto.

[0151] Variants of proximity probe configurations

[0152] As described, the present invention builds on the principle of dual-recognition, which includes allowing two binding molecules to simultaneously bind to an analyte of interest, wherein the binders have nucleic acid domains that interact to form a reporter nucleic acid molecule only when both binders are simultaneously bound to the analyte.

[0153] A number of configurations of dual-recognition probes have been suggested in the art (as cited herein), and such configurations may be adapted for use with the present invention. A representative sample of proximity assay formats is shown schematically in Figure 1 and these embodiments are described in detail below. Versions 1-6 are also described in US patent publication US 2023-0159983, incorporated herein by reference. In general, in a proximity assay, upon binding of a pair of proximity probes to their target analyte the nucleic acid domains of the two probes come into proximity of each other and interact (i.e. directly or indirectly hybridise to one another). The interaction between the two nucleic acid domains yields a nucleic acid duplex comprising at least one free 3’ end (i.e. at least one of the nucleic acid domains within the duplex has a 3’ end which can be extended) in case of a Proximity Extension Assay, or a free 5’ end and a free 3’-end that can be ligated together in case of a Proximity Ligation Assay. Addition or activation of a nucleic acid polymerase or ligase enzyme within the assay mix leads to extension or ligation, as the case may be. The extension / ligation product obtained is a reporter nucleic acid molecule as used herein, comprising a barcode sequence which indicates the presence of the analyte bound by the proximity probe pair from which the extension / ligation product was produced.

[0154] Version 1 of Figure 1 depicts a "conventional" proximity extension assay, wherein the nucleic acid domain (shown as an arrow) of each proximity probe is attached to the analyte-binding domain (shown as an inverted "Y") by its 5' end, thereby leaving two free 3' ends. When said proximity probes bind to their respective analyte (the analyte is not shown in the figure) the nucleic acid domains of the probes, which are complementary at their 3' ends, are able to interact by hybridisation, i.e. to form a duplex. The addition or activation of a nucleic acid polymerase enzyme in the assay mixture allows each nucleic acid domain to be extended using the nucleic acid domain of the other proximity probe as template. As detailed above, the resultant extension product is a reporter nucleic acid molecule which is detected, thereby detecting the analyte bound by the probe pair.Version 2 of Figure 1 depicts an alternative proximity extension assay, wherein the nucleic acid domain of the first proximity probe is attached to the analyte-binding domain by its 5' end and the nucleic acid domain of the second proximity probe is attached to the analytebinding domain by its 3' end. The nucleic acid domain of the second proximity probe therefore has a free 5' end (shown as a blunt arrow), which cannot be extended using a typical nucleic acid polymerase enzyme (which extend only 3' ends). The 3' end of the second proximity probe is effectively "blocked", i.e. it is not "free" and it cannot be extended because it is conjugated to, and therefore blocked by, the analyte-binding domain. In this embodiment, when the proximity probes bind to their respective analyte-binding targets on the analyte, the nucleic acid domains of the probes, which share a region of complementarity at their 3' ends, are able to interact by hybridisation, i.e. form a duplex. However, in contrast to version 1 , only the nucleic acid domain of the first proximity probe (which has a free 3' end) may be extended using the nucleic acid domain of the second proximity probe as a template, yielding an extension product (i.e. reporter nucleic acid molecule).

[0155] In version 3 of Figure 1 , like version 2, the nucleic acid domain of the first proximity probe is attached to the analyte-binding domain by its 5' end and the nucleic acid domain of the second proximity probe is attached to the analyte-binding domain by its 3' end. The nucleic acid domain of the second proximity probe therefore has a free 5' end (shown as a blunt arrow), which cannot be extended. However, in this embodiment, the nucleic acid domains which are attached to the analyte binding domains of the respective proximity probes do not have regions of complementarity and therefore are unable to form a duplex directly. Instead, a third nucleic acid molecule is provided that has a region of homology with the nucleic acid domain of each proximity probe. This third nucleic acid molecule acts as a "molecular bridge" or a "splint" between the nucleic acid domains. This "splint" oligonucleotide bridges the gap between the nucleic acid domains, allowing them to interact with each other indirectly, i.e. each nucleic acid domain forms a duplex with the splint oligonucleotide.

[0156] Thus, when the proximity probes bind to their respective analyte-binding targets on the analyte, the nucleic acid domains of the probes each interact by hybridisation, i.e. form a duplex, with the splint oligonucleotide. It can be seen therefore that the third nucleic acid molecule or splint may be regarded as the second strand of a partially double stranded nucleic acid domain provided on one of the proximity probes. For example, one of the proximity probes may be provided with a partially double-stranded nucleic acid domain, which is attached to the analyte binding domain via the 3' end of one strand and in which the other (non-attached) strand has a free 3' end. Thus such a nucleic acid domain has a terminal single stranded region with a free 3' end. In this embodiment the nucleic acid domain of the first proximity probe (which has a free 3' end) may be extended using the"splint oligonucleotide" (or single stranded 3' terminal region of the other nucleic acid domain) as a template. Alternatively or additionally, the free 3' end of the splint oligonucleotide (i.e. the unattached strand, or the 3' single-stranded region) may be extended using the nucleic acid domain of the first proximity probe as a template.

