Detecting biomolecules in single cells

Abstract

The present invention relates to a method for determining the concentration of at least one target analyte in a sample, comprising: a. Providing at least a first type of binding agent and a second type of binding agent, each binding agent comprising a unique label for said type of binding agent, wherein the first type of binding agent and the second type of binding agent specifically bind to a first target analyte, b. Determining a binding characteristic of the first type of binding agent and the second type of binding agent to the first target analyte, c. Providing a sample comprising a known concentration of cells, d. Contacting the first type of binding agent and the second type of binding agent with the sample wherein the first type of binding agent and the second type of binding agent bind to the first target analyte, thereby forming an analyte-binding agent complex (conjugate), e. Separating a single cell of the sample into a plurality of first partitions, f. Replenishing each of the first partitions comprising single cells with a lysis buffer, thereby lysing the single cells in each of the first partitions, g. Separating a single analyte-binding agent complex (conjugate) into a plurality of second partitions, h. Performing a two-component detection method, and i. Determining the number and / or absolute concentration of conjugates and binding agents in said second partition by taking into account the initial concentration of said cells in said sample in step a., said binding characteristic determined in step b. And the number and / or absolute concentration of conjugates and binding agents determined in step h., and i. Determining the number and / or absolute concentration of conjugates and binding agents in said second partition by taking into account said initial concentration of said cells in said sample in step a., said binding characteristic determined in step b. And said number and / or absolute concentration of conjugates and binding agents determined in step h. An absolute concentration of the first target analyte per cell is determined.

Description

Technical Field

[0001] The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to methods for detecting targets including proteins, post-translationally modified proteins and protein interactions in single cells. The present invention provides such methods with high sensitivity, with a detection limit preferably below 100 or preferably 10 targets / cell, and in absolute molecular numbers. Background Art

[0002] The proteome encompasses areas of the proteome that are equally important for studying proteome function, including the expression proteome involving changes in protein expression levels in different physiological or diseased cellular states, pathway organization determined by protein-protein interactions, enzyme-substrate relationships, and post-translational modifications of proteins, and the structural proteome (complexome) that predicts the three-dimensional structure and higher-order structural assembly of proteins and protein complexes. However, in order to provide a direct link to the role of proteins in the physiological environment of the cell, it is also necessary to evaluate all of the above quantitative information, measuring systematic perturbations or functional inactivation of proteins in their physiological environment to address the potential role of target proteins in cellular processes, similar to genetic disruption that elucidates gene function. Parallel assessment of quantitative protein function at the cellular and molecular levels allows the necessary integrated view to understand the information processing nature of cellular pathways that ultimately determine the work of the cell.

[0003] In the case of single cells, the behavior of individual cells is governed by the following principles: cells are heterogeneous in their work, developmental trajectories, and responses, even if they originally derive from the same ancestor. For example, cells have a certain chance to be in one transcriptional state and a certain chance to be in another transcriptional state, and cells shuttle between states that are only partially controlled by known stimuli. When cells interact with their environment, a range of unusual phenotypes should ensue purely due to these effects. However, observing such cellular behavior amidst overwhelming noise and unsatisfactory analytical capabilities (including low sensitivity measurements) is a daunting task that requires new single-cell analytical methods.

[0004] Existing single cell / cell analysis methods are only partially designed to address this challenge and are defined by collecting the relevant chemical information of the individual members of the large cell population from the comprehensive information processing layer of the cell to the large cell population, including genomic, transcriptomic, and epigenomic methods. However, although proteomic methods have also been developed, these methods are limited in scope compared to more advanced single cell assays based on DNA and RNA. However, no methods are available for single cell interactomes, epi-proteomics (for the purposes of this article, modified proteins (including post-translationally modified proteins) that can be distinguished by antibodies are referred to as epi-proteins (epiproteins)) and complex groups.

[0005] With regard to the sensitivity of single cell methods for the proteome, interactome, epiproteome, and complexome, a sensitivity of 100s or preferably 10s of absolute molecular weight of proteins is required to fully grasp the functional significance of the detected amount. To reach these limits is not possible with contemporary single cell assays, especially for the interactome, epiproteome, and complexome.

[0006] Currently used methods are aimed at capturing genomes, such as MDA-WGA, isothermal amplification (Spits, C. et al. Whole-genome multiple displacement amplification from single cells. Nat. Protoc. 1, 1965–1970 2006), PCR-based DOP-PCR-WGA (Telenius, H. et al. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by asingledegenerate primer. Genomics 13, 718–725 1992), and MALBAC-WGA (Zong, C., Lu, S., Chapman, AR & Xie, XS Genome-wide detection of single nucleotide and copy-number variations of a single human cell. Science 338, 1622–1626 2012). Single-cell genome sequencing reveals the genetic heterogeneity of single cells; applications include the analysis of de novo germline mutations or somatic mutations in normal and cancer cells, however, since the transcriptome also represents a portion of these mutations, transcriptome analysis is also used to elucidate mutation profiles, particularly for B-cell-based somatic hypermutation phenotypes (King HW, Orban N, Riches JC, et al. Antibody Repertoire and Gene Expression Dynamics of Diverse Human B Cell States During Affinity Maturation. SSRN Electron J. 2020; 2: 1-33. doi: 10.2139 / ssrn.3608389).

[0007] Currently used methods also aim to capture the transcriptome, such as Smart-Seq-WTA ( D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30, 777-782 2012), using in vitro transcribed CEL-seq-WTA (Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-Seq: single-cell RNA-seq by multiplexed linear amplification. Cell Rep. 2, 666–6732012), using poly(A)-labeled Quartz-seq-WTA (Sasagawa, Y. et al. Quartz-Seq: a highly reproducible and sensitive single-cell RNA-Seq reveals non-genetic gene expression heterogeneity. Genome Biol. 14, R31 2013), C1-CAGE-5′-end RNA-seq (Kouno, T. et al. C1CAGE detects transcription start sites and enhancer activity at single-cell resolution. Nat. Commun. 10, 360 2019), RamDa-seq-full-length RNA-seq (Hayashi, T. et al. Single-cell full-length total RNA sequencing uncovers dynamics of recursivesplicing and enhancer RNAs. Nat. Commun. 9, 619 2018), droplet-seq-microdroplet-based (Macosko, EZ et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 2015), micropore-seq-micropore-based (Han, X. et al. Mapping the mouse cell atlas by microwell-seq. Cell 172, 1091–1107. e172018).The bulk sample RNA-seq only provides a measurement of the average transcript expression in the cell population. Therefore, in order to accurately describe the transcriptome of a single cell in a heterogeneous cell population, the single-cell RNA-seq method provides a more comprehensive description of the cell state, including the transition between states. scRNAseq transcriptome analysis has been committed to detecting the temporal pattern of the transcriptome to better determine the transition in the pseudo-time space (La Manno G, Soldatov R, Zeisel A et al. RNA velocity of single cells. Nature. 2018; 560(7719): 494-498. doi: 10.1038 / s41586-018-0414-6).

[0008] Currently used methods also aim to capture other aspects of cell physiology (or epigenome), including accessible chromatin, methylation, and histone modifications, such as scBS-seq-DNA methylation (Smallwood, SA et al. Single-cell genome-wide bisulfite sequencing for assessing epigeneticheterogeneity. Nat. Methods 11, 817–820 2014), scRRBS-DNA methylation (Guo, H. et al. Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res. 23, 2126–2135 2013), scAba-seq-DNA methylation 5hmC (Mooijman, D., Dey, SS, Boisset, J.-C., Crosetto, N. & van Oudenaarden, A. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability and enables lineage reconstruction. Nat. Biotechnol. 34, 852–856 2016), scATAC-seq-chromatin accessibility (Buenrostro, JD et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486–490 2015), Drop-ChIP-histone modification ChIP-seq (Rotem, A. et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat. Biotechnol. 33, 1165–1172 2015), scChIC-seq-histone modification (Ku, WLet al. Single-cell chromatinimmunocleavage sequencing (scChIC-seq) to profile histone modification. Nat. Methods 16, 323–325 2019), CUT&Tag-histone modification (Kaya-Okur, HS et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat. Commun. 10, 1930 2019), scHi-C-chromatin structure (Nagano, T. et al. Single-cell Hi-C reveals cell-to-cell variability in chromosome structure. Nature 502, 59–64 2013). .

[0009] Currently used methods also aim to capture combinatorial workflows that measure different aspects of cell physiology in a combinatorial manner, which is driven by the lack of reproducibility of single-cell objects, including combinations of genome, transcriptome, open chromatin, methylation or even surface protein analysis methods, such as G&T-seq-genome and transcriptome: FACS-based MDA / PicoPlex (WGA), SMART-seq2 (Macaulay, IC et al. G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat. Methods 12, 519–522 2015), DR-seq-DNA and RNA sequencing (Dey, SS, Kester, L., Spanjaard, B., Bienko, M. & Van Oudenaarden, A. Integrated genome and transcriptome sequencing of the same cell. Nat. Biotechnol. 33, 285–289 2015), scM&T-seq-DNA methylation and transcriptome (Angermueller, C. et al. Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity. Nat. Methods 13, 229–232 2016), scDam&T-seq-chromatin, transcriptome based on DamID and CEL-seq combined FACS (384-well plate) (Rooijers, K. et al. Simultaneous quantification of protein-DNA contacts and transcriptomes in single cells. Nat. Biotechnol. 37, 766–772 2019), T-ATAC-seq-open chromatin based on scATAC-seq and TCR-seq (Satpathy, AT et al. Transcript-indexed ATAC-seqfor precision immune profiling. Nat. Med. 24, 580–590 2018), SNARE-seq-open chromatin and transcriptome (Chen, S., Lake, BB & Zhang, K.High-throughput sequencing of the transcriptome and chromatin accessibility in the same cell. Nat. Biotechnol. 37, 1452–1457 2019), FACS-based scCAT-seq-open chromatin and transcriptome (Liu, L. et al. Deconvolution of single-cell multi-omics layers reveals regulatory heterogeneity. Nat. Commun. 10, 470 2019), CITE-seq-surface protein and transcriptome (Stoeckius, M. et al. Simultaneous epitope and transcriptome measurement in single cells. Nat. Methods 14, 865–868 2017), REAP-seq-surface protein and transcriptome (Peterson, VM et al. Multiplexed quantification of proteins and transcripts in single cells. Nat. Biotechnol. 35, 936–939 2017). .

[0010] Simultaneous assays have also been designed that are more limited in the scale of measuring transcripts and proteins in single cells, including indexed cell sorting using RNA sequencing combined (Paul, F. et al. Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors. CELL 163, 1663-1677 2015, Wilson, NK et al. Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations. CELL STEM CELL 16, 712-724 2015) or proximity ligation assays (PLA and its variants) combined with PCR (Stahlberg, A. et al. Quantitative PCR analysis of DNA, RNAs, and proteins in the same single cell. Clinical Chemistry 58, 1682-1691, 2012, Genshaft, AS et al. Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single cell) reaction. Genome Biol. 17:188, 2016, Albayrak, C. et al. Digital Quantification of Proteins and mRNA in Single Mammalian Cells. Molecular Cell 61, 914-924, 2016, Darmanis, S. et al. SimultaneousMultiplexed Measurement of RNA and Proteins in Single Cells. Cell Reports 14, 380-389, 2016) or mass cytometry (Frei, AP et al. Highly multiplexedsimultaneous detection of RNAs and proteins in single cells. Nature Methods 13, 269-275, 2016).For example, Albayrak et al., 2016 and Hu et al., 2019 (Hu, Jiumei, et al., "Proximity ligation assays for precise quantification of femtomolar proteins in single cells using self-priming microfluidicd PCR chip", ANALYTICA CHIM-ICAACTA, vol. 1076, 01.10.2019) only optimize their PLA reactions with respect to the optimal concentration of the PLA probe for detecting a certain target concentration. For the final quantification of the analyzed protein in the sample, these methods also only consider the data of the simple calibration / titration curve of the analyzed protein and / or PLA probe (nucleic acid-coupled antibody). Therefore, the quantification performed in the prior art only takes into account the potential bias introduced by different concentrations of target and / or PLA probe during the PLA assay, without analyzing or considering further features of the PLA probe, and therefore absolute quantification is not possible in the method. In addition, these assays are limited in scale and / or can only analyze a few genes and proteins in parallel, and are confined to extracellular proteins, do not have absolute quantification, and cannot process the interactor group, the complex group of single cells, and the apparent proteome is very limited. Conventional techniques such as PLA (adjacent ligation assay) and other applicable methods often cannot accurately detect all molecules, and show background noise, resulting in erroneous detection of non-existent molecules. Therefore, these technologies cannot provide the absolute count of molecules, which is necessary for absolute quantification without reference. The method of the present invention solves these limitations by realizing the accurate and accurate quantification of molecules without the need for reference, thereby overcoming the shortcomings of the prior art.

[0011] Drop-seq, CITE-seq, and REAP-seq apply sequencing-based protein-level readouts by conjugating antibodies to poly-A labeled oligonucleotides (antibody-derived tags-ADTs), which are simultaneously captured by cDNA capture. Antibodies are identified by barcodes contained in the tags. Labeled antibodies bind to their targets in single-cell suspensions, and the cells are washed to remove unbound antibodies and processed for scRNA-seq. Typically, antibody-modified single cells are encapsulated into nanoliter-sized reaction droplets for single-cell lysis, extraction of cellular mRNA as well as antibody tags, and conversion to DNA via their 3′ polyA sequences. The transcribed cDNA and ADT are converted into NGS libraries and sequenced, revealing the quantification of cDNA and ADT. These assays are similar to the widely used FACS (fluorescence activated cell sorting) (Bonner WA, Hulett HR, Sweet RG, Herzenberg LA. Fluorescence activated cell sorting. Rev Sci Instrum. 1972; 43 (3): 404-409. doi: 10.1063 / 1.1685647) and are limited to protein detection using FACS protocols, including washing steps, low specificity and background, and only provide relative semi-quantitative results, which cannot be compared between antibodies classifying these methods as non-absolute quantitative methods. The application of ADT cannot resolve the interactome, complexome of single cells, and the detection of intracellular proteins is severely limited. Therefore, these assays are limited to cell surface proteins because their intracellular application leads to high background and loss of even semi-quantitative results due to difficult to control and unavoidable washing steps.

[0012] Other methods for cell surface protein analysis methods (Labib, M., & Kelley, SO (2020). Single-cell analysis targeting the proteome. Nature Reviews Chemistry, 4 (3), 143–158) include magnetic fractionation cytometry (MagRC) and flow cytometry (FACS). Methods that can be used to analyze the general suitability of proteins for detection include single cell protein blotting, microfluidics proximity ligation assay (PLA), proximity extension assay (PEA), mass cytometry (cytometry by time of flight; CyTOF) and mass spectrometry and single cell barcode chip (SCBC). However, all of these approaches have limitations, particularly in providing absolute quantitative detection and high sensitivity, with the best sensitivity currently limited to a few thousand molecules per single cell, with high background (C. Albayrak, C. A. Jordi, C. Zechner, J. Lin, C. A. Bichsel, M. Khammash, S. Tay, Digital Quantification of Proteins and mRNA in Single Mammalian Cells, Mol. Cell. 61 (2016) 914–924. https: / / doi.org / 10.1016 / j.molcel.2016.02.030.), regardless of cellular compartment, and simultaneously accessing proteome, interactome, epiproteome and complexome targets.

[0013] Therefore, more efficient compositions; high sensitivity to reveal proteome, interactome, epiproteome, and complexome information; access to intracellular targets; and comparable results with universal absolute quantification are necessary to quantify large numbers of single-cell targets for diagnostic and research applications. Summary of the invention

[0014] In light of the prior art, the technical problem underlying the present invention is to provide an improved method for the reliable quantification of target analytes, such as proteins, protein modifications, DNA modifications, protein-protein complexes, protein-DNA complexes or protein-RNA complexes, at the single cell level, regardless of the cellular localization of the target and in an absolutely quantitative manner.

[0015] This problem is solved by the features of the independent claim. The dependent claims provide preferred embodiments of the invention.

[0016] Thus, the present invention relates in one aspect to a method for determining the absolute concentration of at least one target analyte in a sample, comprising:

[0017] a. providing at least a first type of binding agent and a second type of binding agent, each binding agent comprising a label unique to said type of binding agent, wherein said first type of binding agent and said second type of binding agent specifically bind to a first target analyte,

[0018] b. determining the binding characteristics of the first type of binding agent and the second type of binding agent to the first target analyte,

[0019] c. providing a sample containing cells of known concentration,

[0020] d. contacting the first type of binding agent and the second type of binding agent with the sample, preferably under suitable conditions, wherein the first type of binding agent and the second type of binding agent bind to the first target analyte, thereby forming an analyte-binding agent complex (couplex),

[0021] e. partitioning the (single) cells of the sample into a plurality of first partitions, wherein preferably each compartment contains no more than one cell (or one cell or no cell),

[0022] f. supplementing each first partition containing single cells with a lysis buffer, preferably a lysis and / or dispersion buffer, thereby lysing the single cells in each first partition,

[0023] g. partitioning a single analyte-binding agent complex (conjugate) into a plurality of second partitions,

[0024] h. performing a two-component detection method to determine the amount and / or concentration of the conjugate and the binding agent in the second partition,

[0025] i. Determine the absolute concentration of the first target analyte per cell in the first partition by taking into account the initial concentration of cells in the sample in step a., the binding characteristics determined in step b., and the number and / or concentration of conjugates and binding agents determined in the second partition in step h.

[0026] In an embodiment, one or more steps of the aforementioned method may be performed in a different or suitable order. In some embodiments, certain steps are performed in parallel. In an embodiment, for example, depending on the cellular localization of the target analyte, step d. 'contacting the first type of binding agent and the second type of binding agent with the sample' may be performed before or after the first separation step e., wherein preferably, the single cells of the sample are separated (so that the compartments preferably include no more than 1 cell, or one cell or no cells), and after cell lysis and / or adding a lysis or dispersion buffer in each corresponding first compartment (step f). In such embodiments, in particular, the target agent contained in the nucleus and / or other organelles of the cell can be detected / bound by the binding agent because the nucleus or other membranes have been dissolved in the lysis step f. before incubation with the binding agent (in step d.).

[0027] Therefore, in an embodiment, the steps of the aforementioned method can be performed, for example, in the order of step a., step b., step c., step e., step f., step d., step g., step h. and step i.

[0028] Thus, in an embodiment, the method comprises the following steps (in order):

[0029] a. providing at least a first type of binding agent and a second type of binding agent, each binding agent comprising a label unique to said type of binding agent, wherein said first type of binding agent and said second type of binding agent specifically bind to a first target analyte,

[0030] b. determining the binding characteristics of the first type of binding agent and the second type of binding agent to the first target analyte,

[0031] c. providing a sample containing cells of known concentration,

[0032] e. partitioning the sample into a plurality of first partitions, wherein preferably each compartment contains no more than one cell,

[0033] f. supplementing each first partition containing the sample with a lysis buffer, preferably a lysis and / or dispersion buffer, thereby lysing the single cells in each first partition,

[0034] d. contacting the first type of binding agent and the second type of binding agent with the sample in the first partition, preferably under suitable conditions, wherein the first type of binding agent and the second type of binding agent bind to the first target analyte, thereby forming an analyte-binding agent complex (conjugate),

[0035] g. partitioning a single analyte-binding agent complex (conjugate) into a plurality of second partitions,

[0036] h. performing a two-component detection method to determine the amount and / or concentration of the conjugate and the binding agent in the second partition,

[0037] i. Determine the absolute concentration of the first target analyte per cell in the first partition by taking into account the initial concentration of cells in the sample in step a., the binding characteristics determined in step b., and the number and / or concentration of conjugates and binding agents determined in the second partition in step i.

[0038] In some embodiments, the binding reaction d. is performed after the first separation of the sample components (e.g., single cells) and under lysis and / or dispersion buffer conditions, for example, after and / or during the addition of a cell lysis and / or dispersion buffer to each first compartment, and after and / or during incubation with the buffer.

[0039] In embodiments, the steps of cell lysis / permeabilization (step f.) and binding reaction (step d.) are performed in parallel or simultaneously. Thus, in such embodiments, the steps of the aforementioned method may be performed in the order of step a., step b., step c., step e., step f.+step d., step g., step h., and step i. In such embodiments, the combined steps f.+d. include: supplementing each first partition containing the sample with a lysis buffer (preferably a lysis and / or dispersion buffer) and a first type of binding agent and a second type of binding agent, thereby lysing the single cells in each first partition, and preferably contacting the first type of binding agent and the second type of binding agent with the sample in the first partition under suitable conditions, and wherein the first type of binding agent and the second type of binding agent bind to the first target analyte, thereby forming an analyte-binding agent complex (conjugate). In some of the embodiments, the binding agent is added to the first compartment contained in the lysis and / or dispersion buffer (in step f. / d.), or when the binding agent is added to the first compartment, the binding agent is contained in the lysis and / or dispersion buffer (in step f. / d.).

[0040] In some of the described embodiments where the order of method steps is varied, single cell isolation / fractionation is preferably performed prior to cell lysis so that only single cell lysates (rather than bulk lysates) are analyzed.

[0041] Absolute quantification is a key feature of an analytical method, providing a precise measurement of the number of molecules in a sample without reliance on external quantitative standards. In the study of biological systems, achieving absolute quantification is challenging due to the huge differences in protein properties, the wide range of protein abundance, and the complex cellular environment. Existing absolute quantification methods, mainly based on mass spectrometry, are only partially established and lack methods that do not require external standards.

[0042] The two-component method preferably used in the embodiments herein preferably constitutes an external quantitative reference-free protein assay. It preferably utilizes a two-step readout method to achieve high signal-to-noise performance without background noise. In the first step, the partitioning of the conjugate preferably ensures robust signal generation and mathematically traceable results. Forming the conjugate under strict buffer conditions avoids matrix effects and ensures accurate quantification. In the second step, the method of the present invention uses separated single-molecule quantitative digital PCR in the embodiments to accurately quantify the conjugate. This step is preferably separated from the antibody binding process / step to ensure an absolute signal based on the single-molecule sensitivity of the digital PCR reading.

[0043] The currently used two-component detection method provides several advantages over existing assays. It provides a reference-free absolute quantification method, eliminating the need for external standards, making it highly versatile and applicable to a variety of protein analytes. The homogeneous, preferably wash-free workflow of the two-component method of the present invention minimizes signal loss and avoids the challenges associated with the solid phase capture step. In an embodiment, the determination of the reliance on conjugate formation and separation preferably ensures a strict reliance on binding agent binding, thereby preventing dissociation-related problems and providing robust quantification. In an embodiment, by detecting and quantifying the counts of conjugates and individual binding agents, the currently used two-component method enables extensive control of reaction readings, including determination of binding concentrations, normalization of parallel experiments, and offsetting dPCR clustering errors.

[0044] In embodiments, calculations involve determining the correct concentrations of binding reactants (including binding agents and conjugates) in a binding reaction, which values ​​may be corrected in embodiments by labeling ratio (LR), label synthesis error (LE), active fraction (AF) of the binding agent, and / or free label (FL) contamination, and in embodiments, may also be corrected by appropriate compensation of mathematical errors, such as clustering individual compartments according to their positive or negative ranking with respect to containing different molecular entities. In embodiments, the corrected concentrations may be used directly to calculate the absolute concentration of the analyte using, for example, the method described in WO 2020 / 260277A1, which applies chemical equilibrium to determine absolute concentrations, included herein as a reference.

[0045] In the present invention, one aspect of an embodiment of an absolute quantification method can be highlighted, such as saturation conditions. The conditions are preferably able to directly correspond between the concentration of the ternary complex (conjugate) formed during the assay process and the target analyte, preferably without the need to determine the dissociation constant (Kd) of the binding agent (e.g., antibody). In an embodiment, by ensuring saturation of the assay components, the concentration of the conjugate can be reliably measured, thereby providing an accurate and direct method for quantifying the target analyte. This method simplifies the absolute quantification process, preferably eliminates the requirement for determining the Kd value of the binding agent (e.g., antibody) used, and can directly and accurately quantify the analyte of interest.

[0046] The step of double compartmentation (compartments are defined as multiple separate, reproducible partitions with specific conditions) and the use of absolute quantitative methods to deduce the absolute chemical conditions in the first compartment (Partition I) from the second compartment (Partition II) represents a new concept superior to the prior art, as it combines the advantages of two different conditions simultaneously:

[0047] During the first compartment, the binding reaction preferably has a high local target concentration, which can be of two different types:

[0048] (1) It is a suspension of a large number of cells in which the compartments are diffusion-limited and the cells achieve a high local concentration of the target in their own concentrated environment, or

[0049] (2) A few cells or single cells have such a high local target concentration in a small volume.

[0050] In this concept, preferably, the first partition and the second partition are interconnected:

[0051] (3) the absolute chemical correspondence between the first compartment and the second compartment enables the determination of the absolute chemical conditions in the first compartment (first compartment) of single cells from the second compartment (second compartment), without determining the absolute chemical conditions in the first compartment, the absolute target concentration cannot be derived,

[0052] (4) By determining the binding characteristics of the binding agent, a high-precision determination of the absolute amount of a single cell required for a single cell is provided. The single cell binding reaction of the present invention is separated and improved relative to the standard binding reaction in bulk solution, thereby maintaining the separability of the cells, while in the reaction of bulk lysed cells, the results of each cell are inseparable, while achieving absolute chemical correspondence between the compartments.

[0053] For example, in a preferred embodiment, the second partitioning step is used to derive / reveal unknown absolute chemical substances (absolute concentrations of binders and conjugates) present in the first partitions (in each first partition, each partition contains the contents of a single cell and potential binders).

[0054] In an embodiment, a detection unit is defined as a complex of a target and two bound binding agents, referred to as a conjugate. If more than two binding agents are bound, all unique combinations of binding agent pairs are considered different conjugates.

[0055] In a preferred embodiment, the second partition provides precise molecular quantities so that the absolute amount / concentration of the target analyte is derived from the determined absolute chemical substance and is additionally determined by taking into account the volume of the first partition (first compartment) and / or the initial concentration of cells in the initial solution provided to the first partition, and / or the initial concentration of the binding agent in the initial solution provided to the first partition, and most importantly, the previously determined (apparent) binding properties of the binding agent.

[0056] Preferably, in step h., in addition to determining the amount and / or absolute concentration of the conjugate in the second partition, the two-component detection method also determines the amount and / or absolute concentration of the binding agent. Thus, the two-component detection method is also applied to determine the amount and / or absolute concentration of the binding agent in the initial first partition (which is divided into a plurality of second partitions containing its contents).

[0057] In summary, the present invention surprisingly facilitates a reliable and precise quantitative assessment of absolute concentrations (amounts) if different species of binder-targets (target analytes) are present in a single cell.

[0058] The present invention preferably achieves its surprising effects through the following two major improvements over the prior art: (i) using an unknown amount of target material to determine the binding characteristics of a binder such as an antibody, preferably taking into account the apparent dissociation constant of the binder, such as the antibody, taking into account the labeling ratio of the binder, and / or label synthesis errors that render the label undetectable, and / or the active fraction of the binder that has the ability to bind and contamination with free label.

[0059] Subsequently, based on these measurements, an apparent dissociation constant of the binding agent, such as an antibody, is preferably determined. This is followed by (ii) a double partitioning of the single cell components, such that an absolute quantitative correspondence is achieved between a first partitioning condition of the single cells during the binding incubation of the binding agent and a second partitioning condition of the single cell components (e.g., a complex formed by the binding agent and the target molecule), which enables the determination of extremely low (as low as 10 target analytes per single cell) and absolute amounts of target analytes per single cell by taking into account the previously determined binding characteristics of the binding agent.

[0060] In embodiments, the binding characteristics of the active fraction and the contamination with free label are inherently compensated by the second separation method, which represents a surprising discovery and feature of the methods of the present invention.

[0061] Therefore, in a preferred embodiment of the method according to the invention, determining the binding characteristics in step b. comprises determining the following binding characteristics for each binding agent of the first and second type: dissociation constant of the (corresponding) target analyte, labelling ratio and label synthesis error.

[0062] In other words, in a preferred embodiment, in step b., determining the binding characteristics comprises determining the (apparent) dissociation constant for each binding agent of the first type and the second type by taking into account the labeling ratio of the binding agents and the label synthesis error, wherein the active fraction and the free label are self-compensating.

[0063] In a preferred embodiment, the method of the invention uses the labeling ratio of the binding agent and the label synthesis error to determine the apparent dissociation constant of the binding agent, thereby providing an inherently favorable self-compensation for the active fraction of the binding agent and contamination by free label to provide a dissociation constant for the antibody for unbiased determination of absolute quantities in single cells.

[0064] In an embodiment, determining the binding characteristics in step b. includes determining the following binding characteristics of each binding agent of the first type and the second type: dissociation constant with the target analyte required for binding to the target analyte, labeling ratio, labeling error ratio, activity ratio and / or optimal concentration (number of molecules per unit volume).

[0065] In embodiments, determining the binding characteristics in step b. comprises determining a dissociation constant for each binding agent of the first type and the second type to the target analyte.

[0066] In embodiments, determining the binding characteristic in step b. comprises determining a labeling ratio of each binding agent of the first type and the second type.

[0067] In embodiments, determining the binding signature in step b. comprises determining a labeling error ratio for each binding agent of the first type and the second type. In embodiments, determining the binding signature in step b. comprises determining a labeling ratio and / or a labeling error (labeling error; labeling synthesis error (LE)) ratio for each binding agent of the first type and the second type.

[0068] In an embodiment, determining the binding characteristics in step b. comprises determining the free label (FL) contamination of each binding agent of the first type and the second type.

[0069] In embodiments, determining the binding characteristic in step b. comprises determining a labeling error ratio for each binding agent of the first type and the second type.

[0070] In an embodiment, determining the binding characteristics in step b. comprises determining the labeling ratio, labeling error (labeling error; label synthesis error (LE)) ratio and / or free label (FL) contamination of each binding agent of the first type and the second type.

[0071] In embodiments, the determination efficiency and correctness of the binding agent labeling, i.e., the "labeling ratio" and the "labeling error" ratio, but in embodiments, the contamination of the binding agent solution by free labels (after the binding agent is labeled) helps to correct label-related errors from the final result, such as label sequence reading. For example, if the binding agent does not contain a label (labeling ratio) or the label contains a sequence error and / or the binding agent contains an erroneous label, the binding agent cannot be detected and / or identified in subsequent data analysis. Contamination of the sample by free labels can result in false positive detection of conjugates. Therefore, conjugates containing erroneously labeled or unlabeled binding agents cannot be identified in subsequent data analysis. Therefore, predetermining the efficiency and correctness of the binding agent labeling helps to correct the errors subsequently, so that the determination / calculation accuracy of the target analyte concentration in the sample can be further improved.

[0072] In embodiments, determining the binding characteristic in step b. comprises determining a ratio of the activity of each binding agent of the first type and the second type.

[0073] In an embodiment, determining the binding characteristics in step b. comprises determining the optimal concentration (number of molecules per unit volume) of each binding agent of the first type and the second type required for binding to the target analyte.

[0074] In embodiments, determining the binding characteristics in step b. comprises determining the following binding characteristics for each binding agent of the first and second types: dissociation constant, labeling ratio, labeling error ratio and / or activity ratio to the target analyte.

[0075] In embodiments, determining the binding characteristic in step b. comprises determining a labeling ratio, a labeling error ratio and / or an activity ratio for each binding agent of the first type and the second type.

[0076] In embodiments, the binding characteristics of a binder preferably include its (re)activity, specificity, concentration, label ratio (LR), its label synthesis error (LE), active fraction (AF) of the binder and / or free label (FL) contamination,

[0077] Therefore, the determination of the so-called "binding characteristics" of the binding agents herein represents an improved method for optimizing the absolute quantification of the target analyte concentration in the sample. By determining and mitigating the "binding characteristics" associated with the binding agents, the methods of the present invention are surprisingly able to identify specific conditions, wherein the effects of some of these characteristics on absolute quantification preferably become negligible. Specifically, in embodiments, the present invention focuses on establishing saturation conditions, characterized by high concentrations of binding agents, which make the effects of their reactivity on absolute quantitative determination negligible. Applying quantitative methods under these optimized saturation conditions ensures accurate and reliable quantification of target analyte concentrations.

[0078] Therefore, the determination of the so-called "binding characteristics" of a binding agent herein represents an improved method for optimizing the absolute quantification of the target analyte concentration in a sample. This is because additional confounding factors for the calculation of the absolute quantification of the target analyte, such as unlabeled or mislabeled binding agent and / or the dissociation constant between the binding agent and its target analyte, can be identified and can be taken into account during the final calculation of the target concentration.

[0079] In a preferred embodiment, performing step h. of the two-component detection method comprises determining thereby the amount and / or absolute concentration of the conjugate and the binding agent in the second compartment.

[0080] The binding characteristics of a binding agent may also include the reactivity, specificity and / or optimal concentration (number of molecules per unit volume) required for binding to a target analyte. Reactivity is described by the dissociation constant of the binding agent, specificity is described by specific identification of the binding target, and concentration is described by the number of molecules per unit volume. WO 2020 / 260277 A1 describes a method, which is incorporated herein by reference, that applies chemical equilibrium and uses the binding characteristics to determine the dissociation constant of a binding agent.

[0081] The present invention significantly improves upon the method of WO 2020 / 260277 A1 and extends it for highly accurate and sensitive single cell measurements, including additional features such as labeling ratios of antibodies, label synthesis errors that render label undetectable, and / or the active fraction of antibodies that have binding capacity and contamination by free label. Based on these measurements, the method of the present invention then facilitates determination of apparent dissociation constants of binding agents such as antibodies, which, in combination with the described dual compartmentalization of single cell components, provide accurate, highly sensitive detection of target analytes, down to approximately 10 target analytes per single cell.

[0082] In this context, the labeling ratio (LR) of the binding agent describes the ratio of the labeled fraction to the non-labeled fraction of the binding agent. The labeling synthesis error (LE) describes the fraction of undetectable label, for example due to sequence errors of the label. Determining the labeling synthesis error or simply "labeling error" may be related to the accuracy of the embodiments of the method of the present invention, because if the binding agent contains an undetectable label, the binding of the binding agent to the target analyte cannot be detected. The active fraction (AF) of the binding agent describes the ratio of the active fraction to the non-active fraction of the binding agent. Contamination of the binding agent with free label (FL) describes the label fraction that is not bound to the binding agent during the preparation of the binding agent, and there is a risk of false positive results during the measurement of the target analyte by the method of the present invention.

[0083] The present invention describes a method that uses the binding characteristics of a binding agent, preferably its reactivity, specificity, concentration, label ratio (LR), its label synthesis error (LE), the active fraction (AF) of the binding agent and / or contamination by free label (FL) moth, to determine a new calibration constant for the binding agent, such as an apparent dissociation constant, for determining the absolute amount of a target analyte. Because the determined binding characteristics extend the molecular binding characteristics by taking into account practical limitations (such as the active fraction (AF) of the binding agent, etc.), the binding characteristics can also be referred to as extended binding characteristics. In a preferred embodiment, the system has a dispersed, discontinuous concentration of the target analyte, such as a single cell suspension.

[0084] In a preferred embodiment, the invention applies the determination of the (extended) characteristics of binding agents to enable the determination of very low and absolute amounts of a target on a single cell, preferably down to 100s or preferably 10s of target per single cell.

[0085] In an embodiment, the two-component detection method in step h. comprises nucleic acid amplification or a nucleic acid amplification step.

[0086] In an embodiment, the first type of binding agent and the second type of binding agent each comprise a nucleic acid tag that is preferably unique to said type of binding agent, and the two-component detection method in step h. comprises amplifying the nucleic acid sequence tag of the binding agent, preferably within each second partition.

[0087] In an embodiment, amplification includes the connection of nucleic acid sequences of nucleic acid labels of two or more binding agents that form a conjugate, preferably wherein the connection can be the synthesis of a nucleic acid sequence complementary to the single-stranded nucleic acid label of the binding agent. In such embodiments, it is not the label itself that is connected, but its nucleic acid sequence that is connected in the nucleic acid amplification step. Therefore, in an embodiment, the connection (within each second partition) of the nucleic acid sequence label of the first type of binding agent and the nucleic acid sequence label of the second type of binding agent can be understood as being interchangeable / synonymous with the connection / combination of the nucleic acid sequence (nucleotide sequence) of the label. The result of the connection and / or amplification step is preferably a nucleic acid sequence of each nucleic acid sequence of the label of at least two binding agents that form a conjugate, so that the identity of the binding agent can be determined, and therefore the identity of the target analyte in the complex can also be determined.

[0088] In an embodiment, the two-component detection method in step h. comprises combining (merging) a plurality of second partitions.

[0089] In an embodiment of the method of the present invention, the two-component detection method in step h. comprises performing nucleic acid sequence analysis.

[0090] In an embodiment of the method according to the invention, the two-component detection method in step h. comprises performing a nucleic acid sequence analysis, such as nucleic acid sequencing or PCR analysis, to determine the number and / or identity of nucleic acid sequences present in the sample. In an embodiment, a nucleic acid sequence analysis, such as nucleic acid sequencing or PCR analysis, is performed to determine the number and / or identity of nucleic acid sequence tags (of the binding agent). In an embodiment, a nucleic acid sequence analysis is performed to determine the number and / or identity of nucleic acid sequence tags (of the binding agent), and then the number and / or concentration of the conjugate is calculated from the number and / or identity of the preferably unique nucleic acid sequence tags.

[0091] In an exemplary embodiment, when two different binding agents (each binding agent comprises a unique nucleic acid tag, which is also a characteristic of the corresponding type of binding agent) are bound to the target analyte (forming a conjugate) and separated (separated) into the second compartment, the two unique sequences of the nucleic acid tag can be connected / combined by a nucleic acid amplification step. The obtained sequence comprises the connected / combined nucleic acid sequences of the two tags, so that by nucleic acid sequence analysis, the identity of the two different binding agents (based on the connected tag sequences) can be determined, thereby determining the identity of the target analyte forming the conjugate. Since the tag preferably comprises a unique sequence, for example, duplications that occur during amplification and / or sequence analysis can be discarded / ignored during the final analysis. In addition, in an embodiment, the identity and / or number of binding agents that form a conjugate with one or more target analytes can be normalized by the initial concentration of the binding agent in the binding reaction (step d.) and / or one or more binding characteristics of the binding agent (e.g., label ratio, label synthesis error, dissociation constant with the target analyte, etc.), and is included in the calculation of the normalization process described in detail herein.

[0092] In an embodiment of the method of the present invention, the absolute concentration of the first target analyte per cell in the first compartment is calculated from the initial concentration of cells in the sample in the second compartment (as adjusted / determined in step a.), the binding characteristics determined in step b. and the number and / or concentration of conjugates and binding agents in step h., and thereby from the number and / or concentration of conjugates.

[0093] In an embodiment of the method according to the invention, the first type of binding agent and the second type of binding agent each comprise a nucleic acid label unique to said type of binding agent, and the two-component detection method in step h. comprises the following steps:

[0094] - amplifying the nucleic acid sequence marker within each second partition, and

[0095] - combining the plurality of second partitions and subjecting them to nucleic acid sequence analysis in order to determine the number and identity of the nucleic acid sequence tags, and thereby determine the number and / or concentration of the conjugate.

[0096] In an embodiment of the method of the invention, the two-component detection method in step h. comprises a ligation step between the labels of the first type of binding agent and the second type of binding agent in each second partition, which ligation step provides co-partitioning information of the two-component method.

[0097] In an embodiment of the method of the present invention, if the nucleic acid sequence tag of the first type of binding agent and the nucleic acid sequence tag of the second type of binding agent both form a conjugate with the first target analyte, the connection step in step h. includes connecting the nucleic acid sequence tag of the first type of binding agent to the nucleic acid sequence tag of the second type of binding agent in each second partition.

[0098] In some embodiments, the connection step includes nucleic acid amplification (e.g., PCR or digital PCR or linked PCR) for nucleic acid sequence labeling. In other embodiments, the connection step is a classical adjacent ligation reaction involving rolling circle DNA synthesis or rolling circle amplification. In other embodiments, the connection is achieved using a ligase. In subsequent labeled nucleic acid sequencing analysis, the presence of a conjugate in a partition can be demonstrated by a sequence comprising a marker sequence of a first type of binding agent and a marker sequence of a second type of binding agent, both binding agents being specific to the target analyte, and the sequence being related to co-partition information.

[0099] In any case, the ligation step depends on the type of label used. In some embodiments where detection is achieved using fluorescent probes or labels, a ligation step between nucleotide labels may not be substantially required, as the presence of a conjugate can be detected by co-localization of two labels with different fluorescent labels or dyes within one partition or droplet, the fluorescent labels or dyes being used to detect two different types of binding agents in a conjugate comprising two types of binding agents bound to a target.

[0100] In some embodiments, protein or peptide based labels can be cross-linked according to methods known in the art.

[0101] In an embodiment of the method according to the present invention, during the first separation, before contacting the first type of binding agent and the second type of binding agent with the sample, the cells in the sample are permeabilized for the first type of binding agent and the second type of binding agent (preferably antigens are recovered therefrom, in other words, the cells are "antigen recovered"), so that one or more of the first type of binding agent and the second type of binding agent enters one or more of the cells after contacting the sample in step d.

[0102] In embodiments where the target analyte is at least partially located on the surface or membrane or cell wall of the cell, it may be substantially unnecessary to permeabilize the cell to contact the binding agent with the target analyte. However, in embodiments, when the target analyte is located in the cell and the binding agent cannot interact with the target analyte, because they cannot enter the cell, it may be necessary to permeabilize the cells in the sample and / or suspected of containing the target analyte. This permeabilization can also include encapsulating the binding agent into a molecular shuttle such as a liposome so that they can enter the cell. In other embodiments, permeabilization can be achieved using other methods known in the art, such as chemicals (e.g., formaldehyde, triton, tween, NP-40, leucoperm, digitonin, saponin, acetone, methanol, etc.), electric current or (super) ultrasonic treatment. Those skilled in the art know the appropriate method for cell permeabilization, which depends on the binding agent and / or cell used.

[0103] Thus, in one embodiment, the first compartment preferably provides chemical conditions that facilitate detection of a target on the cell surface, within the cell and / or in the cell nucleus.

[0104] In an embodiment of the method according to the invention, the first type of binder and the second type of binder each comprise a nucleic acid marker unique to said type of binder, and wherein the plurality of first partitions in step f. are optionally further supplemented with PCR reagents and optionally nucleic acid amplification primers comprising a barcode unique to each first partition and the single cells therein, and optionally RT-PCR reagents, and wherein within each second partition an additional nucleic acid amplification step is performed after cell lysis, thereby detecting additional nucleic acid targets, in particular specific genomic loci or transcripts or specific conjugates. In an embodiment, during such amplification, the nucleic acid markers of the binders present in the conjugate may be attached and / or barcoded and / or amplified.

[0105] After replenishing the first partitions or droplets with the lysis buffer, the lysis buffer is optionally further supplemented with first-strand cDNA synthesis reagents and optionally nucleic acid amplification primers comprising a barcode unique to each first partition and the single cell therein, and wherein an additional step of first-strand cDNA synthesis is performed after cell lysis within each first compartment.

[0106] In a preferred embodiment, during the amplification, a barcode or UMI unique to each first partition / droplet (or unique to each encapsulated single cell) is added to each amplified nucleic acid sequence, wherein the unique barcode or UMI is preferably included in the sequence of the amplification primer. Based on the barcode or UMI, single-cell specific analysis of the conjugate can be performed during nucleic acid sequencing analysis.

[0107] In an embodiment of the method according to the invention, the plurality of first partitions in step f. and / or preferably in step e. are further supplemented with a protein cross-linking agent and / or a protease inhibitor and / or a nuclease inhibitor.

[0108] In an embodiment of the method according to the invention, the first type of binding agent and the second type of binding agent each comprise a nucleic acid tag that is unique to said type of binding agent, and wherein each nucleic acid sequence tag comprises an identifier sequence (barcode) and a unique molecular identifier (UMI) that identifies the reactivity of the binding agent and its molecular identity.

[0109] If the label of the binding agent is not only specific for the type of binding agent, but also unique for each individual binding agent molecule, then in embodiments the determination of the precise / absolute molecular concentration of the target analyte by the method of the present invention can be even more improved. When the unique label is linked to another unique label of the second binding agent in the conjugate, the use of a unique label for a single binding agent molecule facilitates the determination of the total number of individual conjugates by subsequent nucleic acid sequence analysis.

[0110] In an embodiment of the method according to the present invention, the first type of binding agent and the second type of binding agent each contain a nucleic acid marker unique to said type of binding agent, and wherein the amplification in step h. involves the implementation of PCR and / or droplet PCR and / or digital PCR and / or real-time PCR and / or RT-PCR.

[0111] In an embodiment of the method of the invention, more than one target analyte and corresponding first and second type of binding agents are provided and finally, their absolute concentrations are determined according to the method of the invention.

[0112] In an embodiment of the method according to the present invention, at least one target analyte comprises at least a first target analyte and a second target analyte, and wherein in step a., for each of at least the first target analyte and the second target analyte, at least a first type of specific binding agent and a second type of specific binding agent are provided.

[0113] In an embodiment, step d. is performed under suitable conditions, i.e., the first type of binding agent and the second type of binding agent are contacted with the sample, wherein the first type of binding agent and the second type of binding agent are bound to the first target analyte, thereby forming an analyte-binding agent complex (conjugate). The phrase "under suitable conditions" refers to any conditions described herein, which are suitable for contacting the first type of binding agent and the second type of binding agent with the sample, wherein the first type of binding agent and the second type of binding agent are bound to the first target analyte, thereby forming an analyte-binding agent complex (conjugate). In an embodiment, suitable conditions may be conditions that include or enable "permeabilization" of cells. In an embodiment, suitable conditions may include specific "antigen retrieval" conditions so that one or more antigens can be recovered from cells. In an embodiment, the conditions of step d. are intended to enable the first type of binding agent and the second type of binding agent to contact the first target analyte, thereby forming an analyte-binding agent complex. Preferably, the conditions do not necessarily lyse the cell (release all of its contents), but in embodiments may only permeabilize the cell and / or contact the cell contents with a binding agent, for example by allowing the binding agent to enter the cell to interact with its contents, but in embodiments may only permeabilize the cell and / or contact the cell contents with a binding agent, for example by allowing the binding agent to enter the cell to interact with it, but in embodiments may only permeabilize the cell and / or contact the cell contents with a binding agent, for example by allowing the binding agent to enter the cell to interact with it (if present). In other embodiments, permeabilizing the cell before adding the binding agent can be considered a lysis step that releases the cell contents into the corresponding first compartment and / or contacts the cell contents with a binding agent.

[0114] In embodiments, a lysis and / or dispersion buffer is used to facilitate or facilitate complete dispersion of the cellular material prior to a subsequent partitioning step. In other words, in embodiments, the conditions of step f. are intended to enable lysis of a single cell in each first partition to release (disperse) the entire contents of the cell into the first partition. In embodiments, at least two different lysis (or lysis-like) conditions and / or buffers are applied during the method of the invention, e.g. in steps d. and f., respectively.

[0115] The method of the present invention is also convenient for simultaneously detecting more than one target analyte. In the case of multiple target analytes to be analyzed, for each target analyte, two specific binding agents must be selected, one is the first type and the other is the second type. During the final PCR, qPCR, real-time PCR or dPCR readout, different fluorescent labels, primers or probes can be used in some embodiments to achieve the distinction between different binding agent pairs of different target analytes. In one example, a probe of a connection sequence of two binding agent labels specifically bound to the first target analyte is marked with a fluorescent color, while a probe of a binding agent label with a specific connection to the second target analyte is marked / marked with a different fluorescent color. In other embodiments, when the label of the binding agent in the conjugate is not connected, each binding agent in the conjugate is detected by a specific combination of fluorescent colors, and the specific combination of the fluorescent colors can be distinguished during the final PCR readout.

[0116] In an embodiment of the method according to the invention, the number of different target analytes is less than 5 and wherein, if nucleic acid sequence analysis is performed in step h., said analysis comprises digital PCR and / or real-time PCR.

[0117] In embodiments where the number of target analytes to be analyzed exceeds the number of available fluorescent labels or combinations thereof, nucleic acid sequencing, such as NGS, can be used to specifically analyze the abundance of conjugates of the target analytes.

[0118] In an embodiment of the method according to the invention, the number of different target analytes is greater than or equal to 5, and wherein if nucleic acid sequence analysis is performed in step h., said analysis comprises Next Generation Sequencing (NGS).

[0119] In some embodiments, wherein nucleic acid sequence analysis is performed in step h., it includes next generation sequencing (NGS), preferably using a custom pipeline to process sequencing data. In an embodiment, the custom pipeline includes deconvolution of unique sequences contained in nucleic acid markers (e.g., UMIs). In an embodiment, after describing such deconvolution, the sequences originally contained in the first partition or the second partition (e.g., droplets) can be deconvoluted as described in WO 2020 / 260277A1. In short, in an embodiment, during a ligation reaction (e.g., PCR) step, deconvolution of the partitions is achieved based on unique pairings between the UMIs of each partition, and such pairings randomly constitute UMI pairs within the partition. A unique virtual network can be constructed from UMI pairs (two connected binder tags) that clearly defines the partitions (droplets) of the ligation reaction (preferably the partitions contain only one conjugate), so that the droplet (partition) contents can be reconstructed with reference to the type of binder present in the conjugate / droplet. Based on the contents of the droplets, the distribution of the binding agents can be determined, so droplets with one type of binding agent (partitions) and droplets with two binding agents can be distinguished and counted separately. The total number of droplets for sequence analysis is obtained by direct counting or using, for example, a tracer concept (e.g., pooled / combined before sequencing). The obtained droplet information can be evaluated using the methods described for dPCR readout.

[0120] Advantageously, the inventive method facilitates absolute quantification of target analyte concentrations, and determines the concentrations of multiple target analytes in parallel in a robust and efficient manner. In one embodiment, the inventive method comprises employing an analytical method based on absolute molecular counting. This analytical method based on absolute molecular counting preferably refers to application of digital detection methods, such as digital PCR or NGS analysis.

[0121] In an embodiment, the method of the invention comprises the use of a digital PCR assay. In one embodiment, the method of the invention comprises the use of a target analyte-specific binding agent associated with a unique amplifiable nucleic acid label and employing a partitioned assay, wherein nucleic acid amplification is preferably performed on each partition using a fluorescently labeled amplification product or a fluorescently labeled probe that binds to the amplification product of interest.

[0122] In some embodiments, nucleic acid amplification is PCR, and the fluorescently labeled amplification product is a fluorescently labeled PCR product. In some embodiments, nucleic acid amplification is PCR, and the amplification product of interest (the label of the binding agent forming the conjugate) is detected by a fluorescently labeled probe, such as a hydrolysis or Taqman probe.

[0123] The analyte-specific binding agents may preferably be labeled with a unique PCR-amplifiable DNA marker, wherein two analyte-specific binding agents, such as two antibodies, are preferably labeled with a single-stranded DNA marker that is unique to the binding agent of the type (e.g., the first type of antibody). In an embodiment, the binding characteristics of the labeled binding agents (e.g., antibodies) are first determined, and then they are added to the sample or one or more diluents to form a conjugate. Subsequently, the reactants are preferably highly diluted, such as by a dilution factor greater than 20,000, preferably greater than 50,000, 100,000, to achieve single conjugate separation when first separated into the first partition, such as by emulsification into droplets using methods known in the art.

[0124] In an embodiment of the method according to the invention the binding agent is selected from the group comprising antibodies, engineered protein scaffolds and aptamers.

[0125] In an embodiment of the method according to the invention, the target analyte is selected from the group comprising a peptide, a protein, a chemically modified peptide or protein, a nucleic acid, a chemically modified nucleic acid and any complex thereof.

[0126] In an embodiment, not only can the protein or peptide containing the specific amino acid sequence motif or epitope of the antibody be analyzed, but also the post-translational modification in the protein or peptide can be analyzed, for example, one type of binding agent can recognize chemical modification or post-translational modification, while the second type of binding agent can recognize the specific epitope or domain of the protein. The method can also be used to analyze the absolute amount of protein complexes in a single cell, wherein each type of binding agent recognizes one of the proteins forming the complex. Moreover, nucleic acids or nucleic acid-protein complexes can be recognized by antibodies specific to the members of the complex, for example. For example, one type of binding agent can recognize proteins in DNA-protein complexes, while another type of binding agent recognizes, for example, methylated DNA motifs (which may be in the functional context of the interaction). Therefore, the term "any complex thereof" can refer to any complex comprising proteins, peptides and / or nucleic acids or any combination thereof, because the only requirement of the method of the present invention is that a specific target analyte can be specifically recognized by a pair of two labeled binding agents to form a conjugate.

[0127] In an embodiment of the method according to the invention, the sample is selected from the group comprising tissue samples, biopsies, liquid biopsies, blood samples, plasma samples, urine samples, liquid samples, environmental samples, cell culture derived samples and samples derived from microbial cultures.

[0128] In an embodiment of the method according to the present invention, the sample is provided in step c. after being treated by a method selected from the group comprising: fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), laser capture microdissection (LCM), manual cell picking / micromanipulation, microfluidic separation, and on-demand optical interception and controlled single-cell printing.

[0129] In an embodiment, the present invention relates to a method for determining the absolute concentration of at least one target analyte in a sample, comprising:

[0130] a. providing at least a first type of binding agent and a second type of binding agent, each comprising a nucleic acid sequence tag unique to said type of binding agent, wherein the first type of binding agent and the second type of binding agent specifically bind to a first target analyte, and wherein optionally the nucleic acid sequence tag comprises an identifier sequence (barcode) and / or a unique molecular identifier (UMI),

[0131] b. determining the binding characteristics of the first type of binding agent and the second type of binding agent to the first target analyte,

[0132] c. providing a sample comprising cells of known concentration, optionally wherein said concentration comprises or relates to the number of single cells per volume unit, and / or said concentration is known / given / determined at single cell resolution,

[0133] d. permeabilizing cells in the sample, optionally for a first type of binding agent and a second type of binding agent,

[0134] e. contacting the first type of binding agent and the second type of binding agent with the sample,

[0135] wherein optionally one or more of the first type of binding agent and the second type of binding agent enters the permeabilized cell, and

[0136] wherein the first type of binding agent and the second type of binding agent bind to the first target analyte, thereby forming an analyte-binding agent complex (conjugate),

[0137] f. separating the single cells of the sample into a plurality of first partitions,

[0138] g. replenishing each partition containing single cells with lysis buffer and lysing the single cells in each partition,

[0139] h. partitioning the single analyte-binder complex (conjugate) into a second plurality of partitions,

[0140] i. optionally, if both the nucleic acid sequence tag of the first type of binding agent and the nucleic acid sequence tag of the second type of binding agent form a conjugate with the first target analyte, linking the nucleic acid sequence tag of the first type of binding agent to the nucleic acid sequence tag of the second type of binding agent in each partition,

[0141] j. amplifying the linked nucleic acid sequence label within each partition, optionally thereby determining the amount of conjugate by detecting partitions comprising the first type of binding agent label and the second type of binding agent label,

[0142] k. Merge multiple partitions,

[0143] l. performing nucleic acid sequence analysis to determine the number and identity of the nucleic acid sequence markers connected in step i,

[0144] m. Determine the absolute concentration of the first target analyte per cell by taking into account the initial concentration of cells in the sample in step a., the binding characteristics determined in step b., and the number of linked nucleic acid sequence tags determined in step 1.