[0157] The configuration of nucleic acid domains in Version 3 is in principle the same as in a regular Proximity Ligation Assay shown in Version 10, the difference between the assays being that the splint oligonucleotide is used to bring the nucleic acid domains in proximity to be ligated together using an added ligase.

[0158] As is apparent from the above description, in one embodiment, the splint oligonucleotide may be provided as a separate component of the assay. In other words it may be added separately to the reaction mix (i.e. added separately to the proximity probes to the sample containing the analytes). Notwithstanding this, since it hybridises to a nucleic acid molecule which is part of a proximity probe, and will do so upon contact with such a nucleic acid molecule, it may nonetheless be regarded as a strand of a partially double-stranded nucleic acid domain, albeit that it is added separately. Alternatively, the splint may be pre-hybridised to one of the nucleic acid domains of the proximity probes, i.e. hybridised prior to contacting the proximity probe with the sample. In this embodiment, the splint oligonucleotide can be seen directly as part of the nucleic acid domain of the proximity probe, i.e. wherein the nucleic acid domain is a partially double-stranded nucleic acid molecule, e.g. the proximity probe may be made by linking a double-stranded nucleic acid molecule to an analyte-binding domain (preferably the nucleic acid domain is conjugated to the analyte-binding domain by a single strand) and modifying said nucleic acid molecule to generate a partially doublestranded nucleic acid domain (with a single-stranded overhang capable of hybridising to the nucleic acid domain of the other proximity probe).

[0159] Hence, the extension of the nucleic acid domain of the proximity probes as defined herein encompasses also the extension of the "splint" oligonucleotide. Advantageously, when the extension product arises from extension of the splint oligonucleotide, the resultant extended nucleic acid strand is coupled to the proximity probe pair only by the interaction between the two strands of the nucleic acid molecule (by hybridisation between the two nucleic acid strands). Hence, in these embodiments, the extension product may be dissociated from the proximity probe pair using denaturing conditions, e.g. increasing the temperature, decreasing the salt concentration etc.

[0160] Whilst the splint oligonucleotide depicted in Version 3 of Figure 1 is shown as being complementary to the full length of the nucleic acid domain of the second proximity probe, this is merely an example and it is sufficient for the splint to be capable of forming a duplexwith the ends (or near the ends) of the nucleic acid domains of the proximity probes, i.e. to form a bridge between the nucleic acid domains of the two probes, as is shown in Version 10.

[0161] In another embodiment, shown in Version 11 , the splint oligonucleotide may be provided as the nucleic acid domain of a third proximity probe as described in US patent 8,268,554, which is incorporated herein by reference, which demonstrates that this can further improve the sensitivity and specificity of proximity probe assays. The splint configuration shown in Version 11 may be used both in PEA and PLA.

[0162] Version 4 of Figure 1 is a modification of Version 1, wherein the nucleic acid domain of the first proximity probe comprises at its 3' end a sequence that is not fully complementary to the nucleic acid domain of the second proximity probe. Thus, when said proximity probes bind to their respective analyte the nucleic acid domains of the probes are able to interact by hybridisation, i.e. to form a duplex, but the extreme 3' end of the nucleic acid domain (the part of the nucleic acid molecule comprising the free 3' hydroxyl group) of the first proximity probe is unable to hybridise to the nucleic acid domain of the second proximity probe and therefore exists as a single stranded, unhybridised, "flap". On the addition or activation of a nucleic acid polymerase enzyme, only the nucleic acid domain of the second proximity probe may be extended using the nucleic acid domain of the first proximity probe as template. Version 5 of Figure 1 could be viewed as a modification of Version 3. However, in contrast to Version 3, the nucleic acid domains of both proximity probes are attached to their respective analyte-binding domains by their 5' ends. In this embodiment the 3' ends of the nucleic acid domains are not complementary and hence the nucleic acid domains of the proximity probes cannot interact or form a duplex directly. Instead, a third nucleic acid molecule is provided that has a region of homology with the nucleic acid domain of each proximity probe. This third nucleic acid molecule acts as a "molecular bridge" or a "splint" between the nucleic acid domains. This "splint" oligonucleotide bridges the gap between the nucleic acid domains, allowing them to interact with each other indirectly, i.e. each nucleic acid domain forms a duplex with the splint oligonucleotide. Thus, when the proximity probes bind to their respective analyte, the nucleic acid domains of the probes each interact by hybridisation, i.e. form a duplex, with the splint oligonucleotide.