[0145] Preferably, during step g., incubation between the cell contents and the binding agent is still possible / still occurs, such that the binding agent can also come into contact with (and form a conjugate with) a target agent contained within cells (e.g. within an organelle, such as within the nucleus) which were impermeable (or to a lesser extent permeable) and / or inaccessible (or to a lesser extent accessible) to the binding agent prior to cell lysis.

[0146] In embodiments, the sample comprises cells of a known concentration, and wherein the concentration comprises or relates to the number of single cells per volume unit, and / or the concentration is known / given / determined at single cell resolution, it is particularly advantageous that the method of the invention can also be performed using single cells, rather than using a large number of cells or a suspension of multiple cells.

[0147] In an embodiment of the method according to the invention, the first type of binding agent and the second type of binding agent each contain a nucleic acid marker unique to the type of binding agent, and wherein in step f. / g., the plurality of first partitions containing each partition of single cells and lysing the single cells in each partition are supplemented with lysis buffer, optionally further supplemented with PCR reagents and optionally nucleic acid amplification primers containing a barcode unique to each first partition and the single cells therein, and optionally RT-PCR reagents, and wherein an additional nucleic acid amplification step is performed after cell lysis in each first partition to enable detection of additional nucleic acid targets, in particular specific genomic loci or transcripts or specific conjugates. In an embodiment, during such amplification, the nucleic acid markers of the binding agents present in the conjugate may be connected and / or barcoded and / or amplified. In some embodiments, the sample solution is supplemented with PCR and / or reverse transcription and / or RT-PCR reagents prior to the step of separating the single cells into the first compartments.

[0148] After supplementing the first partitions with lysis buffer, the first partitions are optionally further supplemented with first-strand cDNA synthesis reagents and optionally nucleic acid amplification primers comprising a barcode unique to each first partition and the single cell therein, and wherein an additional step of first-strand cDNA synthesis is performed after cell lysis within each first partition.

[0149] In a preferred embodiment, during the amplification, a barcode or UMI unique to each first partition / droplet (or unique to each encapsulated single cell) is added to each amplified nucleic acid sequence, wherein the unique barcode or UMI is preferably included in the sequence of the amplification primer. Based on the barcode or UMI, single-cell specific analysis of the conjugate can be performed during nucleic acid sequencing analysis.

[0150] In the embodiment, step 1. of performing nucleic acid sequence analysis further includes determining the RNA composition of the single cell from the sequence information. Preferably, in the embodiment, the amplification reaction of the nucleic acid sequence tags connected within each partition in step g. / j. further includes reverse transcribing the RNA into cDNA in the first partition. In other words, in some embodiments of the present invention, the RNA present in each cell can be reverse transcribed during the (initial) amplification step of the nucleic acid tags in the first partition. Therefore, the lysis buffer preferably further comprises a reverse transcription reagent, or the first partition or the initial sample solution is supplemented with a reverse transcription reagent, wherein the generated cDNA can also be preferably labeled with a barcode and / or UMI in the reverse transcription step, so that the obtained RNA sequence can be assigned to the first partition, thereby assigning it to a single cell. In the embodiment, the identity / sequence of the RNA present in each single cell can preferably be obtained by determining the nucleic acid sequence of its corresponding cDNA during the final nucleic acid sequence analysis.

[0151] Therefore, in an embodiment of the method according to the present invention, the sample contains RNA, and wherein the multiple first partitions in steps f. / g. are further supplemented with reverse transcription reagents and the RNA is converted into cDNA, and during any one or all of steps e. / d., the first type and the second type of binding agents are contacted with the sample to lyse the cells, and wherein if nucleic acid sequence analysis is performed in step h. / l., the analysis includes next-generation sequencing (NGS) or qPCR analysis of the cDNA produced in steps e. / d.-g.

[0152] In embodiments, the method of the present invention advantageously uses the method of the present invention that forms a physically separated first compartment to determine the absolute amount of the target analyte. In embodiments that determine the absolute amount of the target analyte, the first partition is implemented as a first partition, wherein the partition is physical, and the binding reaction between the cell and the binding agent is preferably carried out at a high target analyte concentration applied by a low volume containing each single cell. Preferably, the cells are lysed during the first partition. In embodiments, each first compartment is separated for a second time, and a suitable sensitive method is used to count single target analyte molecules, such as nucleic acid sequence analysis. Preferably, the binding characteristics of the binding agent are used to determine the concentration of the target in the first compartment (preferably containing a known volume and a cell), and ultimately lead to the determination of the absolute amount / quantity of the target analyte in each single cell, as described in the present embodiment. In embodiments, the method includes providing a single cell sample; incubating the sample with two target-specific binding agents, wherein the target and at least two binding agents form a conjugate; separating the single cell sample into a plurality of first partitions (each first partition preferably contains a cell or does not contain a cell); and supplementing each first partition with a cell lysis buffer. During the "compartmental two-component method" (in the second partition), the composition of the conjugate contained in the first partition is determined.

[0153] In an embodiment, a binding agent (e.g., an antibody) is conjugated with a DNA amplifiable marker. The labeling efficiency achieved by the binding agent can be determined in an embodiment and confirmed in an embodiment, for example, by SDS-PAGE electrophoresis. In an embodiment, free (unbound) labeling and labeling errors (e.g., see Example 5) can be determined. In an embodiment, a binding agent (e.g., an antibody) may preferably not have overlapping epitopes, and the conjugated binding agent has two different labels. In an embodiment, the dissociation constant of the binding agent can be determined, for example, as described herein or according to Example 6. In an embodiment, a cell is encapsulated in a first partition using a droplet generation technique (e.g., a microfluidic platform). In an embodiment, the resulting emulsion can be collected and incubated. In an embodiment, droplet distribution can be performed at an adjusted droplet concentration, for example, a single first compartment is delivered to a microplate well. In an embodiment, a dPCR reagent is added to the compartment, and a single droplet may preferably be distributed into a hole and loaded into another orifice plate after mixing. Preferably, a negative control (combination control) containing only a labeled binding agent is also analyzed. In an embodiment, dPCR can be performed. In embodiments, the count of conjugates and the count of binding agents can be obtained as described herein (e.g., as in Example 6). In embodiments, the average expected antibody count (copy number) per reaction (corrected antibody labeling ratio and labeling error) and detected coupling (corrected antibody labeling ratio and labeling error) can be determined. In embodiments based on binding agent concentration and a predetermined binding feature including the Kd of the binding agent (e.g., see the apparent kd determined in Example 6), a calibration curve can be constructed. In embodiments, the calculated target concentration per cell can be determined (average).

[0154] In an embodiment, the step of determining the binding characteristics comprises determining the following binding characteristics for each binding agent of the first type and the second type:

[0155] - the dissociation constant with the target analyte, and / or

[0156] - specificity of binding to the target analyte, and / or

[0157] - labeling ratio, and / or

[0158] - flag synthesis errors, and / or

[0159] - Activity score, and / or

[0160] - contamination by free label, and / or

[0161] - The optimal concentration (number of molecules per unit volume) required for binding to the target analyte.

[0162] As described herein, the amount of conjugate can be determined by analyzing the co-localization of a label of a first type of binder with a label of a second type of binder in a partition or emulsion using nucleic acid amplification (e.g., PCR or dPCR) and probes or primers labeled, for example, with fluorescent dyes, and / or alternatively using next generation sequencing (NGS).

[0163] In one embodiment, the present invention relates to a method for processing a sample, the method comprising the following steps:

[0164] A. Antibodies with uniquely identifiable and amplifiable labels are provided, two antibodies per target analyte (ie, first and second type of binders), the label comprising an identifier sequence (barcode) and optionally a unique molecular identifier (UMI).

[0165] B. determining the binding characteristics of the antibody, including determining the labeling ratio of the antibody, the label synthesis error, and the active fraction of the antibody and the contamination by free label, and determining the apparent dissociation constant based on these measurements;

[0166] C. providing a sample containing a certain concentration of cells, wherein the cells contain a target analyte, such as DNA, RNA, protein, epiprotein, or interacting protein;

[0167] D. Optionally, permeabilizing the cells to make them permeable to macromolecules (particularly antibodies);

[0168] E. combining an antibody having a defined binding characteristic with the sample to form a binding reaction between the antibody and the cell containing the target analyte;

[0169] F. First, separate the single cell into multiple first partitions;

[0170] G. combining the separated single cells with a buffer having lysis conditions;

[0171] H. partitioning the antibody-target analyte complex (conjugate) formed by the single target analyte of the target analyte and the single single cell into another plurality of second partitions for a second time;

[0172] I. Optionally, simultaneously during steps EH, converting RNA into cDNA;

[0173] J. amplifying the antibody label, thereby optionally connecting the antibody labels of the two antibodies forming the conjugate to the target analyte and optionally the DNA and the cDNA;

[0174] K. Determine nucleic acid sequence information;

[0175] L. identifying the barcode of the antibody tag from the sequence information determined in step K., thereby identifying the identity of the target analyte of the single cell bound by the two antibodies to form a conjugate,

[0176] and optionally, determining the composition of the first partition by identifying sequence information of the linked antibody tags based on their UMIs;

[0177] M. Optionally, determining the RNA composition of the single cell from the sequence information;

[0178] N. determining the absolute amount of the target analyte in the single cell by taking into account the binding characteristics of the antibody determined in step B., and optionally determining the chemical composition of the first partition based on the antibody label attached in the second partition identified in step L., wherein the determined chemical composition preferably includes the amount, concentration or presence of the target analyte in the first partition.

[0179] Preferably, during step G., incubation between the cell contents and the binding agent is still possible / still occurs, such that the binding agent can also come into contact with (and form a conjugate with) a target agent contained within cells (e.g. within an organelle, such as within the nucleus) which were impermeable (or to a lesser extent permeable) and / or inaccessible (or to a lesser extent accessible) to the binding agent prior to cell lysis.

[0180] In some embodiments, step K. of identifying sequence information comprises collecting sequence information of the linked antibody tag using a separated two-component assay.

[0181] In one embodiment, the present invention relates to a method as described herein, wherein the step of combining the separated single cells with a buffer having lysis conditions comprises exposing the single cells to lysis conditions with cell-specific identifiable barcoded amplification primers to amplify the attached tags and optionally DNA and cDNA in the step of amplifying the attached tags, and optionally, during the second separation to include single cell identifiable barcoding, amplify the DNA and cDNA. In other words, wherein the single cells are exposed to lysis conditions with cell-specific identifiable barcoded amplification primers in step G (as disclosed above) to amplify the attached tags, and optionally, during the second separation to include single cell identifiable barcoding, amplify the DNA and cDNA. In other words, wherein the single cells are exposed to lysis conditions with cell-specific identifiable barcoded amplification primers in step G (as disclosed above) to amplify the attached tags, and optionally, during the second separation to include single cell identifiable barcoding, amplify the DNA and cDNA.

[0182] In one embodiment, the present invention relates to a method, wherein in the step of combining the separated single cells with a buffer having lysis conditions, chemical treatment is applied before the lysis conditions are applied to the single cells, including cross-linking proteins that stabilize protein interactions, inhibitors that prevent sample deterioration, or the labeled antibodies.

[0183] In one embodiment, the present invention relates to a method, wherein in the step of combining antibodies with defined binding characteristics with the sample, the number of antibodies applied is advantageously less than 10, and the method of determining the sequence information of the nucleic acid marker of the binder is similar to the partitioned PCR two-component method described in WO 2016 / 083793 A1, applying the steps from the second partitioning step to the step of optionally determining the RNA composition of the single cell from the sequence information. In other words, wherein before the lysis conditions applied to the single cell in step G. (as disclosed above), a chemical treatment is applied, including cross-linking proteins that stabilize protein interactions, inhibitors that prevent sample deterioration, or the labeled antibodies.

[0184] In one embodiment, the present invention relates to a method, wherein the number of antibodies in step E is advantageously greater than 10, and the method for determining the sequence information of the nucleic acid marker of the binder is a separated NGS two-component method, similar to the method described in WO 2016 / 083793 A1, applied to steps H to M (or L). In other words, wherein the number of antibodies applied in step E (as described above) is advantageously less than 10, and the method for determining the sequence information of the nucleic acid marker of the binder is a separated PCR two-component method similar to the one described in WO 2016 / 083793 A1, applied to steps H to M (as described above).

[0185] In one embodiment, the present invention relates to a method, wherein a plurality of said single cells are subjected to said method, and the information obtained from said single biomolecule, said identity and said absolute amount of said antibody-identifiable biomolecule, said plurality of single cells is determined from the information of step M (as described above; including determining the RNA composition of said single cells from sequence information), optionally combined with the information of said RNA content of said single cells.

[0186] In some embodiments, the first partition preferably provides chemical conditions that allow for the performance of other detection methods including the detection of multiple RNA molecules.Such RNA detection methods for single cells are known in the art.

[0187] In an embodiment, the target or target analyte may comprise many epitopes and may bind to a variety of binding agents. In a preferred embodiment, all bound binding agents are detected in pairs using the method of WO 2016 / 083793 A1 or WO 2020 / 260277 A1.

[0188] The method of the invention is preferably performed under physiological conditions so that the target can be detected in its native environment, ie under conditions identical to those present in living cells. This provides information about the naturally occurring target.

[0189] The method preferably applies a first partition. The chemical composition of the first partition is unknown, but can be inferred from the second partition. Maintaining an absolute quantitative correspondence between the first partition and the second partition is a preferred embodiment of the present invention. Preferably, the first partition is a binding reaction between a binding agent and a single cell target. In an embodiment, the concentration of the target is preferably unknown in the first partition. In addition, in an embodiment, the concentration of the binding agent is unknown, or the concentration of the single cell is unknown, or the volume of the first partition is unknown. In an embodiment, during the second partition, the method determines the count of the binding agent and the count of the conjugate corresponding to the first partition. From the numerical conditions of the method and the determined counts of the first partition, the concentration of the binding agent and the concentration of the conjugate can be derived to determine the chemical composition of the first partition.

[0190] The first compartment preferably provides chemical conditions, concentrations of single cell targets, and binding agents that promote binding reactions. For example, such conditions can be provided by many single cells in a larger volume or a small number of single cells in a smaller volume.

[0191] The step of contacting the binding agent with the target is usually carried out in a known buffer system, for example, in a buffer system that has been used for the target of the binding agent. The reaction can be carried out at room temperature or 4°C or other suitable conditions. In order to obtain a reproducible signal, the optimal time, optimal temperature and other assay conditions, including the binding and detection steps, are determined. The optimal conditions can be determined by those skilled in the art, but are optimized in particular in terms of fixation, permeabilization, incubation time.

[0192] The present invention enables detection of absolute quantities of single-cell targets at previously undetectable levels, as low as 100, 10, or 0 copies of the target per single cell.

[0193] Different concentrations of binding agent and target (variable number of single cells) can be used to calculate quantitative parameters of the target, such as protein-protein interactions, proteins and epiproteins (modified / PTM proteins). Therefore, in a preferred embodiment, the method is performed using different concentrations of binding agent and / or target.

[0194] In another preferred embodiment, during the first partition, the conjugate is formed.

[0195] In conjunction with the second partition, the conjugate needs to be separated, i.e. separated from other conjugates before the tag sequence, i.e. the associated unique nucleotide sequence, is attached. The separation is performed by methods known in the art. For example, separation can be performed by dilution, specific binding, or by physical and / or chemical properties. Preferably, the dilution is a limiting dilution. These conjugates are separated into a second compartment, such as an emulsion, a microcavity, etc., preferably a diffusion-restricted or isolated compartment as described in WO 2016 / 083793 A1.

[0196] In a preferred embodiment, the binding agent and target analyte are non-immobilized and in solution, such that the conjugate is also formed in solution.

[0197] Preferably, the two analyte-specific binding components and the analyte are non-immobilized and in solution, so that the two-component / analyte complex is also formed in solution. In some embodiments, the method includes the step of providing a sample with a known concentration of the analyte. In some embodiments, the method is an in vitro method, wherein preferably, the step of providing a sample does not include surgery or invasive treatment of a living human or animal body.

[0198] Preferably, the separation particularly comprises any one or more of solid surface binding, dilution or phase separation, or more preferably provides diffusion-restricted or separated compartments / partitions. The separation limits the number of unbound binding agents in each compartment. For example, the average number of unbound binding agents in the compartment is one or less, or the average number of unbound binding agents is between one and three. Separation (e.g., effective separation or isolation of a large number of reactions) follows a Poisson distribution. Fully separating the conjugates before further analysis will provide an environment for generating (optionally connected) nucleic acid marker pairs, which is based on the co-localization of the binding agent. Especially when studying complex protein mixtures, separation reduces the number of non-specific co-localization of nucleic acid identity markers and allows identification of specific binding partners.

[0199] On average, the separation may result in a preference for a single conjugate per compartment, where ligation will provide only the ligated nucleotide sequence of the binder, thus reducing the likelihood of random ligation between other members of the binder. This separation is preferred for counting the binder / target complexes in the compartment based on a Poisson distribution and allows determination of their absolute amount, however there are other cases where the mathematical concept fully compensates for other final states of the separation.

[0200] On average, the separation may result in a single unbound binder per compartment, where ligation will provide only self-ligated nucleotide sequences, thus reducing the likelihood of random ligation between other members of the binder. This separation enables counting of binders in a compartment based on a Poisson distribution and allows determination of their absolute amount, and is well known in the art.

[0201] In embodiments where linked nucleotide sequences are generated in compartments (partitions), amplification methods may be used. Compartmental amplification methods are well known to those skilled in the art, such as emulsion polymerase chain reaction (Schütze et al., Anal. Biochem. 2011 March 1; 410(1): 155-7).

[0202] In embodiments where linked nucleotide sequences are generated in compartments, amplification is used to generate covalent bonds between tags and / or to determine colocalization-based linkage of tags in the absence of covalent linkage. In embodiments, generating linked nucleotide sequences in compartments or partitions by amplification may include linking tagged UMIs to generate a unique linked sequence or a simply unique sequence (comprising a unique combination of two tagged sequences).

[0203] As demonstrated herein, in preferred embodiments, an absolute quantitative method for single cell analysis is provided, which can be applied as a novel single cell method, for example, for proteomics and / or interactomes and related fields, and even nucleic acid analysis (essentially any target that can be identified by a binding analyte according to the present invention), the scope of the invention being generally known to those skilled in the art. This method can be used for single cells after different separation methods, including fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), laser capture microdissection (LCM), manual cell picking / micromanipulation, different microfluidic platforms, and on-demand optical interception and controlled single cell printing (A. Gross, J. S. Niekrawitz, W. Streule, L. Riegger, R. Zengerle, P. Koltay, Single-Cell Printer: Automated, On Demand, and Label Free, J. Lab.. Autom. 18 (2013) 504–518. https: / / doi.org / 10.1177 / 2211068213497204.), thereby enabling highly sensitive, absolutely quantitative and parallel proteomic analysis of single-cell targets. The methods of the present invention can also be combined to detect cellular mRNA. Due to the broader detection of different molecular species in single cells, the methods described herein allow for a better understanding of intercellular heterogeneity.

[0204] The methods of the invention can also be used to generate functional quantitative information about the workings of cells, which can help determine functional differences that occur between different cell types, or disease states or species between any cell subjects that have any functional differences.

[0205] The novelty of the present invention enables, in a preferred embodiment, characterization of a binding agent, which can then be used to detect the target of the binding agent, to be analyzed by applying a determined characteristic of the binding agent to a single cell, which has previously been contacted with the binding agent in suspension. In a cell suspension, both advection and diffusion transport rates affect binding in dispersed solutions. In a cell suspension, the applied target concentration can be increased to a level that saturates binding of the binding agent, and the saturation can be demonstrated based on the characteristics of the binding agent, and the absolute amount can be derived. Preferably, binding of the binding agent below the saturation level can also be used to characterize the binding agent, and the absolute amount can be derived. Therefore, the methods described herein are also suitable for generating quantitative proteome and / or interactome or even genomic and / or transcriptome data corresponding to any target of interest that can be identified by the binding agent, and are not limited to highly abundant target analytes, such as highly expressed proteins or RNA.

[0206] The novelty of the present invention lies in novel quantitative assessment of antibody targets in, for example, single cells. In a preferred embodiment, the present invention applies the following two major improvements over the prior art: (i) determining the binding characteristics of a labeled binder (e.g., an antibody), including consideration of the labeling ratio of the binder, label synthesis errors that render the label undetectable, and the active fraction of the binder, the ability of the binder to bind and contamination by free label, preferably using an unknown amount of cellular target material, and determining the apparent dissociation constant of the binder based on these measurements, thereby providing accurate, highly sensitive, low to zero measurements of single cell targets, and (ii) performing a double partition of single cells, enabling absolute quantitative correspondence between the conditions of a first partition of single cells and a second partition of single cells during a binding incubation, determining the absolute amount of very low, 100 or 10s copies / target / single cell of the binder using the determined binding characteristics of the binder, thereby determining the chemical composition of the first partition (e.g., the presence or absence of the target).

[0207] The separation enables a number of separate, unified and specific conditions.

[0208] A preferred embodiment of the first compartment (divided into first sub-regions) comprises a single cell suspension, wherein the compartment is diffusion restricted and the cells themselves are in their concentration environment.

[0209] Furthermore, preferably, the compartmentalization achieves a high overall concentration (enrichment) of the target by isolating single cells, such that individual single cells represent locally different concentrations.

[0210] In another preferred embodiment of the first partition (divided into the first partition), several cells or single cells are contained in a favorable small volume, wherein the partition is physical. Preferably, the partition is achieved by encapsulating a single cell in a preferably small volume to achieve a high total concentration of the target, such volume being in the nanoliter range or picoliter range. The method of encapsulating cells in such small volume compartments is known in the art, including, for example, droplet microfluidics, micropores, and fluid dynamic capture (Murphy, TW, Zhang, Q., Naler, LB, Ma, S. & Lu, C. Recent advances in the use of microfluidic technologies for single cell analysis. Analyst 143, 60 (2018)).

[0211] In a preferred embodiment, after exposure of the single cells to a labeled binding agent (eg, antibody or aptamer), preferably during the first partitioning (partitioning into first partitions), the separation of the single cells is maintained so that the initially partitioned single cells can be analyzed individually.

[0212] The second partitioning (dividing into second partitions) helps to determine the chemical composition (preferably the concentration of binders and conjugates in this context) of each single cell that was separated during the first partitioning by subsequently applying the (apparent) binding characteristics of the binder, so that the absolute amount / concentration of the target can be deduced.

[0213] For example, these methods can be used to detect targets at less than 100 copies or 10 copies per cell, as described in Example 1. The methods can also be used to generate functional quantitative information about the work of cells, which can help determine functional differences that occur between cell types, disease states, or species between any cell subjects with any functional differences.

[0214] The reactivity of a binding agent such as an antibody can be described by the association rate and dissociation rate of the binding agent and its derivatives, and is also referred to in the art as the "dissociation constant". Although reactivity is generally considered to be a constant related to the chemical properties of the binding agent, such as an antibody, the value of the dissociation constant is a function of the chemical environment, including the presence and amount of salts, hydronium ions (pH) and other chemical moieties. In addition, changes in the structure and conformation of the epitope also change the dissociation constant of the binding agent, such as an antibody, and in most cases, since it is also part of a macromolecule, the epitope is also the subject of the same effects of the chemical environment. There are methods to determine, for example, the dissociation constant of an antibody in a given chemical environment to compensate for these effects, including NMR spectroscopy, equilibrium dialysis, dynamic light scattering, analytical ultracentrifugation, ultrafiltration, electrophoretic methods, differential scanning calorimetry, homogeneous time-resolved fluorescence, fluorescence correlation spectroscopy / fluorescence cross-correlation spectroscopy, spectrometry, affinity capillary electrophoresis, biolayer interferometry, dual polarization interferometry, static light scattering, and microthermophoresis (Wilkinson KD. 2004 Quantitative analysis of protein–protein interactions. Methods Mol. Biol. 261, 15–32. doi: 10.1385 / 1-59259-762-9:015).

[0215] In embodiments for determining the dissociation constant of a binding agent such as an antibody, a separated two-component method can be advantageously applied to derive the absolute amount of the conjugate (or ternary complex) formed, thereby facilitating accurate determination of the dissociation constant of the antibody in a complex chemical environment, also in a natural environment, in the absence of an external standard of the target. An embodiment is described in Example 3. The method for determining the dissociation constant of a binding agent is affected by other factors, including the labeling ratio of the binding agent, the labeling synthesis error, the active fraction of the binding agent, and the contamination by free labels, as shown in, for example, Example 4. In such embodiments, only the labeling ratio and / or the labeling synthesis error of the binding agent need to be independently determined to control and compensate for the accuracy of the separated two-component method for determining the dissociation constant of the binding agent. Those skilled in the art can design methods to determine the labeling ratio of the binding agent, the labeling synthesis error of the binding agent, for example, as described in Example 5.

[0216] However, as shown in Example 4, the active fraction of the binder and contamination by free label will be advantageously confounded in the determination of the antibody dissociation constant. Example 6 shows the determination of the antibody dissociation constant for varying the labeling ratio, label synthesis error, and active fraction of the binder. Preferably, a two-component method using separation of the labeling ratio and label synthesis error of the binder is used to determine the apparent dissociation constant of the binder, thereby providing inherent favorable self-compensation of the active fraction of the binder and contamination by free label to provide a dissociation constant of the antibody for unbiased determination of absolute amounts in single cells.

[0217] In one embodiment, preferably, both the determination of the dissociation constant of the antibody (binding agent) and the determination of the absolute amount of said target analyte in said single cell are determined using the same partitioning / zoning two-component method.

[0218] In a preferred embodiment, the two analyte-specific binding components of the two-component method and the analyte are non-immobilized and in solution, so that the two-component / analyte complex (conjugate) is also formed in solution.

[0219] In one embodiment, determining the absolute amount of the target analyte is performed in a first compartment, wherein the compartment is diffusion-restricted, and the binding reaction between the cell and the antibody is performed at a diffusion-restricted concentration, such as shown in Example 1. The concentration of the target protein (target analyte) of the binding reaction is preferably defined by the volume of the cell suspension containing the cell and the total target protein content of the volume. Under suitable conditions and assumptions, with negligible cell volume or by making the cell permeable to antibodies or target protein / analyte and including the cell volume in the entire sample volume, the concentration of the target protein / analyte can be treated like in solution, as described in Example 1. Therefore, in such embodiments, their concentration per cell can be assumed by the concentration calculated for the entire sample volume and the number of cells and binding agents present in the sample volume. Separating each single cell and adopting a suitable sensitive method, the number of targets per cell can be determined by the extrapolated concentration of the target in the suspension volume divided by the number of cells in the suspension. Preferably, the suitable sensitive method is a second compartment method, which can be used to count single molecules. Preferably, cells are lysed before the second compartment. Preferably, the binding signature is used to determine the extrapolated concentration of the target in the suspension volume and ultimately leads to the determination of the absolute amount of target analyte in each single cell, as described in Example 1.

[0220] The present invention discloses a method for determining the absolute amount of a target analyte in each single cell using a partitioning method. In one embodiment, the binding reaction between a cell and an antibody is performed in a first partition. The concentration of the target protein (target analyte) in the binding reaction is defined by the volume of the compartment and the total target protein content within the volume. By taking into account the negligible volume of the cell or making the cell permeable to the antibody or target protein / analyte by lysis, the concentration of the target protein / analyte can be treated as in a solution. Single cell separation is combined with a sensitive second partitioning method capable of counting single molecules, allowing the target quantity of each cell to be determined by extrapolating the target concentration in the first partition volume. The method utilizes lysed cells in the first partition and utilizes the binding characteristics to extrapolate the target concentration, and ultimately is able to absolutely quantify the amount of the target analyte of each single cell, as described in Example 2.

[0221] In one embodiment, the absolute amount of the target analyte (e.g., a biomolecule recognizable by an antibody) is determined in a first compartment, wherein the compartment is physical and the binding reaction between the cell and the binding agent is preferably carried out at a high target analyte concentration imposed by a low volume containing each single cell. Preferably, the cells are lysed during the first compartment. Each first compartment is re-partitioned and a suitable sensitive method is used to count single target analyte molecules. Preferably, the concentration of the target in the first compartment is determined using the binding signature, and ultimately results in determining the absolute amount / quantity of the target analyte in each single cell, as described, for example, in Example 8.

[0222] In one embodiment, a suitable sensitive method is a homogenously partitioned two-component method without washing steps to maintain binding conditions, similar to the method described in WO 2016 / 083793 A1.

[0223] In another embodiment, the two-component method further comprises an absolute quantification capability having a predetermined binding characteristic of the binding agent, having predetermined sequence information (of the attached tag sequence) of said attachment.

[0224] In one embodiment, a suitable sensitive method is a partitioned two-component method, similar to the method described in WO 1999 / 043855A1. This method uses the sequence information of the nucleic acid markers of the two binding agents of the conjugate (referred to herein as the (two) components) to detect the absolute amount of protein in each single cell. This method is suitable for determining its independent parameters because the antibody concentration in the single cell suspension and the number of bound antibodies in the conjugate can be determined based on the solution-based partitioning data of the single cells from the second partition.

[0225] In one embodiment, a suitable sensitive method is a compartmentalized two-component method, similar to the method described in WO 2016 / 083793 A1, wherein for the readout a dPCR-based method using co-compartment-based junction information is applied.

[0226] In one embodiment, a suitable sensitive method is a separated two-component method, similar to the method described in WO 2016 / 083793A1, wherein for readout, an NGS sequencing-based method is applied. In this method, the co-separation-based connection information of the nucleic acid labeling of the binder is based on the determination of the unique molecular identifier (UMI) of the connection, see also this Example 10.

[0227] In one embodiment, a suitable sensitive method is a two-component method using a separation of unique molecular identifiers (UMIs), wherein the UMI sequence comprises at least 2 nucleotides, or more than 2 nucleotides, or at least 4 nucleotides, or at least 6 nucleotides, or at least 8 nucleotides, or at least 10 nucleotides, or at least 12 nucleotides, or at least 14 nucleotides, or at least 20 nucleotides, or at most 8 nucleotides, or more than 8 nucleotides, or at most 10 nucleotides, or at most 14 nucleotides, or at most 20 nucleotides. UMI sequences have been described in the art, for example, by Kivioja et al. (Kivioja et al., 2012, Nat Methods 9:72-74). In a preferred embodiment, the UMI is used in such a way that the molecules into which the UMI is incorporated can be encoded, and the encoded information has a certain amount. The UMI sequence is a random sequence that can be added to other sequences.

[0228] In a preferred embodiment, determining the absolute amount of the biomolecule recognizable by the antibody of the single cell is combined with the method of determining multiple RNA molecules of a single cell as described in Example 7.

[0229] In one embodiment, a suitable sensitive method for detecting the absolute amount of protein per single cell is combined with RNA detection. RNA analysis methods are known in the art and preferably include the use of nucleotide sequences capable of hybridizing to nucleic acids, i.e., nucleotide sequences designed to hybridize to the poly-A tail of mRNA, such as poly-T sequences. In some embodiments, first strand synthesis is primed with an (anchored) oligo dT primer (or possibly a random primer or a combination of both) to which is appended a UMI, an amplification primer binding site, and an optional template switching primer sequence.

[0230] In embodiments of the present invention, nucleic acid sequence barcodes can be preferably used to identify markers, molecules or binding agents derived from single cells. Barcodes can be identified during analysis of nucleic acid sequence information. Unique barcode sequences also allow data to be associated with single cells.

[0231] Additionally or alternatively, the processed RNA may also include a UMI. Thus, the sequence information of the nucleic acid tag of the binder includes a UMI that can be linked and / or the processed RNA may include at least one of a barcode and a UMI. Thus, in some embodiments, oligo dT or random hexamer priming is appended with a UMI. Furthermore, in some embodiments, the template switching oligo also includes a UMI.

[0232] In one embodiment of the invention, the sample is preferably a single cell or an isolated cell nucleus. The invention is not limited to any particular cell type. Preferably, the cell is a nucleated cell. Preferably, the cell is a mammalian cell, preferably a human cell.

[0233] Many variations of single cell cDNA synthesis exist in the literature and are well known to those skilled in the art and can be applied here.

[0234] In embodiments where protein detection of proteins, protein interactions and / or post-translational modifications according to the invention is combined with RNA detection, the readout / analysis is preferably performed by next generation sequencing (NSG), such as provided by Roche, Illumina and Applied Biosystems, or also known in the art as third generation sequencing, as described by David J Munroe & Timothy JR Harris in Nature Biotechnology 28, 426-428 (2010), or such as provided by Pacific Biosciences and Oxford Nanopore Technologies.

[0235] In one embodiment, the method of the invention further comprises depleting ribosomal RNA (rRNA).

[0236] It is immediately understood by those skilled in the art that there may be additional or alternative methods to amplify the obtained cDNA, and the methods applicable here are not limited to the polymerase chain reaction (PCR).

[0237] In different steps of the embodiments of the methods as specified herein, one or more adapters may be attached to the sequences. Additionally or alternatively, during the reverse transcription and / or amplification steps, additional nucleotide sequences may be added by incorporating these sequences into primers for reverse transcription and / or amplification, respectively.

[0238] In one embodiment, the method of the present invention further comprises the step of adding single cell-specific or sequencing method-specific adapters or barcodes.

[0239] In one embodiment, the sequence obtained during the readout according to the method of the present invention, such as the ligated sequence and / or cDNA molecule obtained by the method of the present invention, is flanked by one or more universal sequences. Universal sequences that may be present in different members of a plurality of nucleic acid molecules, and may allow the use of a single universal primer complementary to the universal sequence to replicate or amplify a plurality of different sequences. In an embodiment, the universal sequence may comprise a binding site for a sequencing primer, preferably a binding site for an NGS or deep sequencing primer.

[0240] The embodiments and features of the invention described with respect to any embodiment of the method according to the invention are considered to be disclosed with respect to each and every other aspect of the disclosure, such that features characterizing one embodiment of the method can be used to characterize any other suitable embodiment of the method. The various aspects of the invention are unified in, benefit from, are based on and / or are linked to the common and surprising discovery that the method of the invention enables absolute quantification of target analytes at the single cell level. DETAILED DESCRIPTION

[0241] The present invention provides a method for quantitatively assessing a binder target in a single cell. The present invention preferably applies the following two major improvements over the prior art: (i) using bulk dissolved target material to determine the binding characteristics of a binder such as an antibody, including taking into account the labeling ratio of the binder, label synthesis errors that make the label undetectable, and the active fraction of the binder, the binder has the ability to bind and / or is contaminated by free label, and based on these measurements, determining the apparent dissociation constant of the binder, thereby providing accurate and high sensitivity (down to 10s single cell target) measurement of the target analyte, and (ii) applying 1. a single cell and then 2. a double compartmentalized single cell of its contents, thereby being able to include an absolute quantitative correspondence between the conditions of the first compartment of the single cell (in the first compartment) and the second compartment of the single cell contents (in the second compartment) during the binding incubation of the binder, so as to be able to determine the chemical composition of the binder binding reaction, while using the determined binding characteristics of the binder to determine the absolute amount of the target analyte, even at very low amounts.

[0242] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Those skilled in the art will recognize many methods and materials similar to or equivalent to the methods and materials described herein that can be used to practice the present invention. In fact, the present invention is by no means limited to the methods and materials described.

[0243] The practice of routine techniques in cell culture, biochemistry, molecular biology, recombinant DNA, computational biology, bioinformatics, genomics, proteomics, interactomes, sequencing, and related fields are well known to those skilled in the art and are discussed, for example, in the following references: (Sambrook et al. Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodically updated; and the series Methods in Enzymology, Academic Press, San Diego.)

[0244] Terms in the singular include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a single cell" includes reference to one, two, or more cells, etc., as appropriate.

[0245] As used herein, the term "ligation" is used to describe and illustrate any useful ligation method that provides co-compartmental information about the ligation obtained by labeling the binding agents in the two-component method, the unique nucleotide sequence of the ligating binding agents. The term "ligation" is widely used and describes physical and non-physical ligation methods, and is the same as the term "ligation information" or "ligated sequence information".

[0246] As used herein, the term "approximately" is used to describe and illustrate small variations. For example, the term may refer to less than or equal to 10, such as less than or equal to as low as 1, when appropriate, and may also refer to greater than or equal to 10, such as greater than or equal to 100 or more, when appropriate. It should be understood that the range format is used for simplicity and brevity, and should be flexibly interpreted to include the values ​​explicitly indicated as the boundaries of the range, covering each value and sub-range.

[0247] This article "consider (taking into account)" can preferably refer to one or more parameters are included in the calculation. In other words, according to the corresponding value to be determined, factor or parameter, consideration can refer to certain parameters are included in the corresponding or applicable calculation for determining the value, factor or parameter. In an embodiment, consideration can include the offset of parameters or values ​​from each other. Examples of parameters considered for calculating a certain value or constant are provided herein and in the examples. For example, in an embodiment, during the calculation / determination of the absolute amount / concentration of the target analyte in a sample or cell, the volume of the first partition (first partition) (for calculation) and / or the initial concentration of cells in the initial solution are considered. Or, for example, in an embodiment determining the labeling efficiency of a binding agent such as an antibody, as described herein, the concentration [ng / μl] of the heavy chain (for calculation) of a light chain, a heavy chain and a label is considered.

[0248] In an embodiment, nucleic acid sequencing, such as next generation sequencing (NGS), may be used to analyze the merged droplets / second compartments. The NGS results may be analyzed according to the procedures described in (PCT / EP2020 / 067493). Briefly, in an embodiment, sequencing "separated" dPCR may be used to amplify a binder tag comprising a unique tag (UMI), thereby obtaining a nucleic acid sequence comprising two connected tag sequences in the case of a conjugate. During NGS data analysis, a tag sequence comprising two or more UMIs, such as a "dimerized UMI", may be deconvoluted in an embodiment to reconstruct the contents of each droplet (wherein the tag is ddPCR amplified, preferably only one conjugate is included). That is, in an embodiment, the evaluation of the binding reaction may be based on NGS reads of binder-specific "dimerized" UMI tags generated according to a standard protocol for emulsion coupling (dPCR-based connection of two or more binder tags in a conjugate). In an embodiment, the number of (labeled) binding agents, such as antibodies, in each droplet (preferably forming conjugates) may be determined by counting all unique UMI labels for a given binding agent, such as an antibody (the count is limited to a given binding agent-specific sequence). In an embodiment, during analysis using repeated "dimerization" label sequences, possible PCR or sequencing duplicates may be discarded. In an embodiment, conjugates may be counted based on their dimerization labels (comprising two or more UMIs).

[0249] In an embodiment, once the droplet contents have been identified, a Poisson-based calculation (as described in EP22161450.6) can be performed to determine the number of detected conjugates. In short, the distribution of the binding agent during the separation is controlled by the Poisson distribution, so the occurrence of each binding agent is counted (after the droplet-based PCR amplification, the relative abundance of the binding agent is determined by NGS), and if the number of droplets is known, the background detection of the conjugate can preferably be calculated. A large number of specific binding events will be used to identify the accurate target agent of the binding agent. For example, in a multi-protein complex, the information contained in the connection label of the binding agent is based on the co-localization of the corresponding binding agent on the complex, and indicates the direct interaction between the detected target analytes. In an embodiment, the results obtained can be further normalized using the expected Poisson distribution of the background signal and / or a single binding agent and its label.

[0250] In other words, in embodiments, a ligation reaction (e.g., amplification) of binding agent tags contained in a single conjugate produces a linked nucleic acid tag comprising a linked nucleic acid sequence for each of the corresponding tags, which is itself preferably separated in a second compartment (droplet). For example, if two binding agents bind to a target agent, thereby forming a conjugate, the linked tags comprise the linked nucleic acid sequences of the two binding agent tags. Thus, preferably, the linked tag sequences comprise at least two binding agent type-specific sequences (one sequence from one binding agent) and at least two UMIs, each of which is unique to their binding agent molecule and is unique among the multiple binding agents used in the experiment. In embodiments, nucleic acid sequence analysis (e.g., using NGS) is used to identify the nucleic acid sequences of the linked ("dimerized") tags. Data from sequence analysis of such sequences comprising linked tag sequences can then be analyzed.

[0251] Thus, in embodiments using nucleic acid sequence analysis, linked marker sequences can be identified, and each unique combination of at least two markers (within a sequence, a unique combination of at least two different UMIs) indicates the formation of a conjugate. Thus, in embodiments, during sequencing analysis, each unique sequence comprising at least two marker sequences can be counted, and the total count (of unique linked marker sequences) is preferably equivalent to the total count of conjugates detected / analyzed. In embodiments using binding features described herein, the absolute concentration of the target analyte can be calculated from the counts of the conjugates. In embodiments, the absolute concentration of the first target analyte for each cell in the first partition is determined by the initial concentration of cells in the sample in step a., the binding features determined in step b., and the number and / or concentration of conjugates and binders in the second partition in step h. (taking into account) the offset from each other.

[0252] In embodiments, dilutions of a sample can be measured in the same sequencing reaction using sample-specific DNA barcodes (e.g., barcoded primers), and because different types of binders have distinguishable labels, many measurements (using different antibody pairs for different antigens) can be performed in parallel (multiplexed) in embodiments.

[0253] The terms of linking statements refer to the following situations, in which one or more of the mentioned situations may appear alone, or in combination with at least one of the mentioned situations until all of the mentioned situations appear, including terms such as "including", meaning that the structure is inclusive and open, rather than exclusive.

[0254] Amplification may refer to a variety of amplification reactions, including but not limited to polymerase chain reaction (PCR and its many variants - Saiki, R. et al. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–4., Saiki, RK (1988) Primer-directed enzymatic amplification of DNA with athermostable DNA polymerase. Science 239, 487–91.), in particular fluorescent readout (Holland, PM et al. (1991) Detection of specific polymerase chain reaction product by utilizing the 5′→3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–80., other references: https: / / www.promega.de / resources / guides / nucleic-acid-analysis / pcr-amplification / ).Isothermal amplification reactions include loop-mediated isothermal amplification (Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000). 'Loop-mediated isothermal amplification of DNA'. Nucleic Acids Res. 28(12): 63e–63. doi: 10.1093 / nar / 28.12.e63. PMC 102748. PMID 10871386 and U.S. Patent 6410278, Notomi T, Hase T, 'Process for synthesizing nucleic acid', published on June 25, 2002, assigned to Eiken Kagaku Kabushiki Kaisha), whole genome amplification technology (Zong C, Lu S, Chapman AR, Xie XS. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell.Science.2012;338:1622–6.Pmid:23258894,KleinCA,Schmidt-Kittler O,Schardt JA,Pantel K,Speicher MR,RiethmüllerG.Comparative genomic hybridization,loss of heterozygosity,and DNA sequence analysis of single cells.Proc Natl Acad Sci US A.1999;96:4494–9.Pmid:10200290,Dean FB,Hosono S,Fang L,Wu X,Faruqi aF,Bray-Ward P, et al.Comprehensive human genome amplification using multipledisplacementamplification.Proc Natl Acad Sci US A.2002;99:5261–5266.Pmid:11959976,Langmore JP.Rubicon Genomics,Inc. 2002;3:557–560.,Telenius H,Carter NP,BebbCE,.M, Ponder B a, Tunnacliffe a. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics. 1992; 13: 718–725. Pmid: 1639399, Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci US A. 1992; 89: 5847–51. Pmid: 1631067), strand displacement amplification (GT Walker, MC Little, JG Nadeau and DD Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), helicase-dependent isothermal DNA amplification (Vincent M, Xu Y, Kong H (2004). 'Helicase-dependent isothermal DNA amplification'.EMBO Rep.5(8):795–800.doi:10.1038 / sj.embor.7400200.PMC 1249482.PMID 15247927.), recombinase polymerase amplification (Euler, Milena; Wang, Yongjie; Nentwich, Oliver; Piepenburg, Olaf; Hufert, Frank T.; Weidmann, Manfred (2012).'Recombinase polymerase amplification assay for rapid detection of Rift Valley fever virus'.Journal of Clinical Virology.54(4):308–12.doi:10.1016 / j.jcv.2012.05.006.PMID 22683006, Amer, HM; Abd ElWahed, A.; Shalaby, MA; Almajhdi, FN; Hufert, FT; Weidmann, M. (2013).‘A new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay’. Journal of Virological Methods. 193(2):337–40. doi:10.1016 / j.jviromet.2013.06.027. PMC 7113639. PMID 23811231, Abd El Wahed, Ahmed; El-Deeb, Ayman; El-Tholoth, Mohamed; Abd El Kader, Hanaa; Ahmed, Abeer; Hassan, Sayed; Hoffmann, Bernd; Haas, Bernd; Shalaby, Mohamed A.; Hufert, Frank T.; Weidmann, Manfred(2013). Meng, Xiang-Jin (ed.). ‘A Portable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of Foot-and-Mouth Disease Virus’. PLOS ONE. 8(8):e71642. Bibcode:2013PLoSO...871642A. doi:10.1371 / journal.pone.0071642. PMC 3748043. PMID 23977101, Piepenburg, Olaf; Williams, Colin H.; Stemple, Derek L.; Armes, Niall A. (2006). ‘DNA Detection Using Recombination Proteins’. PLOS Biology. 4(7):e204. doi:10.1371 / journal.pbio.0040204. PMC 1475771. PMID 16756388) or nucleic acid sequence-based amplification (Deiman, Birgit; van Aarle, Pierre; Sillekens, Peter (2002).'Characteristics and Applications of Nucleic Acid Sequence-BasedAmplification(NASBA)'.Molecular Biotechnology.20(2):163–180.doi:10.1385 / mb:20:2:163.ISSN 1073-6085.PMID 11876473.S2CID 28712952), rolling circle amplification (Ali, M. Monsur; Li, Feng; Zhang, Zhiqing; Zhang, Kaixiang; Kang, Dong-Ku; Ankrum, James A.; Le, X. Chris; Zhao, Weian (2014). 'Rolling circle amplification: a versatile tool for chemicalbiology, materials science and medicine'. Chemical Society Reviews.43(10):3324–41.doi:10.1039 / C3CS60439J.PMID 24643375) etc. .

[0255] In the context of the present invention, PCR reagents and / or reverse transcription reagents and / or RT-PCR reagents include one or more reagents selected from the group consisting of DNA polymerase and / or reverse transcriptase (RT-) polymerase and / or dNTPs and / or amplification primers and / or poly-A primers for RNA reverse transcription and / or buffering agents and / or nuclease inhibitors and / or other reagents or enzymes. Those skilled in the art are aware of the components of PCR and reverse transcription reagents required for successful amplification and / or reverse transcription of nucleic acids.

[0256] Digital polymerase chain reaction (dPCR) is a PCR technique that utilizes the enhanced amplification efficiency of low-volume PCR reactions to absolutely quantify target nucleic acids. The quantitative principle of dPCR based on the correction of Poisson distribution of the single-molecule counting and partition reaction of the amplified single molecules (Sykes PJ, Neoh SH, Brisco MJ, Hughes E., Condon J., Morley AA Quantitation of Targets for PCR by Use of Limiting Dilution. Biotechniques. 1992; 13: 444–449.). The key design parameters of the dPCR platform include the number of partitions, the volume of each partition, the total reaction volume, and the change of the partition volume. The statistical accuracy of dPCR is further affected by the variability of sample preparation and the molecular leakage rate and false positive rate. The dPCR platform currently lacks the sample multiplexing of qPCR, but provides a unique assay multiplexing capability. (Quan PL, Sauzade M, Brouzes E. dPCR: A Technology Review. Sensors (Basel). 2018; 18(4): 1271. Published April 20, 2018. doi: 10.3390 / s18041271).

[0257] There are many forms of real-time or qPCR, which differ primarily in the detection chemistry used to monitor the reaction products, e.g., Gelfand et al., U.S. Pat. No. 5,210,015 ('Taqman"); Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecular beacons). Detection chemistries for real-time PCR are reviewed (Mackay et al., Nucleic Acids Research, 30:1292-1305, 2002).

[0258] With regard to the "absolute quantification" of the method, a method based on unique molecular identifiers is described to obtain information from the quantitative composition of single-molecule compartments (WO 2020 / 260277 A1). The term "absolute concentration", "absolute number" or "absolute amount" of a target analyte or biomolecule refers to the total or exact concentration, quantity or amount of the analyte. In other words, absolute quantification determines the quantity or concentration of the analyte in terms of the absolute number of molecules or copies. In contrast, relative quantification determines the fold change in quantity, amount or concentration between two samples. In addition, in contrast, other quantitative methods require external quantitative standards to determine the quantity, which is different from the method described in the present invention, in which the absolute molecular counts in the volume define the molar concentration of the target without comparison with an external (relative) quantitative standard material.

[0259] "Sequencing" refers to determining the order of nucleotides (base sequences) in a nucleic acid sample. Next generation sequencing is described, for example, illustrated by commercial methods such as Roche, 111UMina and Applied Biosystems, Oxford Nanopores, Pacific Biosciences (David J Munroe & Timothy JR Harris in Nature Biotechnology 28, 426-428, 2010). The common feature of these technologies is to determine the sequence of multiple DNA single molecules, effectively and quantitatively revealing the composition of DNA samples in a single run, ranging from hundreds to billions of single readings. Sequencing reaction needs to introduce specific DNA sequences, commonly referred to as adapters, which are different from the technology used. This process is called library preparation, and relates to connection methods (for example), PCR extension or other methods. Adapters can include identifier sequences that distinguish samples. As used herein, "next generation sequencing (NGS)" shall include recently developed nucleic acid sequencing technologies that generally allow much higher throughput than the traditional Sanger method (see Schuster, Next-generation sequencing transforms today's biology, Nature Methods 5:16-18 (2008); Metzker, Sequencing technologies the next generation. Nat Rev Genet. 2010 Jan; 11(1):31-46. These platforms can allow single-molecule sequencing of clonally amplified or unamplified nucleic acid fragments. Certain platforms involve, for example, sequencing by ligating dye-modified probes (including circular ligation and cleavage), pyrosequencing, and single-molecule sequencing. Nucleotide sequence species, amplified nucleic acid species, and detectable products generated therefrom can be analyzed by such sequence analysis platforms. Next generation sequencing can be used in the methods of the present invention, for example, to quantify unique PCR-amplifiable DNA markers to assess the formation of dual component / analyte complexes as described below.

[0260] In embodiments, the method as described herein uses a separated two-component detection method. As used herein, the term "separated two-component detection method" should preferably refer to a two-component detection method in which the sample is separated to quantify the formation of a conjugate after the analyte-specific binding component is contacted with the analyte. In general, the separated two-component detection method is preferably based on separating the solution into small compartments so that in the absence of a conjugate, the presence of two binding components in the compartment is impossible and follows a Poisson distribution. For separation, different determinations can be envisioned. For example, an emulsion method can be used to form droplets in an emulsion (e.g., water-in-oil), wherein each droplet represents a separate compartment. Separation can involve position or physical compartments, or a diffusion-restricted environment.

[0261] As used herein, the term "droplet" preferably refers to a separated portion of a first fluid surrounded by a second fluid. The first fluid preferably includes a hydrophilic fluid, such as water, an aqueous medium or a buffer, and preferably includes a sample solution or one or more diluents thereof to which a two-component detection system or other reagents have been added. The second fluid is preferably a hydrophobic fluid, such as a hydrocarbon, silicone oil, mineral oil, an organic solvent. The emulsion technology of separating sample solutions is well known in the art. In a preferred embodiment, the separated two-component detection method is emulsion coupling, which refers to a digital determination concept based on a double-labeled (two-component) single ternary molecule complex in a detection emulsion, and the complex can be identified, for example, by droplet digital PCR (ddPCR) or next generation sequencing (NGS).

[0262] Preferably, the two analyte-specific binding agents and the analyte are provided in a non-fixed form, i.e. in solution, so that the conjugate is also formed in solution. Thus, the term two-component detection method as used herein is preferably directed to the liquid phase formation of the conjugate and is different from a common sandwich immunoassay involving a (primary) capture binding agent and a (secondary) detection binding agent fixed as a solid phase. Thus, the term "non-fixed" preferably refers to a component, such as an analyte-specific binding component, that can diffuse freely in a (liquid) solution, such that the binding kinetics with the analyte (also freely diffusing in the liquid solution) are governed by the equations of the law of conservation of mass and the law of mass action in solution as described herein.

[0263] In preferred embodiments, determining the binding characteristics comprises determining the following binding characteristics of each binding agent of the first type and the second type: dissociation constant of the target analyte, specificity of binding to the target analyte, label ratio, label synthesis error, activity fraction, contamination by free label and / or optimal concentration (number of molecules per unit volume) required for binding to the target analyte. Thus, in other words, in embodiments, the binding characteristics of a binding agent may include the (re)activity, specificity and / or concentration of the binding agent. Reactivity may be described by the dissociation constant of the binding agent, specificity by identifying the binding target, and concentration by the number of molecules per unit volume.

[0264] In general, the dissociation constant (KD) is an equilibrium constant representing the trend of reversible dissociation (separation) of a binding agent from its target analyte. In other words, the dissociation constant can represent that a binding agent is combined with a target analyte and promotes "reliability" and / or intensity of its detection. In an embodiment, a two-component method using the labeling ratio of a binding agent and / or a labeling synthesis error is used to determine the apparent dissociation constant of a binding agent. Therefore, in an embodiment, the consideration of the dissociation constant for calculating the absolute concentration of a target agent herein is preferably different from the consideration of a simple titration curve in the prior art (for example, in a method for determining the target agent concentration in a sample or cell), and is more complicated than it, because preferably in the context of the inventive method, the labeling ratio of a binding agent and / or a labeling error are additionally (independently) determined, and used to control and compensate (normalize) the accuracy of the method for determining the dissociation constant of a binding agent, thereby achieving the absolute quantitative accuracy of a target agent higher than that which may be achieved by any prior art method.

[0265] In an embodiment, the concentration reference curve of the two-component detection method or the dissociation constant relationship of the two-component detection method can be provided in the form of reference data. In an embodiment, the reference data of the concentration reference curve of the two-component detection method should preferably involve any data that allows a mathematical function to be provided, and the mathematical function reflects the dependence of the signal reflected in the formation of the two-component / analyte complex formed in the solution on the concentration of the analyte. In an embodiment, based on the signal reflecting the concentration of the two-component / analyte complex and the concentration of the analyte and / or the analyte-specific binding component, the reference data of the dissociation constant relationship of the two-component detection method should preferably involve any data that allows a mathematical function to be provided, and the mathematical function is the relationship between the dissociation constant of the analyte-specific binding component (kd1 and kd2, respectively) and the analyte.