[0163] In accordance with Version 3, it can be seen therefore that the third nucleic acid molecule or splint may be regarded as the second strand of a partially double stranded nucleic domain provided on one of the proximity probes. In a preferred example, one of the proximity probes may be provided with a partially double-stranded nucleic acid domain, which is attached to the analyte binding domain via the 5' end of one strand and in which the other (non-attached)strand has a free 3' end. Thus such a nucleic acid domain has a terminal single stranded region with at least one free 3' end. In this embodiment the nucleic acid domain of the second proximity probe (which has a free 3' end) may be extended using the "splint oligonucleotide" as a template. Alternatively or additionally, the free 3' end of the splint oligonucleotide (i.e. the unattached strand, or the 3' single-stranded region of the first proximity probe) may be extended using the nucleic acid domain of the second proximity probe as a template.

[0164] As discussed above in connection with Version 3, the splint oligonucleotide may be provided as a separate component of the assay. On the other hand, since it hybridises to a nucleic acid molecule which is part of a proximity probe, and will do so upon contact with such a nucleic acid molecule, it may be regarded as a strand of a partially double-stranded nucleic acid domain, albeit that it is added separately. Alternatively, the splint may be pre-hybridised to one of the nucleic acid domains of the proximity probes, i.e. hybridised prior to contacting the proximity probe with the sample. In this embodiment, the splint oligonucleotide can be seen directly as part of the nucleic acid domain of the proximity probe, i.e. wherein the nucleic acid domain is a partially double-stranded nucleic acid molecule, e.g. the proximity probe may be made by linking a double-stranded nucleic acid molecule to an analyte-binding domain (preferably the nucleic acid domain is conjugated to the analyte-binding domain by a single strand) and modifying said nucleic acid molecule to generate a partially doublestranded nucleic acid domain (with a single-stranded overhang capable of hybridising to the nucleic acid domain of the other proximity probe).

[0165] Hence, the extension of the nucleic acid domain of the proximity probes as defined herein encompasses also the extension of the "splint" oligonucleotide. Advantageously, when the extension product arises from extension of the splint oligonucleotide, the resultant extended nucleic acid strand is coupled to the proximity probe pair only by the interaction between the two strands of the nucleic acid molecule (by hybridisation between the two nucleic acid strands). Hence, in these embodiments, the extension product may be dissociated from the proximity probe pair using denaturing conditions, e.g. increasing the temperature, decreasing the salt concentration etc.

[0166] Whilst the splint oligonucleotide depicted in Version 5 of Figure 1 is shown as being complementary to the full length of the nucleic acid domain of the first proximity probe, this is merely an example and it is sufficient for the splint to be capable of forming a duplex with the ends (or near the ends) of the nucleic acid domains of the proximity probes, i.e. to form a bridge between the nucleic acid domains of the proximity probes.In another embodiment, the splint oligonucleotide may be provided as the nucleic acid domain of a third proximity probe as described in US patent 8,268,554, which is incorporated herein by reference, which demonstrates that this can further improve the sensitivity and specificity of proximity probe assays.

[0167] Version 6 of Figure 1 is another embodiment of the present invention. As depicted, both probes in a pair are conjugated to partially single-stranded nucleic acid molecules. A short nucleic acid strand is conjugated via its 5’ end to the analyte-binding domain. The short nucleic acid strands which are conjugated to the analyte-binding domains do not hybridise to each other. Rather, each short nucleic acid strand is hybridized to a longer nucleic acid strand (“hybridization oligonucleotide”), which has a single-stranded overhang at its 3’ end (that is to say, the 3’ end of the hybridization oligonucleotide extends beyond the 5’ end of the shorter strand conjugated to the analyte-binding domain. The overhangs of the two hybridization oligonucleotides hybridise to one another, forming a duplex. If the 3’ ends of the two hybridization oligonucleotides hybridise fully to one another, as shown, the duplex comprises two free 3’ ends, though the 3’ ends of the hybridization oligonucleotides may be designed as in Version 4, such that the extreme 3’ end of one of the hybridization oligonucleotides is not complementary to the other, forming a flap, meaning that the duplex contains only one free 3’ end. The two hybridization oligonucleotides which interact with one another may be seen as splint oligonucleotides, in that together they form a bridge between the two short oligonucleotides which are directly conjugated to the analyte-binding domains. Addition or activation of a nucleic acid polymerase results in extension of the free 3’ end or ends of the hybridization / splint oligonucleotides. Notably, extension of either splint oligonucleotide uses the other splint oligonucleotide as template. Thus, when one splint oligonucleotide is extended, the other “template” splint oligonucleotide is displaced from the shorter strand which is conjugated to the analyte-binding domain.