[0266] In the embodiments herein, the separation generation of "connected sequence information" can refer to the physical connection of the labels (preferably nucleic acid labels) of the two binding agents in the case where the two binding agents are co-located in the conjugate within the partition (because they are synchronously bound to their target analytes), thereby forming a conjugate. In other words, under suitable assay conditions, based on the co-localization of these nucleic acid identity labels, the connection process has the potential to form random polymer nucleic acid products. In embodiments, the connection reaction can be based on amplification or involve other technologies. Amplification-based connection can utilize two or more amplification primer pairs, which have the same binding ability but have complementary 5' tags or dimer adapter sequences, resulting in the formation of polymerase-extendable nucleic acid duplexes. The tag or dimer adapter sequence means that the sequence amplified by one primer pair will hybridize with the sequence amplified by the second primer pair. The identity label representing the type of binding agent is therefore connected. The physically connected nucleotide label of the binding agent, if it forms a conjugate with their target analyte, helps to "translate" the information that the binding agent and its target are co-located in the partition into a nucleotide sequence (physical connected nucleotide label). In an embodiment, the identity of the binding agent and the presence of its target can be determined by nucleotide sequence, which can be achieved by nucleic acid sequencing or PCR, such as digital (droplet) or real-time qPCR in an embodiment. One embodiment of the process is described in WO 2016 / 083793 A1. Optionally, before identifying the connected marker, the nucleotide sequences from the connection of more than one conjugate are merged. The identity of the binding agent and / or target can be determined by, for example, sequencing the connected nucleotide marker sequence. This can be achieved using a highly parallel system. The connected sequences can be merged so that a single reaction can be performed to identify all connected sequences. For example, all connected nucleotides can be sequenced in a single reaction. The connected sequences can be quantitatively determined to measure the relative abundance of the connected sequences. In some embodiments, the markers of the co-localized binding agents of the conjugate are not physically connected, but their co-localization in the second partition, so the co-localization in the conjugate is detected by PCR, preferably dPCR and / or real-time qPCR using a fluorescence detection method. This detection method of co-localized nucleic acid markers helps to reliably determine the presence of the conjugate, and thus the presence of the target analyte in the second partition, because the distribution of the components of the single cells of the first partition in the second partition follows a Poisson distribution. Therefore, if the two binding agents do not bind to the target analyte and form a conjugate, the probability that they are present in the same second partition is less than the probability that the two binding agents are co-localized, because they are physically connected by binding to the target analyte, thereby forming a conjugate. Therefore, in an embodiment, their nucleic acid markers can be detected by PCR methods without the need for physical connection of the markers in advance, but for example by droplet PCR with two differently labeled (e.g., two different fluorescent colors) probes.

[0267] Therefore, in this embodiment, during detection, the presence of two different fluorescent colors in the second partition (e.g., emulsion) will indicate the presence of a conjugate in the second partition. In embodiments analyzing multiple target analytes, two specific binding agents should be selected for each target analyte, one of the first type and one of the second type. During the final PCR, qPCR, real-time PCR, or dPCR readout, the distinction between different binding agent pairs for different target analytes can be achieved in some embodiments using, for example, different fluorescent markers, primers, or probes. In one example, a probe of a connection sequence of two binding agent labels specifically bound to a first target analyte is labeled with a fluorescent color, while a probe of a binding agent label that has a connection specific to a second target analyte is labeled / marked with a different fluorescent color. In other embodiments, when the label of the binding agent in the conjugate is not connected, each binding agent pair in the conjugate is detected by a specific combination of fluorescent colors, which can be distinguished during the final PCR readout. In an embodiment, the readout of the marker sequence can be performed by nucleic acid sequencing. In such embodiments, the nucleic acid tags of the binding agents within the conjugate are preferably connected before being read out by sequencing, preferably within the second partition, for example by a ligase reaction or PCR or other methods known in the art, preferably followed by a step of merging all second partitions and a batch sequencing analysis of all tags contained within the partition. During the analysis of the sequence information obtained, the sequence of the connected tags can be determined and assigned to a single conjugate, and thus to a single target analyte molecule.

[0268] Examples of linked sequence information are described in WO 2016 / 083793 A1 and Karakus et al. (Karakus U, Thamamongood T, Ciminski K, Ran W, Günther SC, Pohl MO, Eletto D, Jeney C, Hoffmann D, Reiche S, J,Ulrich R,Wiener J,Hayes MGB,Chang MW,Hunziker A,Yángüez E,Aydillo T,Krammer F,Oderbolz J,Meier M,Oxenius A,Halenius A,Zimmer G,Benner C,Hale BG,García-Sastre A,Beer M,Schwemmle M,Stertz S.MHC class IIproteins mediate cross-species entry of bat influenza viruses. Nature. March 2019; 567(7746):109-112.doi:10.1038 / s41586-019-0955-3. Electronic version February 20, 2019 PMID: 30787439., WO 2016 / 083793A1), for detecting proteins, epiproteins (PTM- / modified proteins) and protein-protein interactions, in particular for labeling, analyzing, detecting and measuring protein-protein interactions with less than 10 antibodies.

[0269] Herein, a "ligation" reaction may also be referred to as "ligation of labels", "linking of labels", "binding agent-labeled ligation", or "antibody-labeled ligation". In some embodiments, the ligation reaction referred to herein is a classical proximity ligation reaction involving rolling circle DNA synthesis or rolling circle amplification. In other embodiments, the ligation is achieved using a ligase. Non-limiting examples of proximity-based two-component methods include methods using resonance energy transfer assays, preferably F6rster resonance energy transfer (FRET) assays or bioluminescence resonance energy transfer (BRET) assays, protein complementation assays (PCA), Alphascreen or DNA-labeled proximity assays, preferably proximity ligation assays (PLA) or proximity extension assays (PEA). These proximity assay techniques are well known in the art. For further references to FRET or BRET, see, for example (Pfleger, Seeber, & Eidne, 2006), for protein complementation assays, Morel! , Ventura, & Aviles, 2009, for Alphascreen Taouji, Dahan, Bosse, & Chevet, 2009 and for proximity methods of DNA labeling such as PLA (Proximity Ligation Assay) or PEA (Proximity Extension Assay) Soderberg et al., 2006.

[0270] As used herein, "barcode" refers to a nucleic acid sequence that is preferably used to identify a single cell during the acquisition of sequence-based linkage information. A unique barcode sequence allows data to be associated with a single cell. As used herein, a barcode may also refer to a nucleic acid sequence that is preferably used to identify a single cell during the acquisition of sequence-based linkage information. A unique barcode sequence allows data to be associated with a single cell.

[0271] The processed RNA may also include a UMI. Thus, in embodiments, the ligated sequence information includes a UMI that may be ligated and / or the processed RNA may include at least one of a barcode and a UMI. Thus, in some embodiments, oligo dT or random hexamer priming has a UMI appended. Furthermore, in some embodiments, the template switching oligo also includes a UMI. In the literature, there are many variations of single cell cDNA synthesis, and these variations are well known to those skilled in the art and may be applied here.

[0272] WO2020260277A1 describes a specific embodiment of using unique molecular identifiers (UMIs) to separate and generate "connected sequence information", wherein UMI is described in Kivioja et al. (Teemu Kivioja, Anna Kasper Karlsson, Martin Bonke, Martin Enge, Sten Linnarsson, and Jussi Taipale. Counting absolute numbers of molecules using unique molecular identifiers. Nat. Methods, 9(1):72–74, January 2012). Various methods have been developed to improve the accuracy of quantification of different polynucleotides in a sample using next-generation sequencing technology, including competitive polymerase chain reaction (PCR) methods as described in U.S. Pat. No. 5,213,961 and deep barcode sequencing using unique molecular identifiers (UMIs), as described in (Smith, AM, Heisler, LE, Mellor, J., Kaper, F., Thompson, MJ, Chee, M., Nislow, C. (2009). Quantitative phenotyping via deep barcode sequencing. Genome Research, 79(10), 1836-1842. https: / / doi.Org / 1 0.110 1 / gr.093955.109).

[0273] "Nested PCR" means a PCR in which the product of a first amplification is used as a template for a second amplification PCR reaction. As used herein, the inner primers in relation to a nested amplification reaction are primers used to generate a first amplicon, and the outer primers are primers used to generate a second or nested amplicon.

[0274] Multiplexing of PCR means a PCR in which multiple target sequences are provided and amplified simultaneously in the same volume (Bernard PS, Ajioka RS, Kushner JP, Wittwer CT: Homogenous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes. Am J Pathol 1998; 153: 1055–1061). Multiplex PCR can be combined with dPCR as long as there is low multiplexity in the compartment and large PCR multiplexity in the total volume of dPCR (Alexandra S. Whale, Jim F. Huggett, Svilen Tzonev. Fundamentals of multiplexing with digital PCR. Biomolecular Detection and Quantification. Vol. 10, 2016, pp. 15-23, ISSN 2214-7535, https: / / doi.org / 10.1016 / j.bdq.2016.05.002).

[0275] In this article, "target analyte" or "target" may also refer to a "biomolecule" or a biomolecule recognizable by an antibody, or simply "target". The terms may be used interchangeably herein. Therefore, these terms may be used interchangeably herein. As used herein, a target may be a single molecule or a group of molecules (e.g., at least two molecules forming a complex, such as a protein-protein complex, a protein-nucleic acid complex, a substance-protein complex, or a substance-nucleic acid complex), which forms a complex with a binding agent, including but not limited to proteins, modified or post-translationally modified (PTM) proteins (epiproteins), including artificial or naturally modified proteins, including phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis, as well as protein interactions, non-protein molecules recognizable by binding agents (e.g., nucleic acids or modified nucleic acids). The complex is usually formed under conditions that maintain the conformation of the target of the organism of interest. The target or target analyte may be selected from the group comprising peptides, proteins, chemically modified peptides or proteins, nucleic acids, chemically modified nucleic acids, substances, and any complex thereof (including any complex of one or more of the listed substances).

[0276] In this article, "conjugate" describes a complex formed by two binding agents (a binding agent pair) or an analyte-specific binding agent and a target analyte itself, and two binding agents specifically bind to the complex. Preferably, in the context of a two-component detection method, such as the method described in WO 2020 / 260277 A1, a conjugate may also be referred to as a "two-component / analyte complex" or a "ternary complex". The conjugate preferably comprises a first type of binding agent and a second type of binding agent, each of which is bound to a different epitope on the same target analyte molecule. In an embodiment, the first type and the second type of binding agent are two different antibodies that recognize different isotopes of the target analyte and can be simultaneously bound to the target analyte. In an embodiment, the combination and detection of two different binding agents (of different types) with the same target analyte has many advantages over the detection by a single binding agent. For example, in an embodiment, a type of binding agent can recognize chemical or post-translational modifications, and a second type of binding agent can recognize specific proteins. In another embodiment, a type of binding agent can recognize a conserved epitope of a protein, and a second type recognizes a specific mutation or variant of a target protein. In another embodiment, the target analyte can be a protein complex or a DNA-protein complex, wherein one type of binding agent recognizes a protein in the complex and another type of binding agent recognizes, for example, a methylated DNA motif, or in the case of a protein-protein complex, each type of binding agent recognizes one of the proteins forming the complex. According to the Poisson distribution, the second partitioning step of the method of the present invention helps to separate single molecules or molecular complexes contained in the lysed cells. Therefore, the presence of two different types of binding agents in a droplet, bead or partition indicates that the two types of binding agents are combined with the target analyte and form a conjugate with it. The (non) probability that two binding agents are simply in the same partition by chance, but not in the conjugate, can be simply calculated.

[0277] In an embodiment, the method of the present invention can be used to identify multiple binding agents that bind to a single target. For example, in an embodiment where the target is a protein, the method can identify antibodies that bind to different epitopes on the protein. Alternatively, the target can be a protein complex, and the method can identify multiple binding agents that bind to different proteins in the complex. For example, in an embodiment, a nucleic acid marker associated with a binding agent in a conjugate can be connected to a nucleotide sequence associated with a second binding agent in the conjugate. Once the identity of the binding agent is known (based on the nucleic acid marker sequence connected), it is possible to identify the components of the target, thereby identifying proteins that interact naturally in the target, for example. For example, in an embodiment where the binding agent is an antibody with known binding characteristics, the protein bound by the antibody can be identified. Therefore, the identity of the protein in the target can be identified. This allows protein-protein interactions in samples to be detected and identified. In addition, once protein-protein interactions are identified, the method can be used to monitor the effects of compounds on the interaction. Alternatively, in an embodiment, a nucleotide sequence associated with a binding agent in a binding agent / target complex can be connected to a nucleotide sequence associated with a target in a binding agent / target complex. In embodiments, the method can be used to identify which binding agent interacts with which target. For example, it can be used to identify which members of a binding agent library can form a complex with a known target. This information can be used to characterize members of a binding agent library to obtain binding signature information. Preferably, in embodiments, the generation of the randomly paired, connected nucleic acid products comprises amplifying identical or different amplicons using at least two pairs of PCR primers; wherein optionally, the PCR primers have sequence tags at the 5' end, wherein randomly paired, connected nucleic acid products are generated by labeled primer amplification. More preferably, the amplification is an emulsion PCR amplification or a digital PCR (dPCR), and the generation of the amplicons and the randomly paired, connected nucleic acid products is a parallel process. Preferably, the sequencing of the connected amplified products is a highly parallel sequencing method (e.g., NGS).

[0278] Preferably, "binding agent" is an antibody, an aptamer or based on an engineered protein scaffold. Preferably, the term "antibody" means any binding agent. Alternatively, a binding agent can be a compound. In this article, binding agents can be distinguished into different "types", wherein a certain type of binding agent preferably recognizes a specific epitope or target binding motif on a target analyte. Therefore, different types of binding agents recognize different motifs or epitopes on a target analyte. Therefore, it is preferred to use two analyte-specific binding agents, both of which show binding potential to the target analyte. Preferably, the two analyte-specific binding agents do not compete with each other to bind to the analyte, but are selected and designed to allow simultaneous binding of components to form a conjugate. Therefore, in this article, two types of binding agents that are specific to a target analyte can bind to the target analyte at different positions of the analyte at the same time. Therefore, a type of binding agent can specifically bind to a domain, chemical modification (such as PTM) or motif of a target analyte, and another type of binding agent can recognize or specifically bind to another domain, epitope or motif of a target analyte. In the case where a molecular complex is a target analyte, different types of binding agents may recognize different members or portions of the complex, thereby facilitating specific detection of the complex rather than its individual members or portions. Such complexes may be protein-protein, protein-peptide, peptide-peptide, protein-nucleic acid, compound-nucleic acid, compound-protein, compound-peptide or compound-compound-complex, or any complex that can be specifically recognized by a binding agent.

[0279] The binding agent can be a member of an antibody display library or an antibody library, wherein each antibody is labeled with a unique nucleotide sequence. The method can use an antibody agent as a binding agent, wherein for example, the binding characteristics of the binding agent and the target binding and the chemical properties of the binding are known, and the chemical properties include the numerical representation of binding affinity, the binding rate and dissociation rate of the binding, and further include the following labeling ratio, labeling synthesis error, activity score, some or all of the numerical considerations of the pollution of the free label, and determine the unique nucleotide sequence associated with multiple displayed antibody agents, and the binding characteristics and the unique nucleotide sequence are correlated. In an embodiment, when two binding agents, preferably a first type of binding agent and a second type of binding agent, are bound to the same target agent molecule, thereby forming a conjugate, the conjugate (comprising two binding agents and target analyte) can also be referred to as "analyte-two component complex". The two binding agents, particularly in the case of a two-component detection method, can also be referred to as "analyte-specific binding component" or simply "binding component".

[0280] The binding agent used in the present invention can be an antibody. As used herein, the term "antibody" refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule containing an antigen binding site that specifically binds to an antigen, whether natural or partially or entirely simulated. The term "antibody" includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanized antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or fully or partially simulated, and any polypeptide or protein having a binding domain that is homologous to or homologous to an antibody binding domain. Therefore, a chimeric molecule comprising an immunoglobulin binding domain fused to another polypeptide or an equivalent is included. The cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023. Examples of antibodies are immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD, and IgA) and their isotype subclasses; fragments comprising an antigen binding domain, such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies can be polyclonal or monoclonal. In addition, fragments of intact antibodies can function to bind to antigens. Examples of binding fragments are (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) a Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of the VH domain (Ward, ES et ai, Nature 341:544-546 (1989)); (v) isolated CDR regions; (vi) a F(ab')2 fragment, which is a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv), in which the VH domain and the VL domain are connected by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al., Science 242:423-426 (1988); Huston et al., PNAS USA 85:5879-5883 (1988)); (viii) bispecific single-chain Fv dimers (PCT / US92 / 09965); and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; P. Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)).

[0281] An "antigen binding domain" is a portion of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. When the antigen is large, the antibody may only bind to a specific portion of the antigen, which is called an epitope. The antigen binding domain may be provided by one or more antibody variable domains. The antigen binding domain may include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0282] Alternatively, the binding agent can be based on an engineered protein scaffold. The protein scaffold is derived from a stable, soluble, native protein structure that has been modified to provide a binding site for a target molecule of interest. Examples of engineered protein scaffolds include, but are not limited to, affibodies, which are based on the Z domain of staphylococcal protein A and provide a binding interface on its two A-helices (Nygren, PA (2008). FEBS J 275 (11): 2668-76); antiphospholipids derived from lipocalin, which incorporate binding sites for small ligands at the open ends of the β-barrel fold (Skerra, A. (2008) FEBS J 275 (11): 2677-83), nanobodies, and DARPins. Engineered protein scaffolds typically target the same antigenic proteins as antibodies. Short peptides can also be used to bind to target proteins. Phylomers are natural structural peptides derived from bacterial genomes. Such peptides represent a range of different protein structural folds and can be used to inhibit / disrupt protein-protein interactions in vivo (Watt, PM (2006). Nat Biotechnol 24(2): 177-83)].

[0283] Alternatively, the binding agent may be an aptamer. An aptamer is a simulated oligonucleotide (DNA or RNA) that recognizes a target molecule with high affinity and specificity through a combination of shape complementarity and non-covalent chemical bonds (Blank & Blind, Current Opin. Chem. Biol., 2005, 9: 336-342). These artificial ligands are easily available in vitro and can be developed to recognize a variety of different molecular categories, from pure ions (e.g., Pb 2+, Liu & Lu, 2003. J Am Chem Soc., 125, 6642-6643) to nucleotides, small molecules, proteins, viruses and cells until the entire organism (Menger et al., 2006. Handbook of Experimental Pharmacology, 359-373). Aptamers with high binding affinity have been selected by the well-known SELEX method (Ellington & Szostak, 1990. Nature, 346, 818-822) for the detection of low molecular weight molecules such as theophylline (Jenison et al., 1994. Science, 263, 1425-1429), L-arginine (Geiger et al., 1996. Nucl. Acids Res., 24, 1029-1036), moenomycin (Schuerer et al., 2001. Bioorg. Med. Chem., 92, 2557-2563), 17b-estradiol (Kim et al., 2007. Biosens. Bioelectron., 22, 2525-2531) and larger molecules such as thrombin (thrombin binding aptamer: 5'-GGTT-GGTGTGGTTGG-3') (Baldrich et al., Anal. Chem. 2004, 76, 23, 7053-63), cholera toxin or HIV-1 tat protein, etc. (for review, see Tombelli et al., 2007, Biomolec Eng., 24, 191-200). Some of the aptamers mentioned above have been used for ELISA-like assays on the surface of microplates or biosensor converters (QCM, SPR). Aptamer-modified AuNP colorimetric systems have also been developed for determining the protein PDGF in sandwich-based assays (Huang et al., 2005, 77, 5735-5741). Recently, aptamer libraries based on modified nucleotides have been applied to measure proteins in a highly parallel manner. Aptamer sequences can be tagged with unique sequences, or they are unique themselves and used as nucleotide tags for binding agents.

[0284] The binding agent can be a library, such as a display binding agent library, such as a part of a bacterial display, mRNA display, phage display, aptamer, ribosome display or yeast display library. Each member of the library has a detectable nucleic acid identity tag, and the tag is preferably unique to a member of the library. Preferably, the unique nucleic acid identity tag is connectable. "Connectable" means that under suitable assay conditions, based on the co-location / separation of these nucleic acid identity tags, the connection process has the potential to form random nucleic acid connection products. Preferably, the product is a dimer or a pair of tags or higher co-separated tags that are not covalently linked.

[0285] Preferably, the binding agent is capable of detecting more than one target, preferably with different apparent affinities. Alternatively, the binding agent is capable of detecting a single target using different epitopes or binding sites, preferably with different apparent affinities.

[0286] Preferably, any binding agent that binds to any target and associates with a unique nucleotide sequence is suitable for use in the present invention.

[0287] A binding agent can recognize a specific target, such as a corresponding protein. This is called specificity. The WO 2016 / 083793 A1 assay used herein as a reference uses two antibodies per detection to enhance these specificities. Alternatively, multiple binding agents can recognize a target, such as a specific target, such as a corresponding protein. This is called redundancy. Similarly, based on the similarity of the target conformation, for example due to protein conformation or protein sequence, a binding agent can recognize more than one target, such as a protein species. This phenomenon is called cross-reactivity. In addition, the recognition of the target protein by the binding agent is based on the conformation of the protein or its protein sequence. This is called its reactivity and its numerical representation, and its affinity is the solubility constant of the binding agent. The protein binding affinity of the binding agent can be calculated from quantitative information, for example, according to WO2020260277A1.

[0288] The predetermined binding characteristics of the binding agent may include reactivity, the active fraction of the binding agent, cross-reactivity with specificity and redundancy, and calculated affinity, such as the apparent dissociation constant of the binding agent. In addition, the binding agent preferably associates with a unique nucleotide sequence, and as described herein, the associated unique nucleotide sequence changes the apparent, measurable dissociation constant of the binding agent, and further includes contamination with free label, labeling ratio of the binding agent, and labeling error as influencing factors.

[0289] In embodiments where the one or more binding agents are antibodies (or fragments thereof), the step of determining the labeling efficiency comprises calculating the labeled fraction (LE) of the binding agent using the concentrations [ng / μl] of the antibody's light chain (lc), heavy chain (hc), and labeled heavy chain (L) as follows:

[0290]

[0291] In an embodiment, determining the concentration of free label (label not bound to the binding agent) after a labeling reaction of the binding agent comprises, for example, performing nucleic acid amplification by dPCR to determine the concentration of free label (label not bound to the binding agent) (e.g., in cp / μl) and the concentration of labeled binding agent (e.g., in cp / μl) in the labeling reaction volume or any other (stock) solution containing the binding agent, and thereby calculating the fraction of free label.

[0292] In embodiments, the binder-label can be amplified using PCT or dPCR (e.g., as described in Example 1) compared to a control sample (e.g., nucleic acid / PCR standard). Preferably, dPCR is capable of determining the exact amplifiable / detectable amount (concentration or percentage) of the binder label. In embodiments, the difference in the two amounts / concentrations (binding agent versus control) determined in the measurement can then be used to calculate the label synthesis error.

[0293] In an embodiment, a two-component method using labeling ratios and / or label synthesis errors of the binding agent is used to determine the apparent dissociation constant of the binding agent. This can preferably provide an inherently favorable self-compensation of the active fraction of the binding agent and contamination by free label to provide a dissociation constant of the antibody for unbiased determination of absolute amounts in single cells.

[0294] In an embodiment, for each sample, a calibration curve can be calculated as described herein. However, the calibration curve can be calculated with an adjusted binding feature as described in WO 2020 / 260277 A1. Using the individually determined labeling ratio and / or labeling error of the antibody, the calibration curve is corrected, for example, with the correction values ​​of the single positive value and the double positive value of dPCR, resulting in correction of antibody and conjugate concentrations. This constitutes a significant improvement over the prior art, in which the concentration of the target analyte in the sample can usually be determined using only an uncorrected calibration curve. However, the calibration curve of the prior art does not take into account any error or deviation caused by the unlabeled binding agent and / or the error in the labeling synthesis, etc. The method of the present invention surprisingly overcomes this shortcoming of the prior art, wherein in an embodiment, the labeling error or deviation from the wrongly labeled binding agent is taken into account during the quantification of the target analyte, in other words, it is used to normalize the data obtained from, for example, the calibration curve and / or sequence analysis.

[0295] In embodiments, assuming that all samples contain the same concentration of target analyte, the apparent dissociation constant of the binding agent may be recursively calculated so as to minimize the standard deviation of the recalculated target analyte concentrations for all measurements.

[0296] In chemistry, biochemistry, medical technology and pharmacology, the dissociation constant (Kd) is a specific type of equilibrium constant that measures the tendency of a larger complex to reversibly dissociate into smaller components (Friguet, Chaffotte, Djavadi-Ohaniance, & Goldberg, 1985). The dissociation constant is the reciprocal of the association constant. In the special case of an embodiment of a two-component measurement method, the dissociation constant is the dissociation constant between the two components (binding agents) and the target analyte. Because the components can be antibodies, the determination of the dissociation constant has a wide range of significance. In the prior art, some methods are known for determining the dissociation constant.

[0297] Thus, the term "two-component detection method" or "bimolecular detection method" refers to a method of using two analyte-specific binding components and determining a signal reflecting the formation of a two-component / analyte complex (conjugate) when the two analyte-specific binding components are contacted with a solution containing the analyte. The term "two-component detection system" or "bimolecular detection system" preferably refers to the components or reagents required to perform a two-component detection method. This preferably includes two analyte-specific binding components (binding agents), preferably provided as a single or separate solution of known concentration and optionally a sample solution containing the analyte to be analyzed.

[0298] Preferably, the two-component detection method is compartmentalized. The compartmentalization is diffusion limited, physical. Preferably, the compartmentalized two-component detection method is in solution, or preferably involves surface bound molecular components.

[0299] In a two-component detection method, two analyte-specific binding components are preferably used, both of which show binding potential to the analyte. Preferably, the two analyte-specific binding components do not compete with each other for binding to the analyte, but are selected and designed to allow the components to bind simultaneously to form a two-component / analyte complex. As previously described, the analyte can refer to an aggregate or complex formed by multiple entities, such as a protein-protein complex. One of the two analyte-specific binding components can be bound to an entity of the analyte complex, such as a first protein, while the other analyte-specific binding component is bound to a second entity of the analyte, such as a second protein. Therefore, a two-component / analyte complex is formed only when two proteins interact, and the two proteins are labeled with the binding components.

[0300] In one embodiment, determining the "dissociation constant" of a binding agent may include preparing one or more dilutions of a sample or binding agent with a known dilution factor, and applying a two-component detection method to the sample and one or more dilutions, wherein applying a two-component detection method to determine the dissociation constant of an analyte-specific binding agent preferably means contacting two target analyte-specific binding agents (two components) of known concentration with a sample of known concentration to generate a signal that depends on the concentration of the two binding agents / target analyte complexes (conjugates or two-component / analyte complexes) formed in the solution. The concentration of the analyte-specific binding agent or sample is changed by dilution, providing a dilution with a known dilution factor. The signal detected in the sample and in one or more dilutions and the concentration of the target analyte and / or target analyte-specific binding agent can be used as a constraint input for a mathematical fit. The signal can take a variety of forms and depends on the two-component detection method used. In the case of separated two-component methods, such as the conjugation described in WO 2020 / 260277 A1, the signal can also refer to the signal generated by digital droplet PCR (dPCR) and / or NGS sequencing, which indicates the presence of a two-component (two binders) / target analyte complex (conjugate) based on deviations from the expected Poisson distribution in the separated droplets.