[0168] In a preferred embodiment, the short nucleic acid strand (“conjugation oligo”) conjugated directly to the analyte-binding domain is a “universal strand”. That is to say, the same strand is conjugated directly to every proximity probe used in the multiplex detection assay. Each splint oligonucleotide therefore comprises a “universal site”, which consists of the sequence which hybridises to the universal strand, and a “unique site”, which comprises a barcode sequence unique to the probe. Such proximity probes, and methods for making them, are described in US10781473, incorporated by reference herein.

[0169] The probes of Version 6 can easily be adapted for use in a PLA setting, as shown in Version 12. In this version, the sequences of the hybridization oligonucleotides are designed so that they can both hybridize to a splint oligonucleotide that facilitates ligation as described above.Version 7 of Figure 1 can be considered a variant of Version 6, wherein the universal site, i.e. the site where the hybridization oligonucleotide and the conjugation oligonucleotide can hybridize, is located internally within the hybridization oligonucleotide so that the hybridization oligonucleotide has both a 3’-overhang and a 5’-overhang. The sequences of the 5’-ends of the hybridization oligonucleotides will template the sequences of the 3’ and 5’-ends of generated reporter molecules. It is thus convenient to add sequence elements that are required at the ends of reporter molecules at the 5’-ends of the hybridization oligos, e.g. sequencing adapters such as P5 and P7 The 5’-overhang in this version may also contain an identification sequence, e.g. an analyte or sample identification sequence. The 3’-overhang in this version may also contain an identification sequence, e.g. an analyte or sample identification sequence. In one embodiment, the 5’-overhang contains a sample identification sequence and the 3’-overhang contains an analyte identification sequence. In one embodiment, the 5’-overhang contains an analyte identification sequence and the 3’-overhang contains a sample identification sequence.

[0170] Similar to Version 6, also the probes of Version 7 can easily be adapted for use in a PLA setting, as shown in Version 13. In this version, the sequences of the hybridization oligonucleotides are designed so that they can both hybridize to a splint oligonucleotide that facilitates ligation as described above. As can be seen, the sequence elements templating the ends of the reporter molecule will be located at the 5’-end of one hybridization oligonucleotide and at the 3’-end of the other hybridization oligonucleotide. Placement of the desired sequence elements, e.g. identification sequences and sequencing adapters, thus needs to be adjusted accordingly.

[0171] Versions 8, 9, and 14-16 illustrates further embodiments, based on Versions 7 and 12, adapted for capture of one of the probes, or the probe-analyte complex, on a solid phase. These versions make use of a further “anchor oligonucleotide” comprising an anchor group at its 5’-end, wherein the anchor group can attach to a corresponding capture group on a solid phase, as known in the art. In Version 8, the anchor oligonucleotide is able to hybridize to a single stranded part of one of the hybridization oligos. The anchor oligonucleotide can be hybridized to the hybridization oligonucleotide already at manufacture of the probes, or added at a later stage of the method in which the probes are used. The anchor oligonucleotide can also be provided pre-attached to the solid phase to hybridize to the hybridization oligonucleotide when the probes are combined with the solid phase. In Version 14, the anchor oligonucleotide and the splint oligonucleotide is embodied in the same molecule, whereas Version 15 shows an embodiment where the anchor oligonucleotide and the splint oligonucleotide are separate molecules. In Versions 9 and 16, the anchor moiety is attached to the 5’-end of one of the hybridization oligos. Version 17 is an illustration of howthe present invention may be implemented in a PLA method as generally described in US12105084.

[0172] Figure 2 discloses some of the probe configurations shown in Figure 1 in greater detail. Figure 2A corresponds to Version 1 of Figure 1 , and is also shown in Wik et al., 2021 , Mol Cell Proteomics 20, 100168. Following the nomenclature adopted in Wik et al., the left hand probe is termed the “forward probe” and the right hand probe is termed the “reverse probe”. The nucleic acid moiety of the forward probe comprises the following sequence elements in 5’-3’ direction: a=lllumina P5 sequencing adapter; b=lllumina read 1 sequencing primer site (Rd1SP); fa=forward analyte identification sequence (termed “forward barcode” in Wik et al.); and h=hybridization sequence. The nucleic acid moiety of the reverse probe comprises the following sequence elements in 3’-5’ direction: h’=hybridization site able to hybridize to sequence element h of the forward probe; c= spacer; ra= reverse analyte identification sequence (termed “reverse barcode” in Wik et al.); and d=primer binding site for incorporating a sample index sequence and the Illumina P7 sequencing adapter in a separate amplification reaction.

[0173] The configuration in Figure 2B corresponds to Version 7 in Figure 1. The sequence element references in Fig 2B refer to equivalent sequence elements as in Fig 2A. In the configuration of Fig 2B, the forward probe has a conjugation oligonucleotide comprising the sequence b’ coupled (covalently or non-covalently) to it at its 5’-end. b' is the reverse complement of sequence element b, so that b and b’ can hybridize to form a duplex. The hybridization oligonucleotide of the forward probe comprises the same sequence elements a, b, fa, and h as the nucleic acid moiety of the forward probe in Fig 2A, and in addition a sequence element fs=forward sample identification sequence. In Fig 2A, fs is placed between a and b, but it may also be positioned between b and fa, or between fa and h. Correspondingly, the reverse probe has a conjugation oligonucleotide comprising the sequence d’ coupled (covalently or non-covalently) to it at its 5’-end. d' is the reverse complement of sequence element d, so that d and d’ can hybridize to form a duplex. The hybridization oligonucleotide of the reverse probe comprises the same sequence elements d, ra, c and h’ as the nucleic acid moiety of the reverse probe in Fig 2A, and in addition a sequence element rs= reverse sample identification sequence and e=the P7 sequencing adapter. In Fig 2A, rs is placed between e and d, but it may also be positioned between d and ra, or between ra and h’. The configuration in Figure 2C also corresponds to Version 7 in Figure 1 but includes a further sequence element allowing hybridization of an anchor oligonucleotide to arrive at Version 8. The sequence element references in Fig 2C refer to equivalent sequence elements as in Fig 2B. In the configuration of Fig 2C, the forward probe further has asequence element g, placed between fa and h, comprising a hybridization site for an anchor oligonucleotide. As in the previously described configurations, sequence element g can be placed in other locations as well, such as between a and fs, between fs and b, or between b and fa, or at corresponding locations in the hybridization oligonucleotide of the reverse probe.

[0174] Figure 2D shows the configuration of Fig 2C with an anchor oligonucleotide hybridized to the hybridization oligonucleotide of the forward probe. The anchor oligonucleotide comprises, from 3’-5’, the following sequence elements: t= a toehold sequence enabling toehold mediated strand displacement, and g'=the reverse complement of sequence element g in the hybridization oligonucleotide. The anchor oligonucleotide also has an anchor group at its 5’-end such that it can be immobilised on a solid-phase. A spacer element may be included between sequence element g and the 5’-end, to provide adequate flexibility and accessibility for the anchor group to attach to a capture group on a solid-phase, as applicable. The spacer may be a stretch of nucleotides, or another type of spacer as routinely used in organic chemistry.

[0175] Figure 3 illustrates some steps of one embodiment of the method as disclosed herein. In step I, two sample-specific probe compositions are incubated with two samples (Sample 1 and Sample 2), to form sample-probe combinations. This is done in separate reactions, as indicated by the dashed boxes. The samples contain analytes (3102, 3104, 3202, 3204) and matched pairs of forward (306) and reverse (308) probes, wherein both probes in a matched pair bind the same analyte. At least one of the forward and reverse probes, preferably both, carry a sample identification sequence identifying the sample-specific probe composition as either “Sample 1” or “Sample 2”, respectively. If the method is intended to measure only one analyte (i.e. 3102 and 3104 are two identical molecules, as are 3202 and 3204), then no analyte identification sequences are necessary as the detected reporter molecules comprising the sample identification sequence(s) to 100% originate from detection of that same analyte. If the method is intended to measure more than one analyte, at least one of the forward and reverse probes, preferably both, comprise analyte identification sequences as well, as described in figure 2. The forward probes have an anchor oligonucleotide (310) attached to the nucleic acid moiety. As shown in this figure, the anchor oligonucleotide can be attached already at the start of the protocol, or even at manufacture. However, it is equally possible to add the anchor oligonucleotide at a later stage, such as in step II or step III of Figure 3.

[0176] In step II, the probes are allowed to bind to their respective analyte molecules in separate reactions for each sample. When the probe-analyte complexes have formed, all samples arepooled in step III. In step IV, the pooled sample-probe combinations are brought into contact with a solid phase (312) comprising a capture group (314) to which the anchor group of the anchor oligonucleotide can bind. Unbound probes and other reagents are washed away in step V. In step VI, a displacer oligonucleotide (316) is added to displace the forward probe from the anchor oligonucleotide, which is left attached to the solid phase. The so released probe-analyte complexes can be eluted and reporter molecules generated and readout performed as described elsewhere herein. Only probe-analyte complexes with two bound proximity probes will generate a reporter molecule. In the figure, the probes bound to analytes 3104, 3202, and 3204 will generate reporter molecules. Single probes, both bound and unbound to an analyte, will not have a matching probe to interact with, and will thus not generate reporter molecules. In the figure, analyte 3102 is bound to a single probe, which cannot on its own generate a reporter molecule.

[0177] Production of proximity probes

[0178] Proximity probes, or detection probes, as used in the present invention are generally composed of an analyte-binding domain and a nucleic acid domain.