[0301] "Emulsion" herein refers to a water-in-oil emulsion that is used to separate a single cell or molecule into a single droplet or compartment containing a very small volume. Those skilled in the art are familiar with various methods for producing water-in-oil emulsions, such as microfluidic devices in some embodiments. Droplet-based microfluidics manipulate discrete volumes of fluid in immiscible phases to produce (micro) droplets containing tiny volumes (μl to fl). Alternatively, in other embodiments, the partitions can be emulsified gel beads produced using emulsified gel beads (GEM) technology, such as 10XGenomics.

[0302] In this article, "partition" refers to the emulsion and physically separated partitions. Those skilled in the art are familiar with various methods for generating partitions, such as microfluidic devices in some embodiments. Alternatively, in other embodiments, the partition can be a nanopore, such as Qiagen's QIAcuity nanopore.

[0303] In the context of the present invention, a "first partition" is a partition, compartment or emulsion that contains a single cell or does not contain a cell. In the method of the present invention, it is preferred that the sample is diluted and separated in such a way that a single first partition or droplet can contain a single cell, or does not contain a cell, preferably no more than one cell. This separation and separation in the first partition is necessary to facilitate n analysis at the single cell level. In some embodiments of the method of the present invention, each single cell is lysed in its first partition to release the cell components into the volume of the first partition or droplet, thereby facilitating the subsequent further separation of the single molecules of each cell into the second partition. Therefore, in the context of the method of the present invention, the "second partition" preferably contains only one molecule, preferably only one target analyte, which is preferably combined with a first type and a second type of binding agent to form a conjugate. Therefore, in the context of the present invention, it is preferred that the compound of the single cell in the first partition is diluted and separated into the second partition in a way that each conjugate formed is contained in a separate second partition or droplet. This separation enables the accurate determination of the number of single conjugates formed during the method of the present invention. In the context of the present invention, the "first partition" may also be referred to as the "first compartment", and the "second partition" may also be referred to as the "second compartment", so the terms "partition" and "compartment" may be used interchangeably herein. The same applies to "partition" and "partition".

[0304] "Emulsion conjugation" or "conjugation" or "protein conjugation" or "PICO" is a digital assay concept based on the detection of dual-labeled (ternary) single molecule complexes (e.g. conjugates) in emulsions, which are identified, for example, by dPCR or next generation sequencing (NGS).

[0305] Preferably, by using unique nucleotide-associated binders, particularly RNA and DNA targets, the target can be assessed simultaneously with other targets having different properties that are not suitable for the assay. In these cases, other methods can be advantageously combined with the present invention, particularly methods for detecting RNA expression of single cells.

[0306] Preferably, the target comprises a protein. More preferably, the target is part of a protein sample. Optionally, the target can be cross-linked with multiple targets, such as other targets within the protein sample. For example, a protein within a sample can be cross-linked with one or more other proteins within the sample and form a ternary or higher order complex target as a new entity of the cross-link.

[0307] The target can be associated with a unique nucleotide sequence. "Association" means that the presence of the target in the binding agent / target complex can be detected by the presence of the nucleic acid sequence in the connection sequence produced by the method. The nucleotide sequence can be attached to the target as a label, or present in the target, such as a nucleic acid in a bacteriophage, or the nucleotide sequence is associated using a cross-linking process, such as a DNA binding protein is DNA, such as DNA is cross-linked to histones. Alternatively, the nucleotide sequence can be a part of an aptamer known to be bound to the target. The binding agent / target complex can be contacted with an aptamer to identify the presence of the target by connecting a unique nucleotide sequence (including an aptamer). However, many alternative ways to associate a label are known in the art.

[0308] The present invention can be applied to any protein sample derived from a single cell. Single cells can be derived from any biological sample, including but not limited to tissue, cytological samples, body fluids, cell cultures or any other single cell-containing material. Body fluid samples include blood, saliva, urine, cerebrospinal fluid and buffy coat. Single cells can be prepared from samples using standard methods known in the art (Hu, P., Zhang, W., Xin, H., & Deng, G. (2016). Single cell isolation and analysis. Frontiers in Cell and Developmental Biology, 4 (OCT), 116. https: / / doi.org / 10.3389 / FCELL.2016.00116 / BIBTEX). Among them, fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), laser capture microdissection (LCM), manual cell picking / micromanipulation and different microfluidic platforms are more frequently used. Recently, on-demand optical interception and controlled single cell printing (A. Gross, J. S. Niekrawitz, W. Streule, L. Riegger, R. Zengerle, P. Koltay, Single-Cell Printer: Automated, On Demand, and Label Free, J. Lab. Autom. 18 (2013) 504–518. https: / / doi.org / 10.1177 / 2211068213497204.).

[0309] As used herein, the term "single cell" refers to a cell. Single cells that can be used in the methods described herein can be obtained from tissues of interest, or from biopsies, blood samples, or cell cultures. In addition, cells from specific organs, tissues, diseased tissues, etc. can be obtained and used in the methods described herein. In addition, in general, cells from any population can be used in the methods, such as prokaryotic or eukaryotic unicellular organism populations, including bacteria, fungi, or yeast. Therefore, preferably, the sample herein is selected from the group comprising: tissue samples, biopsies, liquid biopsies, blood samples, plasma samples, urine samples, liquid samples, environmental samples, cell culture-derived samples, and microbial culture-derived samples.

[0310] In some embodiments of the methods described herein, obtaining a sample can include the steps of first obtaining single cells and then lysing the cells.

[0311] Single cell suspensions can be obtained using standard methods known in the art, including, for example, enzymatic use of enzymes on tissue samples or release of mechanically adhered cells in culture. Single cells can be placed in any suitable reaction vessel, wherein the single cells can be processed individually. For example, a 96-well plate, so that each single cell is placed in a single well, which can also be referred to as separation. For example, single cells can be selected individually based on features detectable by different analytical methods, based on measurable parameters such as position, morphology or gene expression.

[0312] Cell lysis methods are well known in the art. Lysis can be achieved, for example, preferably by using detergents or other chemical methods, or by a combination of these methods.

[0313] Single cell samples can be chemically treated prior to the second partition, for example, different fixation chemicals or cross-linking agents such as BS3-(bis(sulfosuccinimidyl) suberate) or formaldehyde and glutaraldehyde can be used to form bonds between lysine residues, thereby producing cross-linked proteins. Formaldehyde and glutaraldehyde are typically used at a concentration of 0.5-4% in PBS depending on the sample. Single cell samples can be cross-linked or non-cross-linked. Depending on the purpose of the experiment and the type of target being studied, single cell targets can be analyzed in their denatured or non-denatured form and / or in cross-linked or non-cross-linked form. Single cell protein samples can be analyzed under a variety of conditions to collect information about the quantitative characteristics of multiple targets. For example, the concentration or amount of the binding agent can be varied to determine the dissociation constant and other kinetic parameters.

[0314] A separate permeabilization step can also be performed on a single cell to approach the intracellular target. This allows the binding agent to enter the intracellular structure while keeping the morphology of the cell intact. Triton, digitonin and saponin are examples of permeabilization agents that work by destroying the cell membrane. The level of permeabilization is very important because the acquisition of epitopes may require different levels of permeabilization (e.g., cytoplasm versus nuclear epitopes). Such techniques are commonly used in fluorescence activated cell sorting studies and are well known in the art. There are also many commercially available kits that provide reagents for fixation and permeabilization. However, the present invention preferably does not need to remove unbound antibodies, because the preferred technique described in WO 2016 / 083793 A1 is a homogeneous assay, which means that it does not require a washing step.

[0315] In embodiments, the present invention relates to a method for performing a homogeneous assay using the binding agent in a first partition of a cell material exposed to lysis. In some embodiments, wherein step d. (binding reaction) is performed after steps e. and f. (partitioning and lysis), an effective and reliable analysis can be performed by the additional binding of the binding agent under lysis conditions. This preferably prevents binding interference from cellular components. By adopting such methods, the assay preferably achieves enhanced sensitivity and accuracy when detecting and quantifying analytes present in dissolved cell material.

[0316] Single cells can be pre-selected. For example, single cells can be enriched for specific proteins present, such as proteins from specific cell locations, proteins from specific cell types, proteins with similar size or electrostatic charge, proteins with similar binding characteristics, similar sequence characteristics or similar functions, such as enzymes (Current Protocols in Molecular Biology (2006) 20.0.1-20.0.6 CHAPTER 20 Analysis of Protein Interactions.). Preferably, specific proteins include phosphoproteins, membrane proteins or natural proteins, post-translationally modified proteins, and artificially modified proteins.

[0317] Single cells can present protein displays either extracellularly or intracellularly. Examples are well known in the art and described in [Galán, A., Comor, L., A., J., Guillemin, N., Mrljak, V., & Bhide, M. (2016). Library-based display technologies: where do we stand? Molecular BioSystems, 12(8), 2342–2358. https: / / doi.org / 10.1039 / C6MB00219F].

[0318] The target may contain many epitopes and may bind to a variety of binding agents. In a preferred embodiment, all bound binding agents are detected in pairs using the method of WO 2016 / 083793 A1 or WO2020260277A1.

[0319] A detection unit is defined as a complex of target and two bound binding agents, called a conjugate. If more than two binding agents are bound, all unique combinations of binding agent pairs are considered different conjugates.

[0320] "Label" or unique label herein comprises or consists of a unique DNA, RNA and / or protein sequence. Any suitable label known in the art may be used as a label of a binding agent herein. The label may be covalently or non-covalently attached to, connected to or bound to a primer, probe, molecule or binding agent. In some embodiments, the label may also comprise a fluorophore and / or a quencher, or consist of a fluorophore and / or a quencher.

[0321] For the next generation sequencing detection in the embodiment, the binder may preferably be labeled with a unique PCR amplifiable nucleic acid (preferably a DNA marker) comprising a specific marker for the type of binder (e.g., antibody) and preferably also comprising an individual marker for each binder molecule, i.e., a unique molecular barcode or a unique molecular identifier-UMI (see Parekh et al., 2017). In a preferred embodiment, the labeled binder can be added to the sample or its dilution.

[0322] For embodiments including PCR, qPCR or dPCR detection, PCR reagents can be added before sample emulsification, or added separately to each emulsion (partition), such as by droplet fusion or microinjection. PCR or dPCR can be performed using standard dPCR protocols known in the art. The evaluation of the reaction can be based on the partitions marked in the dPCR reaction using fluorescently labeled PCR products (e.g., real-time PCR probes labeled with FAM or VIC). According to dPCR, standard evaluation can determine droplet clusters according to the fluorescent signal of droplets. In this article, the number of labeled binding agents (e.g., antibodies) is determined in each reaction (all labeled positive droplets of a given label are counted, and the same droplet cluster definition is used for all reactions). In addition, the number of dual colors (with two different binding agent labels, such as different types of binding agents, or binding agents for different target analytes) is also determined. In the absence of a ternary complex (conjugate), the partition of the labeled antibody follows a Poisson distribution, and a calculable number of dual-color droplets is generated (based on pure chance, with two binding components in one compartment, but not bound to the target of the present invention). In the case of the presence of a ternary complex in the reaction, the number of detected, for example, two-color droplets (with an attached ternary complex) is greater than the number expected by the Poisson distribution. Based on this measurement, the number of conjugates can be calculated (for further reference, see, for example, EP 20 3224360 or Karakus et al., 2019). This results in an absolute (molecular count) quantification of the ternary analyte complex (conjugate).

[0323] In embodiments where multiple target analytes should be detected, a library comprising binding agents that are specific for different analytes can be provided, wherein each binding agent comprises a label that is unique not only for the type of binding agent, but also for the specific analyte to be detected. For a small amount of different target analytes to be detected, the uniqueness can be encoded by different fluorescent labels with different colors. For a large number of different target analytes, the readout / analysis can be performed by nucleic acid sequencing, such as NGS, in which case the label should be a nucleic acid sequence that comprises a unique sequence for each target analyte and for each type of binding agent. In embodiments, during the initial binding agent labeling reaction or at a later time point in each emulsion (partition), a droplet-specific or molecule-specific barcode is additionally added to the label of each binding agent.

[0324] Depending on the labeled molecule, one or more labels may be used. Preferably, all labels are unique to the molecule as described in WO2012042374A2, but are read out using WO2020260277A1. Preferably, a labeled molecule has multiple labels and all labels are unique to the molecule.

[0325] A unique tag may also be an identity tag, i.e. a unique sequence of interest used in a binder and / or target to unambiguously identify all members of the binders and targets of the invention, the identity not being the molecular unique identity of the tag. Preferably, the tag is both an identity tag and a molecular unique tag.

[0326] In embodiments, the unique tags can be different in their biological context, thus based on the amplification and physical ligation process of biologically specific different primer pairs, for example one or two or more primer pairs amplify the target sequence. The ligation of different tags allows the binding agent specific information to be linked to the target information.

[0327] Nucleic acid amplification for each compartment or partition to generate a connected nucleic acid barcode can be achieved by highly diluting the sample before nucleic acid amplification, for example, the dilution factor is greater than 20,000, preferably greater than 50,000, and more preferably greater than 100,000. In a preferred embodiment, nucleic acid amplification is PCR. In other preferred embodiments, nucleic acid amplification is any suitable method known in the art. PCR reagents can be added to achieve single complex separation and nucleic acid amplification of each emulsion or partition. For this purpose, digital PCR (dPCR) standard methods may be particularly suitable. WO 2016 / 083793 A1 describes a method for using linkage PCR or only generating linkage information at the same time. Afterwards, compartments, such as emulsions, can be re-merged in a common collection, and parallel nucleic acid sequencing technology can be used to evaluate antibody-specific dimerization markers, such as UMIs. In this article, the number of labeled binders, such as antibodies, can be determined in each reaction by counting all unique markers (e.g., UMIs) for a given binder, such as an antibody. The conjugate can be counted based on a PCR product labeled with a dimerization double (UMI) containing two different binder-specific markers. In the absence of formation of ternary complexes (conjugates), the partitioning of the labeled binder follows a Poisson distribution and produces a calculable number of conjugate droplets. In the presence of conjugates in the reaction (target analyte present in the sample), the number of conjugates detected is greater than that expected by a pure Poisson distribution.

[0328] Methods for attaching nucleotides to binding components such as antibodies or compounds are known in the art. Alternatively, if the binding component library is a phage display library, the unique nucleotide sequence can be a sequence encoding one or more CDR regions or a displayed binding domain. For example, a display library can be generated by inserting a sequence encoding an amino acid sequence to be displayed into a known position of a phage. Universal primers that will amplify the inserted sequence can then be used to identify the binding sequence.

[0329] Alternatively, in an embodiment, wherein the binding agent is an aptamer and the aptamer itself can be a unique nucleotide sequence. The nucleotide sequence can be an oligonucleotide, and can include single-stranded or double-stranded RNA or DNA. The nucleotide length for labeling a binding agent or a target can be preferably 5-150 bases, for example, 10-40 or 40-80 bases in length. The nucleotides forming the nucleic acid can be chemically modified to increase the stability of the molecule, to improve its bioavailability or to give it additional activity. For example, pyrimidine bases can be modified at 6 or 8 positions, and purine bases can be modified at 5'-position by CH3 or halogens such as I, Br or Cl. Modification or pyrimidine bases also include 2-NH3, O-CH3, N-CH3 and N2-CH3. The modification at the 2'-position is a sugar modification, and generally includes NH2, F or OCH3 groups. Modification can also include 3' and 5' modifications, such as capping. Alternatively, modified nucleotides such as morpholino nucleotides, locked nucleic acids (LNA) and peptide nucleic acids (PNA) can be used. Morpholino oligonucleotides are assembled from different morpholino subunits, each containing one of the four genetic bases (adenine, cytosine, guanine and thymine) linked to a 6-membered morpholine ring.

[0330] They are linked by nonionic phosphodiamidate intersubunit bonds to form morpholino oligonucleotides. LNA monomers are characterized by the furanose ring conformation being constrained by a methylene linker connecting the 2'-0 position to the 4'-C position. PNA is a DNA analog in which the backbone is a pseudopeptide rather than a sugar. BRIEF DESCRIPTION OF THE DRAWINGS

[0331] Figure 1 : dPCR readouts of single cells with appropriate controls. NTC - no template control containing labeled antibody or HER2 protein; ABC - containing the same concentration of antibody as samples (labeled trastuzumab, pertuzumab), but without HER2 protein; BT474sc - single cell PICO reaction; direct rHER2 - direct recombinant HER2 PICO reaction.

[0332] Figure 2 :Graphical description of simulated equimolar titration data (re-logarithm) (see Table 1). The X-axis represents the logarithm of the target molar concentration, and the Y-axis is the logarithm of the conjugate molar concentration, both of which are under the binding conditions of the two-component method. The simulated preset concentration of the target is 1.0E-11M (isoCC), and the preset Kd is 5.0E-11 (Kdx1) and 1.0E-10 (Kdx2), and the simulation is performed under the data of the labeling score value of 1, the active score value of 1, no labeling error and free labeling, respectively. The antibody concentration is shown on the curve and is set to 4.0E-11, 1.0E-11, 4.0E-10, 1.1E-9 and 5.0E-9. The concentration of the conjugate is obtained by solving the equation described in WO2020260277A1. By setting Kd to a preset value, the recursive minimization standard deviation of antigen concentration was 0.18% (STDp) or 1.81E-14 (STD), and isoCC was also reproduced.

[0333] Figure 3 : Graphical description (logarithm) of simulated equimolar titration data (see Tables 1 and 3) when the activity fraction value is 0.5, and other conditions are the same as Figure 2 The X-axis represents the logarithm of the molar concentration of the target and the Y-axis represents the logarithm of the molar concentration of the conjugate, both under the binding conditions of the two-component method. The preset concentration of the target is 1.0E-11M (isoCC). Figure 3 A shows the data in Table 3. The apparent Kd is 1.0E-10 (Kdx1) and 1.0E-10 (Kdx2), respectively. The recursive minimization standard deviation of the antigen concentration is 0.12% (STDp) or 1.16E-14 (STD). It is significantly different from the preset value of Kd (see Figure 2 ) and indicates the new apparent Kd. It is noteworthy that the preset isoCC value is restored. Figure 3 B shows the data in Table 2, the recursive minimization standard deviation of the antigen concentration is 2.28% (STDp) or 2.33E-13 (STD), where the (apparent) Kd is 5.0E-11 (Kdx1) and 1.0E-10 (Kdx2), respectively. These values ​​are consistent with Figure 2 The values ​​of are the same. (A) Describes the situation when the activity fraction is unknown and the active concentration of the antibody is incorrectly determined based on the labeled antibody (the actual concentration is lower since the inactive antibody is non-reactive). The results show that after recursively minimizing the standard deviation of the antigen concentration, the isoCC is identical to the original isoCC, but the apparent Kd is different. (B) Control simulation. The antibody concentration is adjusted according to the activity fraction and the original Kd is reproduced together with the original isoCC.

[0334] Figure 4 : Graphical depiction (relogarithm) of simulated equimolar titration data (see Table 4) at a labeling fraction value of 0.5. The X-axis represents the logarithm of the molar concentration of the target and the Y-axis represents the logarithm of the molar concentration of the conjugate, both under the binding conditions of the two-component method. The preset concentration of the target is 1.0E-11 M (isoCC). Figure 4 A depicts changes in apparent antibody and conjugate concentrations according to simulated values ​​for a labeling fraction of 0.5, and the indicated antibody concentrations are 2.0E-11, 5.0E-11, 2.0E-10, 5.5E-10, and 2.50E-9 (half the amount, because only the labeling fraction is detected). It is worth noting that in the absence of recursive minimization, where Kd 5.0E-11 (Kdx1) and 1.0E-10 (Kdx2) are unchanged, the standard deviation of the antigen concentration is 17.11% (STDp) or 1.04E-12 (STD). This is because the simulated target concentrations are not exactly consistent. Figure 4 B shows that after recursive minimization, the standard deviation of the antigen concentration is 0.37% (STDp) or 1.84E-14 (STD), and the apparent Kd 2.60E-11 (Kdx1) and 1.0E-10 (Kdx2) are respectively reproduced original values, however, the antigen (isoCC) concentration is 5.0E-12. Since the labeling fraction is 0.5, the antigen concentration needs to be adjusted to obtain 1.0E-11M, which is the same as the original antigen concentration.

[0335] Figure 5 : This figure shows the Agilent Protein 230 chip with the labeled antibody against GAPDH on the 2100 Bioanalyzer electropherogram. The labeling fraction was determined according to Example 5 and was 97.6%.

[0336] Figure 6 :The figure shows a graphical description (re-logarithm) of the apparent dissociation constants for trastuzumab and pertuzumab. The X-axis represents the logarithm of the target molar concentration, and the Y-axis is the logarithm of the conjugate molar concentration, both of which are under the binding conditions of the two-component method. The sample has an equimolar concentration, but the exact concentration of HER2 is unknown (the sample is obtained from lysed HER2 cells), and the concentration of the antibody is different (the concentration of the antibody is 5.5E-9, 1.50E-9, 4.0E-10, 1.2E-10, 3.5E-11 and 1.0E-11). The red line is the calibration curve of the HER2-specific two-component assay at these antibody concentrations and apparent Kd, and the assay is similar to the method described in the WO2020260277A1 assay. Assuming that all samples contain the same concentration of HER2, the latter is recursively determined to minimize the SD of the recalculated HER2 concentrations of all measured values. The molar HER2 concentration is designated as MeanHER2 (same as isoCC in other depictions), STD and pSTD are the standard deviation of the measurements and their percentage of their mean, respectively. The dashed line is the mean of the HER2 measurements at different antibody concentrations. Finally, the dissociation degrees of the antibodies (Kd1TTZ and Kd2PTZ) were recursively determined to be 2.9E-11 and 9.3E-11, respectively.

[0337] Example

[0338] Example 1

[0339] The present embodiment provides an example of the first separation of the binding reaction between cells and antibodies, which can preferably be carried out under high cell diffusion-limited concentrations. The concentration of the target protein (target analyte) of the binding reaction is preferably defined by the volume of the cell suspension and the total target protein content in the volume. Under suitable conditions and assumptions, the concentration of the target protein / analyte can be processed as in a solution, as shown in the present embodiment. Therefore, in such embodiments, the concentration of each of their cells can be assumed by their concentration calculated for the entire sample volume and the number of cells in the sample volume. Separate each single cell and have a suitable sensitive method, the number of targets per cell can be determined by the extrapolated concentration of the target in the suspension volume multiplied by the number of cells in the suspension. Preferably, the method includes a second separation, and the second separation can be used to count the single molecules including conjugates and binding agents. Preferably, the cells are lysed before the second separation. Preferably, the extrapolated concentration of the target in the suspension volume is determined using a binding feature, and ultimately results in determining the absolute amount of the target analyte in each single cell, as described in the present embodiment. This example further demonstrates that the methods of the present invention surprisingly facilitate the detection of target analyte concentrations below 1000 or 100 copies or 10 copies per cell due to the dual partitioning described herein.

[0340] The method used in this embodiment

[0341] MCF7( HTB-22) and BT-474( HTB-20 cells were obtained from the BIOSS Centre for Biological Signaling Studies (Freiburg, Germany). TM ), MCF7 cells were cultured in DMEM, GlutaMAX Supplement (31966021, Gibco), and BT-474 cells were cultured in DMEM / F12, GlutaMAX Supplement (31331028, Gibco). Both culture media were supplemented with 10% FBS (10270106, Gibco) and 1% Pen / Strep (15140122, Gibco). The cells were cultured in a cell culture incubator (Heracell TM 150i CO2 Incubator, 50116048, Thermo Scientific TM ) in a 5% CO2 atmosphere at 37°C until the cells were approximately 90% confluent. The cells were harvested by scraping. The cells were washed twice with DPBS (14040133, Gibco) and counted ( II Automated CellCounter,Invitrogen TM ), including the use of trypan blue (T10282, Invitrogen TM ) for live / dead staining.

[0342] 0.5 μl of LBTW was dispensed into 384-well V-bottom plates (0030623304, Eppendorf) using instant droplet-on-demand technology (I.DOT One; Dispendix, Stuttgart, Germany) [50, 51] with an I.DOT PURE plate 90 μm orifice (Dispendix, Stuttgart, Germany). Prior to dispensing, the I.DOT was calibrated for the applied liquid to ensure reliable dispensing. Diluted crude cell lysate ("cl") or DPBS was dispensed using liquid class "H2O".

[0343] In this embodiment, the binding agent is an antibody conjugated with a DNA marker using a proprietary procedure of Actome GmbH, Germany. The labeling efficiency achieved is between 80-100% and is confirmed by SDS-PAGE electrophoresis. The antibody preparation has 3% free labeling and 25% labeling error (see Example 5). Trastuzumab (TTZ) and Pertuzumab (PTZ) are recombinant humanized monoclonal antibodies, both of which target the extracellular region of the HER2 tyrosine kinase receptor and have no overlapping epitopes. The conjugated antibody has two different labels, namely, trastuzumab BL and Pertuzumab P8 labels. The apparent dissociation constant of the antibody is determined according to Example 6, and is TTZ=0.49E-10M and PTZ=0.93E-10, respectively.

[0344] The two-component assay used was similar to that described in WO 2016 / 083793A1, which is incorporated herein by reference. Briefly, MCF7 and BT-474 cell concentrations were adjusted to 1×10 7 Cells / ml were added and 100 μL of cell suspension was incubated with conjugated antibody (ABX-antibody cocktail) overnight, or cell suspension was lysed using proprietary lysis buffer provided by Actome GmbH, Germany, and the lysate was incubated with antibody (supplemented with ABX) overnight.

[0345] Without lysing the cells, the cell distribution procedure was performed as previously described (Groβ, C. Jeney, D. Halm, G. Finkenzeller, G. B. Stark, R. Zengerle, P. Koltay, S. Zimmermann. Characterization of CRISPR / Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning, BioRxiv. (2020) 1–19. https: / / doi.org / 10.1101 / 2020.08.17.253559.). Briefly, the single-cell printer (SCP) (A. Gross, J. S. Niekrawitz, W. Streule, L. Riegger, R. Zengerle, P. Koltay, Single-Cell Printer: Automated, OnDemand, and Label Free, J. Lab. Autom. 18 (2013) 504–518. https: / / doi.org / 10.1177 / 2211068213497204.). Both MCF7 and BT-474 cells were adjusted to 1x10e6 cells / ml and loaded into a dispensing cassette (CYTENA GmbH, Freiburg, Germany). The settings for MCF7 cells were a cell size of 10 to 25 μm (BT-474: 10 to 30 μm) and a circularity of 0.5 to 1 (also for BT-474). F.SIGHT enables reliable dispensing of single cells with minimal liquid volume. The single cell partitioning efficiency (successful single cell isolation events divided by target events) is typically about 90% and is additionally controlled by the cell images that are clearly assigned to each partitioning event. Thus, in addition to single cell partitioning, events such as doublets or empty droplets can be excluded.

[0346] QIAGEN's QIAcuity Probe Mastermix (41 μL) was added, and the samples (0.5 μL for lysed bulk samples and single cell samples) were loaded onto a QIAcuity Nanoplate 26k 24-well plate. Digital PCR was performed using the QIAGEN QIAcuity digital PCR system, where the cycle parameters were based on the following PCR conditions: hot start at 95°C for 2 minutes, 40 cycles, denaturation at 95°C for 15 seconds, and annealing at 58°C for 30 seconds. Imaging conditions P8 labeling-FAM green channel, 500ms integration time, gain 6, and BL labeling-HEX yellow channel, 400ms integration time, gain 6.