[0179] 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 (Uhlen et al., Nat Methods, 2016 Oct; 13(10), 823-827, incorporated herein by reference).

[0180] 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.

[0181] 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 include, among others, ThermoFisher 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, 1995, 37(3): 93-118), llgu and Nilsen-Hamilton (Analyst. 2016, 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.

[0182] 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.

[0183] 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.

[0184] As described further above, 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, IQ-15 or 15-20 nucleotides. The oligonucleotide may also contain sequences related to primer sites and / or sequencing adaptors for readout, 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.

[0185] 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 availablefrom 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).

[0186] 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.

[0187] 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.

[0188] Specific linker groups that may find use in the presently disclosed proximity probes include heterofunctional compounds, such as azidobenzoyl hydrazide, N-[4-(p-azidosalicylaminojbutylj-S'-^'-pyridyldithiojpropionamide, 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.

[0189] 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.

[0190] 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 of such detection probes are described i.a. in international patent publication WO 2017 / 068116 (US2018312901A1), incorporated by reference in its entirety herein.

[0191] In an embodiment of the present invention, one or both members of the hybridizable oligonucleotide pair is attached at its 5’-end to its respective analyte-binding moiety. In another embodiment, one or both members of the hybridizable oligonucleotide pair is hybridized to a partially complementary oligonucleotide, which is attached (at its 5’-end or 3’-end) to its respective analyte-binding moiety, as described in WO2017 / 068116

[0192] (US2018312901 A1), incorporated by reference in its entirety herein.

[0193] All embodiments as described herein are applicable in all aspects of the invention as well as in any combination.

[0194] All prior publications cited in the present specification are incorporated by reference in their entirety.

[0195] The invention will be further described in the following illustrative example(s). The example(s) are merely for facilitating understanding of the invention and shall not be construed as limiting the scope of the invention, which is that of the appended claims.EXPERIMENTAL SECTION

[0196] Example 1

[0197] This example relates to an implementation of the invention describing the advantages of proximity probes of a proximity probe pair comprising sample identification sequences, wherein the sample identification sequences have been introduced already at manufacturing of the probes.

[0198] Material and methods

[0199] A general reference is made to Wik et al., 2021 for details regarding common reagents and process steps referred to in the below.

[0200] Protocol

[0201] 24 different batches of a nine-plex set of PEA probe pairs (forward and reverse) targeting the proteins ROBO1, FABP1, CST7, LTBR, PLAUR, TNSFS13, IL1R2, SIRPB1 and SCGB3A2 were produced to also carry a batch specific sample identification sequence on each probe in the pairs (dual sample indexing), in a similar manner as described elsewhere (see e.g. WO2017 / 068116).

[0202] The probes were also designed to include the Illumina P5 and P7 sequences according to the manufacturer’s instructions to facilitate sequencing of reporter molecules on Illumina instruments.

[0203] The oligonucleotides of the forward probes were further allowed to hybridize to biotinylated DNA linkers carrying a non-matching toehold sequence at its 3’-end (cf. Figure 2D) to enable capture on a streptavidin coated solid phase and release using toe-hold mediated DNA strand displacement (as illustrated in Figure 3). Immune reactions (n = 24) were set up by mixing 7.9 pl of Olink Explore Incubation solution, 1 pl forward probes (1.33 nM), 1 pl reverse probes (1.33 nM) and 3.3 pl sample. The samples used are different concentrations of the nine antigens in purified form (diluted in Olink Sample Diluent) or buffer (Olink Sample Diluent). Each sample was incubated with PEA probes from a separate batch, so that the batch specific index sequences were used as sample identification sequences.

[0204] The immune reactions were incubated 18 hours in fridge before being separated into two different downstream protocols with twelve incubations each.

[0205] Protocol 1:

[0206] 10 pl from each immune reaction (n = 12) was pooled into a reagent reservoir that was prefilled with 1 ml of wash buffer (PBS, 0.1% BSA, 0.05% Tween20). The total pooled anddiluted volume (1120 pl) was then mixed with 100 pl pre-washed streptavidin coated magnetic beads (ThermoFisher MyOne Streptavidin T1) and incubated at room temperature for 5 min. The beads were then washed twice with 1 ml wash buffer followed by addition of 30 pl release buffer (PBS, 0.1% BSA, 100 nM DNA displacer) and incubation for 5 min at room temperature.

[0207] 20 pl of eluate were then used as input into two parallel 100 pl extension and PCR reactions according to standard Olink PEA protocol. Extension and PCR was performed using Pwo DNA polymerase. PCR was performed using P5 and P7 primers for amplification of all extension products.

[0208] The extension mix (comprising ultrapure water, DMSO, Pwo DNA polymerase and PCR solution) was added to the eluate, which was 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). The forward primer comprised the Illumina P5 sequencing adapter sequence and the reverse primer comprised the Illumina P7 sequence.

[0209] PCR solution

[0210] Component Concentration

[0211] Tris base 168.40 mM

[0212] Tris-HCI 31.47 mM

[0213] MgCk hexahydrate 10.00 mM

[0214] dATP 2.00 mM

[0215] dCTP 2.00 mM

[0216] dGTP 2.00 mM

[0217] dTTP 2.00 mM

[0218] Forward "P5" primer 10.00 pM

[0219] Reverse “P7” primer 10.00 pM

[0220]

[0221] Protocol 2:

[0222] 4 pl from each of the other immune reactions (n=12) was sequentially pooled directly into a single tube, yielding a final volume of 48 pl. This is referred to as “limited dilution” below. Thepooled immune reactions (48 pl) were mixed and then 10 pl were used as input for two parallel 100 pl extension and PCR reactions as in Protocol 1.

[0223] Pooling

[0224] After amplification, all the PCR reactions from Protocols 1 and 2 (n = 4) were pooled together and sequenced using Illumina NextSeq 550 high output sequencing kit.

[0225] Results

[0226] Sequencing reads matching the expected barcode combinations (matched / interacting proximity probes of a proximity probe pair) were assigned to different samples and counted using either one of the two sample identifying sequences of the first and second proximity probe (“single indexing forward and single indexing reverse”) or using matched sample identifying sequences of the first and second proximity probe (“forward and reverse sample indices”, dual indexing) resulting in three different variants of quantitation for each sample and protocol condition. Log2-values of the counts for these different quantitation methods were then individually plotted against the input concentration of the nine different antigens (Figure 4).

[0227] Conclusions

[0228] The dynamic range of a detection assay is the range of analyte concentrations where the assay can adequately measure the concentration of the analyte. The lowest concentration that the assay can reliably measure is the Lower Limit of Detection, (LLOD).

[0229] When samples are pooled between the incubation step and the extension step, individual proximity probes may dissociate from its target analyte molecule and re-bind to analyte molecules from a different sample. Count values taking only one sample identification sequence (originating from either the forward or reverse probe) into account correspond to a probe design wherein only one of the probes in a probe pair carries a sample index.

[0230] The data in Figure 4 clearly shows that implementing the dual index design (wherein both proximity probes of a proximity probe pair comprises a sample identification sequence) expands the dynamic range for the assay using limited dilution and no solid phase, as the curve for the dual index design (light grey line, upper panel, also marked as A) carries useful information (i.e. a given signal can be correlated to a certain concentration) over a wider range as compared to the single sample ID sequence designs (light grey lines in the two lower panels, also marked as A).It is also shown in the data that the assay using limited dilution and no solid phase, but only the two sample ID feature, is capable of measuring lower concentrations of analyte, i.e. has a lower (improved) LLOD.

[0231] It is also highly beneficial to include a combination of dilution in the pooling reaction followed by a solid phase capture to further limit signal cross-over between samples (dark grey as compared to light grey lines in the upper panel in figure 2, also marked as •). This most preferred set of conditions ensure maintained low background signals which results in a broad dynamic range and high sensitivity like current Olink PEA protocols with individual extension- and amplification reactions for each sample (data not shown).

Claims

AMENDED CLAIMSreceived by the International Bureau on 25 May 2026 (25.05.2026)1. A method for detection of at least one analyte of interest in multiple samples simultaneously, said method comprising the steps of:(i) providing multiple sample-specific probe compositions, each sample-specific probe composition comprising at least one proximity probe pair, each proximity probe pair comprising:a) a first proximity probe comprising a first analyte binding domain coupled to a first nucleic acid domain, and;b) a second proximity probe comprising a second analyte binding domain coupled to a second nucleic acid domain,wherein the first and second analyte binding domains of said proximity probe pair are capable of simultaneously and specifically binding to a specific analyte of interest present, or suspected of being present, in the samples,wherein the first and second nucleic acid domain are capable of directly or indirectly interacting to generate a reporter nucleic acid molecule when said first and second proximity probes are both bound to the same analyte;wherein the first and / or second nucleic acid domain(s) comprise(s) a sample identification sequence identifying each sample-specific probe composition,(ii) bringing each sample separately into contact with a sample-specific probe composition to form a sample-probe combination, and allowing the proximity probes of said sample-specific probe composition to bind to the analytes in each sample-probe combination;(iii) pooling all sample-probe combinations of step (ii);(iv) generating reporter nucleic acid molecules from interacting first and second nucleic acid domains; and(v) detecting the at least one analyte of interest through detection of the generated reporter nucleic acid molecules of step (iv) comprising the respective sample identification sequences.

2. The method of claim 1 , wherein both of the first and second nucleic acid domains of each proximity probe pair comprise a sample identification sequence.