[0347] Cells were treated according to the following steps: (a) Single cells were incubated with labeled PTZ and TTZ at 1x10e6 cells / 50μl, respectively. (b) After overnight incubation, cells were printed into 0.5μL (I-DOT dispensed) lysis buffer. (c) QIAcuity Probe Mastermix (41μL) was added and samples (0.5μL for lysed bulk and single cell samples) were loaded onto the QIAcuity. Negative controls (antibody binding controls) containing only labeled trastuzumab and pertuzumab antibodies were also set up, which were derived from cell-free buffer or from ABX antibody mixture.

[0348] The calibration curves referred to herein are derived similarly to those described in WO 2020 / 260277 A1, but applying the binding characteristics described herein.

[0349] In the case of bulk lysate, calibration curve shows the molar concentration of target analyte (HER2 protein in the present embodiment).Directly read out the number of conjugates of the lysate of the single cell equivalent amount of distribution in the dPCR reaction, multiplied by the dilution factor of the binding reaction, to obtain the molar concentration of the conjugate in the binding reaction, wherein the dilution factor of the binding reaction is determined by the fraction of the binding reaction volume measured in the dPCR reaction.Also similarly determine the molar concentration of the antibody, directly read from the dPCR reaction and similarly apply the dilution factor of the binding reaction, obtain initial counts.Previously determined the apparent dissociation constant of the antibody.Utilize the compensation value of the absolute concentration of the absolute concentration of the analyte in the binding reaction of the binding feature-determine the bulk.

[0350] In the case of single cells, the calibration curve shows the number of target analytes (HER2 protein) determined in each single cell. The dilution factor of the binding reaction is similarly applied to determine the molar concentration of antibodies and conjugates in the binding reaction. However, the dilution factor is calculated differently, because the single cell represents the single cell fraction of the target protein (analyte), and the number of conjugates of the measured single cells is multiplied by the number of cells in the suspension volume, so that the number of cells in the suspension volume is regarded as the dilution factor of the binding reaction. The apparent dissociation constant of the antibody was previously determined. The absolute number of target analytes in the suspension (sample) is determined using the compensated molar input and apparent dissociation constant of the binding feature-antibody. In short, all cells in the suspension are considered to calculate the molar concentration of the conjugate and the adhesive, and the suspension is assumed to be based on the conditions of the solution during the calculation. Chemical calculations are performed, similar to those described in WO 2020 / 260277 A1, to determine the absolute molar concentration. Given that the molar concentrations of the conjugate and the binder are derived from each single single cell, this is a mathematical compensation for the formation of targets by unbound non-conjugates, thereby obtaining absolute concentrations. The absolute (compensated) molar concentration of the target is obtained and taking into account the number of cells in suspension, the absolute count of the target analyte per single cell is determined. The amount of target protein per single cell is derived by dividing the absolute concentration of the target by the number of cells in suspension.

[0351] For the validity of this method, it is assumed that the target concentration on a single cell is lower than the antibody concentration in the cell suspension and does not significantly reduce the free antibody concentration. However, at physiological concentrations of proteins in cells, this assumption generally holds and it is known to those skilled in the art that recursive calculations can compensate for this.

[0352] Notably, bulk lysates of cells contained more detectable HER2 molecules compared to single cells, since single-cell detection is limited to the cell surface in the absence of permeabilization.

[0353] In the case of MCF7 cells, the average number of HER2 proteins in each single cell is 4500 ± 1,060 molecules, while the expected number under bulk conditions is 10,150 ± 1,260 molecules. Similarly, BT474 cells show approximately 208,000 ± 1,260 HER2 proteins per single cell compared to the bulk condition results of 2.3E+6 ± 8.E+5 molecules. These differences in the number of HER2 proteins are attributed to the inherent differences between the surface accessible and bulk accessible protein collections. The method disclosed herein provides a reliable means of quantifying the HER2 proteins in single cells and comparing them with bulk conditions, thereby contributing to a comprehensive understanding of the protein distribution at the cell and population levels.

[0354] Example 2

[0355] Plate-based "scPICO" method according to the present invention

[0356] Reagents and buffers

[0357] On the day of the experiment, prepare reagents and buffers as listed below.

[0358] Additive C (5x stock solution)

[0359] 500 μl PBS

[0360] BSA (5x stock solution)

[0361] 20mg BSA

[0362] 400 μl PBS

[0363] EDTA-free Protease Inhibitor Cocktail (PIC) (25x stock)

[0364] cOmplete Protease TM 1 tablet of inhibitor cocktail

[0365] Dilute to 2 ml with PBS

[0366] Cell Lysis Buffer (LBT) (2x stock)

[0367] 200 μl Additive T (10x)

[0368] 400 μl dissolved Additive C (5x)

[0369] 80μl PIC (25X)

[0370] 200 μl Additive L

[0371] 120 μl PBS

[0372] Cell Lysis Buffer (LBTW) (1x)

[0373] 300 μl LBT buffer (2x stock solution)

[0374] 300 μl PBS

[0375] Antibody Binding Control (ABC) Buffer (1x)

[0376] 250 μl LBT buffer (2x stock solution)

[0377] 100 μl BSA (5x)

[0378] 150 μl PBS

[0379] Oligonucleotides

[0380]

[0381]

[0382] Antibody cocktail

[0383] To detect HER2 in single BT474 cells, two anti-HER2 humanized monoclonal antibodies, pertuzumab (PTZ) and trastuzumab (TTZ), were used. Both antibodies were covalently modified with DNA oligonucleotide labels using PICOglue technology. The labels contained a 10-base-pair random nucleotide sequence (UMI) for binding to P8 and NOS-6, respectively, and a TaqMan hydrolysis probe annealing site (PTZ-P8 and TTZ-NOS6) (see below). The labeled antibody stock solution was diluted to a molar concentration of 5.00x10 -10 The antibody cocktail was prepared by eluting 1 μM of each antibody. The antibody cocktail was then diluted 1:2 in LBTW and stained with fluorescein to a final concentration of 1 μM. The concentrations applied were designed to provide saturating conditions.

[0384] Cell culture

[0385] Breast ductal carcinoma BT474 cells were grown in DMEM F12 culture medium supplemented with GlutaMax+10% FCS+1% P / S in T175 flasks and split at 1:2 to 1:3 every 5-7 days at 80-90% confluence. After washing with 10ml PBS, cells were harvested by incubating with 5ml Versene non-enzymatic cell dissociation reagent (Thermo) for 3-5 minutes at room temperature. When cells began to separate, the reaction was terminated by diluting with PBS at 1:2, and then the cell suspension was centrifuged at 400g for 5 minutes. The cells were then resuspended in 40ml fresh culture medium in each T175 flask and inoculated.

[0386] For single cell analysis according to the present invention ("single cell PICO method"), BT474 cell cultures from T175 flasks were harvested as described above and resuspended in 1000 μl PBS and subsequently filtered through a 30 μm cell strainer. Cells were counted, washed again with 1 ml PBS, centrifuged at 400 g for 2 minutes, and resuspended in PBS stained with 100 μM fluorescein to a density of approximately 0.5 x 10 -6 The cells were kept on ice until use.

[0387] 384-well plate preparation and single cell isolation

[0388] The wells of the 384-well Eppendorf microplate 384 / V-PP were prefilled with 4 μl Vapor-Lock PCR overlay (Qiagen) and centrifuged briefly at 1000 g. 12 nl of antibody mixture (1:2 dilution in LBTW; 1 μM fluorescein) was dispensed into each well using an I.DOT liquid handler (Dispendix). The plate was then centrifuged at 2000 g for 2 minutes to ensure that the droplets were positioned in the center of the wells under the Vapor-Lock overlay. Successful droplet distribution and positioning were controlled under the microscope using a Dino-Light fluorescence microscope (AM4115T-GFBW).

[0389] Single BT474 cells were isolated using the f.sight single cell dispenser (Cytena) with automated offset correction (AOC) enabled. Single cells with a size of 15-25 μm and circularity set to 0.6-1.0 were selected, and only reactions with one single cell were accepted as valid for downstream processing.

[0390] Merging of the antibody mixture droplet and the droplet containing the single cells was considered successful when an increase in fluorescence signal was observed compared to reactions that did not receive single cells. Additional reactions that did not receive single cells were also used as intraplate antibody binding controls (ip ABC) (see below).

[0391] After single cell isolation, the plate was sealed with PCR foil (4titude) and incubated at 4°C for about 24 hours, so that both cell lysis and binding reaction between HER2 and both antibodies (conjugate formation) could occur during the incubation period.

[0392]

[0393] Preparation of control binding reactions and sample dilutions

[0394] Before being used as the input of dPCR, 12nl single cell binding reaction was diluted as follows in PBS. In order to achieve 1:5000 pre-dilution, 60μl PBS was added directly to the single cell reaction, followed by repeated mixing by pipetting and centrifugation at 2000g for 2 minutes. 5μl pre-dilution was transferred to 42.5μl PBS (1:9.5), and then mixed by repeated pipetting. 1μl and 1.32μl of this dilution were used as input for Stilla and QIAcuity dPCR master mixes, respectively. For the partition sizes of 0.78nl and 0.59nl on Stilla Sapphire chip and QIAcuity system, respectively, the input amount was correct to achieve a lambda of 0.15.

[0395] This corresponds to 1.19x10 6 and 1.52x10 6 The total dilution factor.

[0396] dPCR Master Mix

[0397]

[0398] Partitioning and dPCR reactions were performed on a Naica Geode system with a Sapphire chip (Stilla) for coupled PCR and on a QIAcuity system (Qiagen) using a Nanoplate 24 26k.

[0399] Naica Sapphire System-dPCR Parameters:

[0400] 1. Partition Sapphire V1 at 40°C

[0401] 2. Temperature is 95℃ for 10 minutes

[0402] 3. Start loop 40

[0403] - Temperature 95°C, last 30 seconds

[0404] -Temperature 60℃, duration 15 seconds

[0405] 4. Start loop 20

[0406] - Temperature 95°C, last 30 seconds

[0407] -Temperature 50℃, duration 15 seconds

[0408] 5. Close the loop

[0409] 6. Release P, Sapphire V1

[0410] QIAcuity System-Nanoplate2426k-dPCR Parameters:

[0411] 1. QIAcuity Standard Startup

[0412] 2. Temperature 95℃, last 2 minutes

[0413] 3. Start loop 40

[0414] - Temperature 95°C, last 30 seconds

[0415] -Temperature 60℃, duration 15 seconds

[0416] 4. Start loop 20

[0417] - Temperature 95°C, last 30 seconds

[0418] -Temperature 50℃, duration 15 seconds

[0419] 5. Close the loop

[0420] dPCR reaction

[0421] Emulsion, ddPCR and reading were performed using the Stilla Naica system following standard dPCR protocols. As a negative control, antibodies were mixed in the absence of antigen and treated in the same manner as the samples (antibody binding control, ABC).

[0422] The amount of HER2 protein is measured by detecting the difference in partitioning induced by simultaneous binding of antibodies to HER2. The measured values ​​are normalized to an ABC control, which defines zero-level detection by the ABC signal. Figure 1 Shown are dPCR results of single cell analysis with appropriate controls.

[0423] The NTC and ABC readings were zero, indicating a contamination-free dPCR reaction and a well-balanced dPCR reading, and considering the number of conjugates detected using the applied dilution and other factors affecting the conjugate count (as described in detail in Karakus et al. 2019, EP22161450.6, which is incorporated herein by reference), the BT474sc sample indicated an average of approximately 987,000 (±7.6%) HER2 molecules detected per cell, while the rHER2 sample indicated approximately 876,000 (±9.8%) HER2 molecules considering a similar procedure.

[0424] Droplet recovery and DNA extraction from Sapphire chips

[0425] First, the inlet and outlet of the sapphire chip were completely emptied with a pipette and the collected oil was transferred to a collection tube. An Eppendorf Combitip advanced (1,0 mL, yellow) was then firmly inserted into the outlet. Each inlet was then filled with 50 μl of Novec 7500. Subsequently, the emulsion inside the chip was recovered by slowly pulling the Combitip piston until the liquid level in the inlet dropped to the bottom. The process was repeated again and the collected liquid was transferred to a collection tube. Next, the tube was centrifuged for 2 minutes and the oil phase was removed from the bottom of the tube with a pipette. Demulsification was performed by adding 60 μl of 20% PFO (v / v) in Novec 7500, vortexing for 10 seconds and centrifuging at 1000 g for 2 minutes. Separation of the aqueous and oil phases was achieved by slowly flipping the tube upside down and collecting the aqueous phase from the bottom of the tube.

[0426] NGS library preparation

[0427] For NGS library preparation, NEBNext UltraII Library Prep was used according to the manufacturer's protocol with the following settings and modifications. All reaction volumes were reduced by 50% and approximately 5 ng of unpurified amplicon DNA was used as input. NEB adapters were diluted 25-fold (1:25) to 0.6 μM in 10 mM Tris-HCl pH 8.0 with 10 mM NaCl and no size selection was performed prior to PCR. Library DNA was purified according to the protocol with 22.5 μl (0.9X) resuspended beads (NEB).

[0428] NGS on the Illumina MiSeq Platform

[0429] Libraries were pooled in equimolar amounts and subjected to NGS using the MiSeq platform using the MiSeq reagent kit v2 with 300 cycles of paired-end according to the manufacturer's protocol. After denaturation, the library pool was diluted 5 pM and a 10% PhiX control was added.

[0430] The NGS results are calculated according to the procedure described in (PCT / EP2020 / 067493), in short, separated dPCR is performed using molecular unique markers (UMIs), and dimers are deconvoluted to reconstruct the contents of the droplet (where ddPCR is performed, preferably containing only one conjugate). That is, the evaluation of the reaction can be based on the NGS readings of the binder (e.g., antibody, specific dimerization UMI markers generated according to the standard protocol of emulsion coupling). The number of labeled binders, such as antibodies, can be determined in each reaction by counting all unique UMI markers for a given binder, such as an antibody (the count is limited to a given binder). Possible multiple markers of the same binder, such as an antibody, can be eliminated using its preferential dimerization sequence (multiple markers of each binder will always produce dual UMI-labeled dimers with a given antibody-specific labeling background because they are co-localized in the same droplet). The conjugates are counted based on the PCR products of the dual UMI markers of dimerization (in the case of two different binder type-specific markers (e.g., antibodies against a specific HER2 antigen)).

[0431] Once the droplet contents are obtained, a Poisson-based calculation (as described in EP 22161450.6) is performed to confirm the number of conjugates detected. In short, the distribution of the binding agent during the separation is controlled by the Poisson distribution, so the occurrence of each binding agent is counted by direct dPCR (or after droplet-based PCR amplification, the abundance of the binding agent is determined by NGS), and if the number of droplets is known, the background detection of the conjugate can be calculated. A large number of specific binding events will be used to identify the accurate target agent of the binding agent, which can be performed in the same way. For example, in a multi-protein complex, the information contained in the connection label of the binding agent is based on the co-localization of the corresponding binding agent on the complex, and indicates the direct interaction between the detected target analytes. With respect to each protein / protein complex, when calculating the background detection, any detected changes in the protein / protein complex are due to different interactions, which can be calculated by removing the calculated background detection of the protein / protein complex (or Poisson-corrected subtraction, because the protein / protein complex bound to the binding agent changes the total number of binding agents). The ABC-compensated number is for the BT474sc sample, indicating that approximately 943,000 (±14.8%) HER2 molecules were detected per cell.

[0432] Example 3

[0433] This example provides an example of how the dissociation constant (Kd) of a binder, or binding characteristics of labeling ratio, label synthesis error, active fraction and contamination with free label under various conditions, can be determined for subsequent application in a method according to the invention to deduce the absolute amount of conjugate (or ternary complex) formed, thereby facilitating the determination of the dissociation constant of antibodies in complex chemical environments, but also in natural environments, in the absence of target standards.

[0434] name Conjugate ableft bright ABC 7.06E-12 4E-10 4E-10 ABC 7.06E-12 4E-10 4E-10 ABC 7.06E-12 4E-10 4E-10 ABX#1 9.71E-12 5E-09 5E-09 ABX#1 9.71E-12 5E-09 5E-09 ABX#1 9.71E-12 5E-09 5E-09 ABX#1 9.71E-12 5E-09 5E-09 ABX#2 8.76E-12 1.1E-09 .1E-09 ABX#2 8.76E-12 1.1E-09 .1E-09 ABX#2 8.76E-12 1.1E-09 .1E-09 ABX#2 8.76E-12 11E-09 .1E-09 ABX#3 7.06E-12 4E-10 4E-10 ABX#3 7.06E-12 4E-10 4E-10 ABX#3 7.06E-12 4E-10 4E-10 ABX#3 7.06E-12 4E-10 4E-10 ABX#4 3.18E-12 1E-10 1E-10 ABX#4 3.18E-12 1E-10 1E-10 ABX#4 3.18E-12 1E-10 1E-10 ABX#4 3.18E-12 1E-10 1E-10 ABX#5 1.13E-12 4E-11 4E-11 ABX#5 11.13E-12 4E-11 4E-11 ABX#5 1.13E-12 4E-11 4E-11 ABX#5 1.13E-12 4E-11 4E-11 NTC 0 0 0

[0435] Table 1 Simulated equimolar titration data. Column headings are: “Name” – sample name, “Conjugate” – two antibody-target complexes, “ableft” – concentration of antibody x1, “right” – concentration of antibody x2, all concentrations are in M. ABX means “antibody cocktail” and they contain equimolar concentrations of the two specified antibodies.

[0436] Figure 2 A graphical description of the simulated equimolar titration data is shown (see Table 1). The preset concentration of the target is 1.0E-11M (isoCC), the preset Kd is 5.0E-11 (Kdx1) and 1.0E-10 (Kdx2), and the data have a labeling score value of 1, an active score value of 1, no labeling error, and a free label, respectively. The antibody concentration is shown on the curve and is set to 4.0E-11, 1.0E-11, 4.0E-10, 1.1E-9, and 5.0E-9. The concentration of the conjugate is obtained by solving the equation described in WO2020260277A1. The recursive minimization standard deviation of the antigen concentration is 0.18% (STDp) or 1.81E-14 (STD).

[0437] name Conjugate ableft abright ABC 5.27E-12 2E-10 4E-10 ABC 5.27E-12 2E-10 4E-10 ABC 5.27E-12 2E-10 4E-10 ABX#1 9.65E-12 2.5E-09 5E-09 ABX#1 9.65E-12 2.5E-09 5E-09 ABX#1 9.65E-12 2.5E-09 5E-09 ABX#1 9.65E-12 2.5E-09 5E-09 ABX#2 8.42E-12 5.5E-10 1.1E-09 ABX#2 8.42E-12 5.5E-10 1.1E-09 ABX#2 8.42E-12 5.5E-10 1.1E-09 ABX#2 8.42E-12 5.5E-10 .1E-09 ABX#3 6.38E-12 2E-10 4E-10 ABX#3 6.38E-12 2E-10 4E-10 ABX#3 6.38E-12 2E-10 4E-10 ABX#3 6.38E-12 2E-10 4E-10 ABX#4 2.38E-12 5E-11 1E-10 ABX#44 2.38E-12 5E-11 1E-10 ABX#4 2.38E-12 5E-11 1E-10 ABX#4 2.38E-12 5E-11 1E-10 ABX#5 7.4E-13 2E-11 4E-11 ABX#5 7.4E-13 2E-11 4E-11 ABX#5 7.4E-13 2E-11 4E-11 ABX#5 7.4E-13 2E-11 4E-11 NTC 0 0 0

[0438] Table 2: Simulated equimolar titration data, changing the active concentration x1 of the antibody to 0.5 of the fractional active value x1, used to determine a new conjugate concentration using a preset Kd for the antibody. Column headings are: "Name" - sample name, "Conjugate" - conjugate, "ableft" - concentration x1 of the antibody, "right" - concentration x2 of the antibody, all concentrations in M. ABX means "antibody mixture" and they contain the two specified antibodies at the stated concentrations. The data shows that if the amount of "ableft" is half of the original value, the corresponding conjugate value is also lower (the amount of conjugate is determined by simulation).

[0439] name Conjugate ableft abright ABC 5.27E-12 4E-10 4E-10 ABC 5.27E-12 4E-10 4E-10 ABC 5.27E-12 4E-10 4E-10 ABX#1 9.65E-12 5E-09 5E-09 ABX#1 9.65E-12 5E-09 5E-09 ABX#1 9.65E-12 5E-09 5E-09 ABX#1 9.65E-12 5E-09 5E-09 ABX#2 8.42E-12 1.1E-09 1.1E-09 ABX#2 8.42E-12 1.1E-09 1.1E-09 ABX#2 8.42E-12 1.1E-09 1.1E-09 ABX#2 8.42E-12 1.1E-09 1.1E-09 ABX#3 6.38E-12 4E-10 4E-10 ABX#3 6.38E-12 4E-10 4E-10 ABX#3 6.38E-12 4E-10 4E-10 ABX#3 6.38E-12 4E-10 4E-10 ABX#4 2.38E-12 1E-10 1E-10 ABX#4 2.38E-12 1E-10 1E-10 ABX#4 2.38E-12 1E-10 1E-10 ABX#4 2.38E-12 1E-10 1E-10 ABX#5 7.4E-13 4E-11 4E-11 ABX#5 7.4E-13 4E-11 4E-11 ABX#5 7.4E-13 4E-11 4E-11 ABX#5 7.4E-13 4E-11 4E-11 NTC 0 0 0

[0440] Table 3Simulated equimolar titration data, using the conjugate concentrations for the antibody x1 activity fraction value of 0.5 in Table 2, were used to determine the new apparent Kd for the antibodies. The conjugate concentrations are the same as in Table 2. The values ​​represent 0.5 effective fraction, but the effective fraction is considered unknown and "ableft" is not compensated as in Table 2. The column headings are: "Name" - sample name, "Conjugate" - the two antibody-target complexes, "ableft" - concentration x1 of the antibody, "Right" - concentration x2 of the antibody, all concentrations are in M. ABX means "antibody mixture" and they contain the two specified antibodies at the stated concentrations. If the activity fraction is 1, the "ableft" value is adjusted, but the conjugate concentration is based on the activity fraction value of 0.5.

[0441] Figure 3 Graphical depictions of simulated equimolar titration data at an activity fraction of 0.5 are depicted (see Tables 2 and 3). The preset concentration of the target was 1.0E-11 M (isoCC). A. Data from Table 3. The apparent Kd is 1.0E-10 (Kdx1) and 1.0E-10 (Kdx2), respectively. The standard deviation of the recursive minimization of the antigen concentration is 0.12% (STDp) or 1.16E-14 (STD). B. Data from Table 2, the standard deviation of the recursive minimization of the antigen concentration is 2.28% (STDp) or 2.33E-13 (STD), where the (apparent) Kd is 5.0E-11 (Kdx1) and 1.0E-10 (Kdx2), respectively. These values ​​are consistent with Figure 2 The values ​​of are the same, thus reproducing the preset values. (A) Describes the situation when the activity fraction is unknown and the active concentration of the antibody is incorrectly determined based on the labeled antibody (the actual concentration is lower because the inactive antibody is non-reactive). The results show that after recursively minimizing the standard deviation of the antigen concentration, the isoCC is the same as the original isoCC, but the apparent Kd is different. (B) Control simulation. The antibody concentration is adjusted according to the activity fraction and the original Kd is reproduced together with the original isoCC.

[0442] name Conjugate ableft abright ABC 3.53E-12 2E-10 4E-10 ABC 3.53E-12 2E-10 4E-10 ABC 3.53E-12 2E-10 4E-10 ABX#1 4.855E-12 3E-09 5E-09 ABX#1 4.855E-12 3E-09 5E-09 ABX#1 4.855E-12 3E-09 5E-09 ABX#1 4.855E-12 3E-09 5E-09 ABX#2 4.38E-12 6E-10 1.1E-09 ABX#2 4.38E-12 6E-10 1.1E-09 ABX#2 4.38E-12 6E-10 1.1E-09 ABX#2 4.38E-12 6E-10 1.1E-09 ABX#3 3.53E-12 2E-10 4E-10 ABX#3 3.53E-12 2E-10 4E-10 ABX#3 3.53E-12 2E-10 4E-10 ABX#3 3.53E-12 2E-10 4E-10 ABX#4 1.59E-12 5E-11 1E-10 ABX#4 1.59E-12 5E-11 1E-10 ABX#4 1.59E-12 5E-11 1E-10 ABX#4 1.59E-12 5E-11 1E-10 ABX#5 5.65E-13 2E-11 4E-11 ABX#5 5.65E-13 2E-11 4E-11 ABX#5 5.65E-13 2E-11 4E-11 ABX#5 5.65E-13 22E-11 4E-11 NTC 0 0 0

[0443] Table 4 Simulated equimolar titration data, using corrected antibody x1 and conjugate concentrations at a labeling fraction value of 0.5, for determination of apparent Kd of antibodies. Column headings are: "Name" - sample name, "Conjugate" - conjugate, "ableft"' - concentration of antibody x2, "right" - concentration of antibody x1, all concentrations are in M. ABX means "antibody cocktail" and they contain the two specified antibodies at the stated concentrations. Compared to Table 1, "ableft" and "conjugate" values ​​are adjusted due to the labeling fraction of 0.5.

[0444] Figure 4 A graphical depiction of simulated equimolar titration data at a labeling fraction of 0.5 is depicted (see Table 4). The preset concentration of the target was 1.0E-11 M (isoCC). Figure 4 A shows that the apparent antibody and conjugate concentrations were adjusted based on the simulation and the antibody concentrations were assigned to be 2.0E-11, 5.0E-11, 2.0E-10, 5.5E-10, and 2.50E-9 (half amount because only the labeled fraction was detected). Sub-figure A. shows no recursive minimization. The Kds were 5.0E-11 (Kdx1) and 1.0E-10 (Kdx2), respectively. The standard deviation of the antigen concentrations was 17.11% (STDp) or 1.04E-12 (STD). Figure 4 B shows that after recursive minimization, the standard deviation of the antigen concentration is 0.37% (STDp) or 1.84E-14 (STD). The apparent Kd is 5.0E-11 (Kdx1) and 1.0E-10 (Kdx2), respectively, and the antigen concentration (isoCC) is 5.0E-12. Since the labeling fraction is 0.5, the antigen concentration needs to be adjusted to 1.0E-11M, which is the same as the original antigen concentration.

[0445] The simulations show that the labeling ratio of the antibody and the active fraction of the antibody achieve absolute quantitative determination of the analyte concentration, and they have different effects on it. The active fraction has a self-compensation effect, the apparent Kd will confuse the effect of the active component, and the labeling ratio needs to be determined separately and needs to be numerically compensated in the absolute quantitative results. The numerical adjustment based on the labeling ratio and active fraction of the antibody improves the accuracy of absolute quantitative determination of the analyte concentration using the method.

[0446] Example 4

[0447] The present embodiment provides an example of determining the dissociation constant of a binding agent, which is affected by additional factors, including the labeling ratio of the binding agent, the labeling synthesis error, the active fraction of the binding agent and the pollution of the free label. As shown in the present embodiment, the active fraction of the binding agent and the pollution of the free label will be advantageously confused in the determination of the antibody dissociation constant. As shown herein, it is preferably necessary to independently determine the labeling ratio and labeling error of only the binding agent to control and compensate for the accuracy of the method for determining the dissociation constant of the antibody (the method is, for example, a separated two-component method). For the method of the present invention, this method advantageously determines the binding characteristics of the binding agent.