3. The method of claim 1 or 2, wherein the method is for detecting a plurality of analytes of interest, and wherein the first and / or second nucleic acid domain of each proximity probe pair comprise an analyte identification sequence identifying the specific analyteof interest.

4. The method of claim 3, wherein both of the first and second nucleic acid domains of each proximity probe pair comprise an analyte identification sequence identifying the specific analyte of interest.

5. The method of any preceding claim, wherein each sample-probe combination is diluted at least 1:10, preferably at least 1 :20, 1:30, 1 :40, 1:50, 1 : 100 or 1 :200, when the sample-probe combinations are pooled in step (iii).

6. The method of any preceding claim, wherein pooling of the sample-probe combinations in step (iii) is performed sequentially or simultaneously, optionally into a dilution buffer.

7. The method of any preceding claim, wherein step (iv) is initiated no more than 60 minutes after initiating step (iii), such as after no more than 1, 2, 3, 4, 5, 10,15, 20, 25, 30, 45 minutes after initiating step (iii).

8. The method of any preceding claim, wherein step (iv) is preceded by a step of capturing the first or second proximity probe of all sample-specific probe compositions comprised in the pooled sample-probe combinations on a solid phase and subjecting the captured proximity probes to one or more wash steps.

9. The method of claim 8, wherein the proximity probes are released from the solid phase prior to step (iv).

10. The method of claim 8 or 9, wherein the step of capturing the first or second proximity probe of all sample-specific probe compositions comprised in the pooled sampleprobe combinations is initiated no more than 60 minutes after initiating step (iii), such as after no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 45 minutes after initiating step (iii).

11. The method of any preceding claim, wherein step (v) is preceded by a step of amplifying the reporter nucleic acid molecules of step (iv).

12. The method of claim 11, wherein said amplification is performed by Polymerase Chain Reaction (PCR) or by an isothermal amplification reaction.

13. The method of any preceding claim, wherein step iv) of generating the reporter nucleic acid molecules comprises hybridization between the first and second nucleic acid domain of each proximity probe through paired hybridization sequences present in said first and second nucleic acid domain of said probes or hybridization through a common splint oligonucleotide, said common splint oligonucleotide comprising hybridization sequences complementary to each of the paired hybridization sequences of the first and second nucleic acid domain of said probes, wherein said hybridization is followed by an extension or a ligation reaction, respectively.

14. The method of any preceding claim, wherein the first and / or second analyte-binding domain of each proximity probe is or comprises a binding moiety selected from the group consisting of monoclonal, recombinant monoclonal and polyclonal antibodies and antigen-binding antibody derivatives and fragments, 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 nucleic acid molecules comprising the complementary sequence for a target nucleic acid, or combinations thereof.

15. A sample-specific probe composition comprising at least one proximity probe pair, each proximity probe pair comprising:a) a first proximity probe comprising a first analyte binding domain coupled to a first nucleic acid domain, and;b) a second proximity probe comprising a second analyte binding domain coupled to a second nucleic acid domain,wherein the first and second analyte binding domains of said proximity probe pair are capable of simultaneously and specifically binding to a specific analyte of interest; wherein the first and second nucleic acid domain(s) are capable of interacting to generate a reporter nucleic acid molecule when said first and second proximity probes are both bound to the analyte of interest; andwherein the first and / or second nucleic acid domain(s) comprise(s) a sample identification sequence identifying the sample-specific probe composition.

16. The sample-specific probe composition of claim 15, wherein both the first and second nucleic acid domains comprise a sample identification sequence.

17. The sample-specific probe composition of any of claims 15 to 16, wherein the first and / or second nucleic acid domain(s) of each proximity probe pair further comprises an analyte identification sequence identifying the specific analyte of interest.

18. The sample-specific probe composition of any of claims 15 to 17, wherein both of the first and second nucleic acid domain(s) of each proximity probe pair comprise an analyte identification sequence identifying the specific analyte of interest.

19. The sample-specific probe composition of any of claims 15 to 18, wherein at least one member of each proximity probe pair comprises an anchor moiety for capturing the proximity probe or a proximity probe-analyte complex on a solid phase.

20. The sample-specific probe composition of any of claims 15 to 19, wherein the first and / or second analyte-binding domain of each proximity probe is or comprises a binding moiety selected from the group consisting of monoclonal, recombinant monoclonal and polyclonal antibodies and antigen-binding antibody derivatives and fragments, 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 nucleic acid molecules comprising the complementary sequence for a target nucleic acid, or combinations thereof.

21. A kit comprising multiple sample-specific probe compositions of any one of claims 15 to 20, said kit optionally further comprising one or more reagents and optionally instructions for use.

22. The sample-specific probe composition of any of claims 15 to 20, or a kit of claim 21, wherein said sample-specific probe composition and / or said reagents are provided in dried form.

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