[0448] Labeling efficiency and labeling error

[0449] The "labeling fraction" or "labeling efficiency" or "labeling fraction" of a binding agent such as an antibody is an independent parameter for determining the absolute quantitative concentration of a target analyte using the method. To mathematically demonstrate b10 (absolute concentration of antibody 1), b20 (absolute concentration of antibody 2), and c12 (absolute concentration of the conjugate formed in the reaction), the following formula can be used:

[0450] b 10 ←lf1b 10

[0451] b 20 ←lf2b 20

[0452] c 12 ←lf1l f2 c 12 ,

[0453] Where lf1 and lf2 are the labeling fractions of the two antibodies (first type and second type of binder), respectively. lf1 and lf2 are independent variables and correlated with the other parameters. It should be noted that if the exact values ​​of lf1 and lf2 are determined, it is easy to reverse this conversion and reconstruct the absolute concentration of the antibodies.

[0454] Labelling errors which can be considered as unlabelled or comprising mislabelled binding agents such as antibodies have the same analytical effect on the absolute quantification of the analyte and can be derived in the same mathematical form.

[0455] Active fraction and free label

[0456] The active fraction of a binding agent such as an antibody is a dependent parameter for the absolute quantitative determination of the target analyte concentration. To mathematically prove that b1 (free absolute concentration of antibody 1), b2 (free absolute concentration of antibody 2) and c12 (absolute concentration of the conjugate formed in the reaction), a (free absolute concentration of the analyte), c1 and c2 are the absolute concentrations of the bimolecular complex (conjugate) of the analyte and the corresponding antibodies b1 and b2, the following formula can be used:

[0457]

[0458] where af1 and af2 are the active fractions of the two antibodies. After some rearrangement, the equation can be transformed into one with the original chemical equilibrium term on the right and a constant correction term on the left, demonstrating that changing the active fraction is self-compensating for changing the measured apparent dissociation constant and can therefore be eliminated:

[0459]

[0460] It should be noted that these derived dissociation constants no longer conform to the original definition of the dissociation constant, however, represent the apparent effect of the antibody in the reaction. It is not possible to use the original chemical constants describing the chemical reaction and thus obtain the correct values.

[0461] The presence of free label, which can be considered as inactive antibody, has the same analytical effect on the absolute quantification of the analyte, since it can be derived in the same mathematical form.

[0462] Example 5

[0463] In a preferred embodiment, the labeling ratio and / or labeling synthesis error of the binding agent need to be determined independently to control and compensate for the accuracy of the separated two-component method for determining the dissociation constant of the binding agent. This embodiment provides an example of a method for determining the labeling ratio of the binding agent, the labeling synthesis error of the binding agent. For the method of the present invention, this method advantageously determines the binding characteristics of the binding agent, thereby determining the labeling fraction of the binding agent.

[0464] The labeling efficiency of the antibody was analyzed using the Agilent Protein 230 chip on the 2100Bioanalyzer from Agilen. To this end, 4 μl of labeled and unlabeled (as a control) antibody solution was loaded according to the manufacturer's instructions. In order to determine the labeling efficiency, the concentrations [ng / μl] of the light chain (lc; 26 ± 3 kDa), heavy chain (hc; 54 ± 4 kDa) and labeled heavy chain (L; 75 ± 5 kDa) were recorded. The labeling score (LE) was calculated as follows using the concentration values:

[0465] Figure 5 An Agilent Protein 230 chip with labeled antibody against GAPDH on a 2100 Bioanalyzer electropherogram is shown. The labeling fraction was determined according to the text and was 97.6%.

[0466] The labeled antibody was purified by several washing steps using an agarose A / G affinity matrix. The concentration of the labeled antibody was determined by digital PCR (dPCR) using a QIAcuity dPCR device. Methods described elsewhere (see Example 1). The last wash sample and antibody were measured at appropriate dilutions. The free fraction of the label was calculated by dividing the concentration of the free label by the concentration of the labeled antibody. The dPCR determined the concentration of the free label (3.42E9 cp / μl) and the concentration of the labeled antibody (4.94E10 cp / μl), and the fraction of the free label was calculated according to this example, and it was 14.4%.

[0467] Marking Error

[0468] BL and P8 markers were synthesized according to the proprietary DNA sequence of Actome GmbH (described in Example 1) and these markers were amplified using the dPCR protocol (described in Example 1). dPCR enables the determination of the exact amplifiable / detectable amount of a marker.

[0469] For all reactions, 100 μL aliquots of labeled samples and standards were pipetted into microplate wells and 100 μL of OliGreen working solution was added to each well. OliGreen working solution was freshly prepared by diluting OliGreen concentrate reagent 1:200 with TE as described by the manufacturer. The samples were incubated at room temperature for approximately 5 minutes and their fluorescence was determined using a Bio-Tek Instruments FL600 fluorescence plate reader with an excitation filter of 485 nm, 20 nm bandwidth and an emission filter of 530 nm, 25 nm bandwidth. Sensitivity settings were changed as needed and data were collected from the top with a 3 mm probe using static sampling with a 0.35 second delay, 50 readings per well.

[0470] The difference between the measurements of the two methods defines the marker synthesis error.

[0471] Example 6

[0472] This example shows the determination of the dissociation constant (Kd) of an antibody taking into account the labeling ratio, label synthesis error and active fraction of the binder, see Example 3. Preferably, a two-component method using separation of the labeling ratio and label synthesis error of the binder is used to determine the apparent dissociation constant of the binder, thereby providing an inherently advantageous self-compensation of the active fraction of the binder and contamination by free label to provide the dissociation constant of the antibody for unbiased determination of absolute amounts in single cells. For the methods of the present invention, this method advantageously determines the binding characteristics of the binder, thereby determining the label fraction of the binder.

[0473] The antibody was conjugated to a DNA amplifiable marker using a proprietary procedure of Actome GmbH, Germany. The labeling efficiency achieved was between 80-100% and confirmed by SDS-PAGE electrophoresis (Example 5). The antibody preparation had 3% free labeling and 25% labeling error (see Example 5). Trastuzumab (TTZ) and Pertuzumab (PTZ) are recombinant humanized monoclonal antibodies, both of which target the extracellular region of the HER2 tyrosine kinase receptor and have no overlapping epitopes. The conjugated antibody has two different labels, namely, Trastuzumab BL and Pertuzumab P8 labels.

[0474] The two-component assay used is similar to that described in WO 2016 / 083793 A1, which is included herein as a reference. Briefly, BT-474 cells are optionally cross-linked in 5mM BS3 in PBS at 4°C for 1 hour. The BT-474 cell concentration is adjusted to 1x10e7 cells / ml and lysed in 100 μL of a proprietary lysis buffer provided by Actome GmbH, Germany, and the lysate is incubated with antibodies overnight at different concentrations of two antibodies for concentrations of 1.00E-09, 2.00E-10, 4.00E-11, 8.00E-12 and 1.60E-12M, respectively.

[0475] For higher concentrations of the antibodies used, the incubated lysates were diluted in PBS to reach a concentration of 0.15λ in the final dPCR. Typically, the antibody concentrations in the ABX were equal. Negative controls containing only labeled trastuzumab and pertuzumab antibodies were also set up using antibodies at a concentration of 4.00E-11 M (antibody binding control).

[0476] QIAGEN's QIAcuity Probe Mastermix (41 μL) was added, and the sample (0.5 μL of lysed bulk) was loaded into a QIAcuity Nanoplate 26k 24-well plate. Digital PCR was performed using the QIAGEN QIAcuity digital PCR system, where the cycle parameters were based on the following PCR conditions: hot start at 95°C for 2 minutes, 40 cycles, denaturation at 95°C for 15 seconds, and annealing at 58°C for 30 seconds. Imaging conditions P8 labeling-FAM green channel, 500ms integration time, gain 6, and BL labeling-HEX yellow channel, 400ms integration time, gain 6.

[0477] The original dPCR parameters (different positive rates) measured from dPCR are obtained as antibody concentration (monochrome positive rate) and dual-color positive rate (confusing statistical overlap and two-component complex, referred to as conjugate). According to each of the three parameters measured individually and using the two-component determination theory (see WO 2020 / 260277 A1), the concentration of the conjugate is obtained. In short, the resulting fluorescence readout of the compartment is thresholded and aggregated into empty single positive and double positive groups. dPCR evaluation is based on general dPCR theory (Basu, AS (2017) Digital Assays Part 15I: Partitioning Statistics and DigitalPCR.doi: 10.1177 / 2472630317705680), providing positive compartments (antibody A, B) quantity, dual-color compartment quantity and empty compartment quantity based on monochrome determination. The evaluation of the reaction is based on the partitions marked in the dPCR reaction. Ordinary dPCR evaluation determines the absolute number of labels in each reaction, which in turn can be used to calculate the number of double positive droplets (spontaneous overlap of labels) based on the statistical probability of co-segregation (Poisson background). The Poisson background is compared to the number of double positive droplets detected (including conjugates), and a statistical model is developed to calculate the number of molecular complexes to explain the number of double positive droplets on the Poisson background. The concentration of the conjugate is determined and multiplied by the previously obtained dilution factor to obtain the molar concentration of the conjugate in the binding reaction. The molar concentration of the antibody is also determined similarly, and the concentration is obtained by directly reading from the dPCR reaction and applying the dilution factor of the binding reaction similarly. For a given antibody concentration, four parallel experiments are performed. The ABC control has an expected zero conjugate value, so its deviation from zero shows the evaluation (clustering) and other deviations, and the non-zero conjugate of ABC (if measured) is used to normalize all measurements.

[0478] Figure 6 The calculated calibration curve for each sample is shown. The calibration curve was derived using a method similar to that described in WO 2020 / 260277 A1, but with adjusted binding characteristics. Using the individually determined labeling ratio and labeling error of the antibody, the corrected calibration curve was corrected with the correction values ​​of the single positive and double positive values ​​of the dPCR, resulting in corrected concentrations of the antibody and conjugate.

[0479] Assuming that all samples contain the same concentration of HER2, the apparent dissociation constant of the antibody is calculated recursively to minimize the standard deviation of the recalculated HER2 concentrations for all measurements.

[0480] Figure 6It is a graphical description of the apparent dissociation constants for trastuzumab and pertuzumab. The samples have equimolar concentrations, but the exact concentration of HER2 is unknown (the samples are obtained from lysed HER2 cells), and the concentrations of the antibodies are different (the concentrations of the antibodies are 5.5E-9, 1.50E-9, 4.0E-10, 1.2E-10, 3.5E-11, and 1.0E-11). These lines represent calibration curves for HER2-specific two-component assays at these antibody concentrations and apparent Kd, and the assay is similar to the method described in WO 2020 / 260277A1 assay. Assuming that all samples contain the same concentration of HER2, the latter is determined to minimize the SD of the recalculated HER2 concentrations of all measured values. The molar HER2 concentration is designated as MeanHER2, and STD and pSTD are the standard deviations of the measured values ​​and the percentages of their mean values, respectively. The dotted line depicts the mean values ​​of HER2 measurements at different antibody concentrations. Finally, the dissociation of the antibodies (Kd1TTZ and Kd2PTZ) were recursively determined to be 2.9E-11 and 9.3E-11, respectively.

[0481] Example 7

[0482] This method advantageously uses the method of the invention to determine the RNA content of a sample and simultaneously determine the absolute amount of a target analyte.

[0483] Simultaneous RNA detection

[0484] For simultaneous RNA detection, the DNA-dependent DNA polymerase activity of MMLV reverse transcriptase was exploited, described as the SMART library preparation protocol (Clontech - Opening the mechanism at the 5' end of the RNA template). Briefly, MMLV reverse transcriptase converts the end of RNA-based DNA synthesis starting from the 3' end of the mRNA into a TSO (Template Switching DNA Oligonucleotide) (which in turn can introduce a 5' PCR primer site, commonly called a PCR handle), and introduces a similar PCR handle at the 3' end at the beginning of the first strand synthesis, which forms a universally amplifiable amplicon. Briefly, the MMLV reverse transcriptase terminal transferase activity adds several additional nucleotides (mainly deoxycytidine) to the 3' end of the newly synthesized cDNA chain. These deoxycytidine bases form the annealing site of the template switching (TS) oligomer (TSO). The reverse transcriptase recognizes this new template strand, formed as a TSO from the cellular RNA, and continues to synthesize the second strand incorporating the universal sequence, i.e., the PCR handle. The single-cell RNA-seq protocol applies this concept [ D., Luo, S., Wang, YC, Li, R., Deng, Q., Faridani, OR, Daniels, GA, Khrebtukova, I., Loring, JF, Laurent, LC Activity(2012).Full-length mRNA-seq from single-cell levels of RNAand individual circulating tumor cells.Nat.Biotechnol.30,777–782].

[0485] Template switching oligomer (TS) structure. The simplest form of TSO is a DNA oligomer sequence carrying three riboguanosines (rGrGrG) at its 3' end [Zhu YY, Machleder EM, et al. (2001) Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction Biotechniques, 30 (4): 892-897.]. The complementarity between these consecutive rG bases and the 3'dC extension of the cDNA molecule enables template switching [Turchinovich A, Surowy H et al. (2014) Capture and Amplification by Tailing and Switching (CATS). An ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA. RNA Biol, 11 (7): 817-828.]. A method has been reported to reduce library background and thus improve cDNA yield by incorporating isomeric nucleotides into TS oligomers [Kapteyn J, He R et al. (2010) Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples. BMC Genomics, 11: 413.]. In this study, two modified bases, iso-dC and iso-dG, were appended to the 5' end of the TS oligomer, forming hydrogen bonds with each other but not with the naturally occurring C and G nucleotides, effectively locking the 5' end of the TSO, demonstrating its efficacy in minimizing the tandem of TS oligomers, which is usually caused by the cycling of reverse transcriptase activity [Saliba AE, Li L et al. (2016) Single-cell RNA-seq ties macrophage polarization to growth rate of intracellular Salmonella. Nat Microbiol, 2: 16206.].

[0486] MCF7 ( HTB-22) cells. In a cell culture incubator (Heracell TM 150i CO2 Incubator, 50116048, Thermo Scientific TM ) in a 5% CO2 atmosphere at 37°C until the cells were approximately 90% confluent. The cells were harvested by scraping. The cells were washed twice with DPBS (14040133, Gibco) and counted ( II Automated Cell Counter,Invitrogen TM ), including the use of trypan blue (T10282, Invitrogen TM ) for live / dead staining.

[0487] The antibody was conjugated to a DNA amplifiable marker using a proprietary procedure of Actome GmbH, Germany. The labeling efficiency achieved was between 80-100% and confirmed by SDS-PAGE electrophoresis. The antibody preparation had 3% free labeling and 25% labeling error (see Example 5). Trastuzumab (TTZ) and Pertuzumab (PTZ) are recombinant humanized monoclonal antibodies, both of which target the extracellular region of the HER2 tyrosine kinase receptor and have no overlapping epitopes. The conjugated antibody has two different labels, namely, Trastuzumab BL and Pertuzumab P8 labels. The dissociation constant of the antibody was determined according to Example 6, and was TTZ=0.49E-10M and PTZ=0.93E-10, respectively.

[0488] 0.5 μl of LBTW was dispensed into 384-well V-bottom plates (0030623304, Eppendorf) using instant droplet-on-demand technology (I.DOT One; Dispendix, Stuttgart, Germany) with an I.DOT PURE plate 90 μm orifice (Dispendix, Stuttgart, Germany). Prior to dispensing, the I.DOT was calibrated for the applied liquid to ensure reliable dispensing. Diluted crude cell lysate ("cl") or DPBS was dispensed using liquid class "H2O".

[0489] Without lysing the cells, the cell distribution procedure was performed as previously described (T. Groβ, C. Jeney, D. Halm, G. Finkenzeller, G. B. Stark, R. Zengerle, P. Koltay, S. Zimmermann, Characterization of CRISPR / Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning, BioRxiv. (2020) 1–19. https: / / doi.org / 10.1101 / 2020.08.17.253559.). Briefly, the F. SIGHT single-cell dispenser (CYTENAGmbH, Freiburg, Germany), an improved version of the single-cell printer (SCP) (A. Gross, J. S. Niekrawitz, W. Streule, L. Riegger, R. Zengerle, P. Koltay, Single-Cell Printer: Automated, On Demand, and Label Free, J. Lab. Autom. 18 (2013) 504–518. https: / / doi.org / 10.1177 / 2211068213497204), MCF7 and BT-474 cell concentrations were adjusted to 1 × 106 cells / ml and loaded into a dispensing box (CYTENA GmbH, Freiburg, Germany). The settings for MCF7 cells were a cell size of 10 to 25 μm (BT-474: 10 to 30 μm) and a circularity of 0.5 to 1 (the same for BT-474) ( Figure 1 a, S3a and S3b). F.SIGHT enables reliable single cell dispensing with minimal liquid volumes. Single cell dispensing efficiency (successful single cell isolation events divided by target events) is typically around 90% and is additionally controlled by the cell image that is clearly assigned to each dispensing event. Thus, in addition to single cell dispensing, events such as doublets or empty droplets can be excluded.

[0490] Samples (0.5 μL single cell samples) were added and a master mix was prepared as follows: 1X qScript XLT 1-Step RT-qPCR ToughMix (95132, Quantabio) containing (1.3 mM GTP, 1.3 U / μL RNase inhibitor, 12.5 μM TSO, PCR handle-tagged random hexamer) and loaded onto four Crystal Digital PCR System Sapphire Chips were reacted and read with a Prism3 reader (Stilla Technologies, Villejuif, France). Digital PCR was performed using cycling parameters (5 cycles, incubation at 42°C for 10 minutes and incubation at 25°C for 15 seconds, followed by hot start at 95°C for 2 minutes, 40 cycles, denaturation at 95°C for 15 seconds, and annealing at 58°C for 30 seconds). Imaging conditions P8 labeling-FAM green channel, 500ms integration time, gain 6, and BL labeling-HEX yellow channel, 400ms integration time, gain 6).

[0491] The two-component assay signal was evaluated as described (Example 6).

[0492] After the cycle, the Sapphire chip was removed from the cycler and the blue cover was removed, and the emulsion was recovered using 20% ​​perfluorooctanol (PFO) in chloroform or hydrofluoroether (HFE), the emulsion was destroyed and the cDNA library was purified. In brief, 125 μL of recovery agent (20% PFO in HFE), 55 μL GITC buffer (5M GITC, 25mM EDTA, 50mM Tris-HCl pH 7.4) and 5 μL 1M DTT were added to each individual aliquot of 50 μL emulsion and incubated on ice for 5 minutes. 99 μL of Ampure XP beads were added to the aqueous phase and incubated for 10 minutes. The Ampure beads were precipitated on a Nd magnet and washed twice with 80% EtOH. Elution was performed in 30 μL T8.5-DTT-Tween (10 mM DTT, 0.1% Tween-20 in T8.5 (10 mM Tris-HCl pH 8.5)).

[0493] Linear PCR amplification was performed (40 μL library, 50 μL 2×KAPA HiFi (Roche), 10 μL 10 μM TSO-PCR handle primer). PCR cycles were performed according to (3 min at 95°C, 13 cycles of [20 sec at 98°C, 20 sec at 63°C, 3 min at 72°C], 5 min at 72°C), followed by a final hold at 4°C. 2 μL 1 M DTT was added and 0.6× Ampure XP purification was performed according to the manufacturer's recommendations.

[0494] Final sequencing library is prepared according to the following customized NEB Ultra II FS scheme (NEB E7805S).Use thermal cycle scheme by 80ng amplified cDNA in Ultra II fragmentation mixture (26 μ L amplified cDNA, 7 μ L NEBNext Ultra II Fs reaction buffer, 2 μ L NEBNext Ultra II FS enzyme mixture) fragmentation: 10 minutes at 37 ℃, 30 minutes at 65 ℃ and finally remain at 4 ℃.Add 15 μ L T8.5, and carry out 0.8 × Ampure purification according to the advice of manufacturer, and elute in 50 μ L.Use NEBNext Ultra II adapter connection mixture (35 μ L fragmentation library, 30 μ L NEBNext Ultra II connection master mixture, 1 μ L NEBNext connection enhancer, 2.5 μ L NEBNextAdapter for Illumina) fragmentation library is carried out adapter connection, 15 minutes at 20 ℃, remain at 4 ℃. 28.5 μL T8.5 was added and 0.8×Ampure purification was performed according to the manufacturer's recommendations and eluted in 30 μL. The eluted library was amplified in a PCR master mix (50 μL 2×KAPA HiFi, 10 μL 10 μM Hy-i7 primer, 10 μL 10 μM Hy-i5 primer, 30 μL eluted library) with the following thermal cycle program: 3 minutes at 95°C, 13 cycles of (20 seconds at 98°C, 30 seconds at 64°C, 30 seconds at 72°C), 5 minutes at 72°C, and finally held at 4°C. The sequencing-ready library was purified using 0.8×Ampure purification and eluted in 30 μL T8.5.

[0495] The library was sequenced on an Illumina NextSeq2000 system using 50 cycles of read 1 (3' end of cDNA), 10 cycles of index 1 (sample index, custom i7 read primer), 10 cycles of index 2 (sample index), 58 cycles of read 2 (5' end of cDNA).

[0496] Data processing of the libraries was performed using a custom pipeline, but according to the main guidelines of scRANseq. In brief, STARsolo was used to map the reads and demultiplex the cells (Kaminow B, Yunusov D, Dobin A. 2021. Genomic SuperSignature: interpretation of RNA-seq experiments through robust, efficient comparison to public databases. Bioinformatics (Oxford, England) 1: e55. DOI: https: / / doi.org / 10.1101 / 2021.05.05.442755), and cells were filtered based on the expression of up to 4000 genes. The filtered expression matrix was converted to total counts and logarithmically normalized.

[0497] Example 8

[0498] This method advantageously determines the absolute amount of a target analyte using the method of the invention forming a physically separated first compartment.

[0499] Determine the absolute amount of the target analyte, first partitioned into first partitions, wherein the partition is physical, and the binding reaction between the cells and the binding agent is preferably carried out at a high target analyte concentration imposed by the low volume containing each single cell. Preferably, the cells are lysed during the first partition. Each first compartment is partitioned a second time, and a suitable sensitive method is used to count single target analyte molecules. Preferably, the concentration of the target in the first compartment is determined using the binding characteristics, and ultimately leads to the determination of the absolute amount / number of the target analyte in each single cell, as described in this embodiment.

[0500] Providing a single cell sample, including: during the separation of the single cell sample into a plurality of first partitions, incubating the sample with two target-specific antibodies, wherein the target and the antibody form a conjugate, and supplementing each first partition containing a single cell with a lysis buffer. During the two-component method of separation (second partition), determining the condition of the conjugate in the first partition.

[0501] MCF7 ( HTB-22) cells. In a cell culture incubator (Heracell TM150i CO2 Incubator, 50116048, Thermo Scientific TM ) in a 5% CO2 atmosphere at 37°C until the cells were approximately 90% confluent. The cells were harvested by scraping. The cells were washed twice with DPBS (14040133, Gibco) and counted ( II Automated Cell Counter,Invitrogen TM ), including the use of trypan blue (T10282, Invitrogen TM ) for live / dead staining.

[0502] The antibody was conjugated to a DNA amplifiable marker using a proprietary procedure of Actome GmbH, Germany. The labeling efficiency achieved was between 80-100% and confirmed by SDS-PAGE electrophoresis. The antibody preparation had 3% free labeling and 25% labeling error (see Example 5). Trastuzumab (TTZ) and Pertuzumab (PTZ) are recombinant humanized monoclonal antibodies, both of which target the extracellular region of the HER2 tyrosine kinase receptor and have no overlapping epitopes. The conjugated antibody has two different labels, namely, Trastuzumab BL and Pertuzumab P8 labels. The dissociation constant of the antibody was determined according to Example 6, and was TTZ=0.49E-10M and PTZ=0.93E-10, respectively.

[0503] Cells were encapsulated in HFE-7500 Novac oil with EA-008 surfactant (RAN Biotech) in (PBS, 25 mM DTT, 15% Optiprep, 1.3 U / μL RNase inhibitor, cOmplete protease inhibitor cocktail tablet 1xcc, 4.4% PEG-8000) using a droplet generation chip (LabSmith) on an Onyx microfluidic platform (Droplet Genomics) and microinjected using the Onyx system, achieving 1x LBTW with labeled trastuzumab (TTZ) and pertuzumab (PTZ) (both at a concentration of 1.e-11 M) in the first partition. The resulting emulsion was collected in aliquots of 50 μL total volume and incubated overnight at 4C.

[0504] Perform droplet distribution and adjust the droplet concentration to 1×10 6Droplets / ml, a single first compartment is delivered to a 96-well microplate well. QIAGEN's QIAcuity Probe Mastermix (42 μL) is added to a 96-well microplate, a single droplet is dispensed into the well, and loaded into a QIAcuity Nanoplate26k 24-well plate after mixing. A negative control containing only labeled trastuzumab and pertuzumab antibodies was also established using an antibody (antibody binding control) at a concentration of 4.00E-11M. Digital PCR was performed using the QIAGEN QIAcuity digital PCR system, where the cycle parameters were based on the following PCR conditions: hot start at 95°C for 2 minutes, 40 cycles, denaturation at 95°C for 15 seconds, and annealing at 58°C for 30 seconds. Imaging conditions P8 labeled-FAM green channel, 500ms integration time, gain 6, and BL labeled-HEX yellow channel, 400ms integration time, gain 6.

[0505] As described elsewhere (Example 6), conjugate counts and antibody counts were obtained. Antibody counts are expected to average 4320 (corrected antibody labeling ratio and labeling error) copies per QIAcuity Nanoplate 26k 24-well plate reaction, and the detected conjugate averages 40 (corrected antibody labeling ratio and labeling error). If the antibody concentration is 4.e-11M, then the 4320 antibody copies detected correspond to a droplet size of 0.18nL, so the conjugate series (average) is 3.7e-13M. Based on the antibody concentration, including the predetermined binding characteristics of the antibody kd (exemplary Kd is PTZ=7.4E-11 and TTZ=1.2E-10, the apparent Kd determined, see Example 6), a calibration curve can be constructed. However, the range of the conjugate can be 10 to 150, with corresponding repetitions of 9.2e-14 and 1.3e-12M. The calculated concentration of HER2 protein target was 4.4e-12 M (average), which is 476 copies in 0.18 nL, i.e., 476 HER2 copies per cell (average), with a range of 114-1992 HER2 copies per cell.

[0506] Example 9

[0507] This method advantageously determines the absolute amount of a target analyte using the method of the invention forming a physically separated first compartment, but using a protein marker instead of a nucleic acid marker,

[0508] Protein labeling

[0509] An exemplary embodiment of the present invention is implemented using protein labeling, and an antibody capture agent is attached to the surface of 2.7-μm diameter paramagnetic beads, each of which contains 250,000 attachment sites. Typically, 500,000 beads will be added to a 100-μL sample. At low target concentrations, when the number of beads is greater than the number of target molecules in the sample, each bead will capture 0 or 1 target molecules. The fraction of beads that capture target molecules in a given sample follows a Poisson distribution. Single beads are captured in femtoliter-sized holes on the Simoa Disc, which allows a "digital" readout of each individual bead to determine whether it is bound to the target analyte.

[0510] The beads are washed to remove non-specifically bound proteins and incubated with two detection antibodies labeled with beta-galactosidase and alkaline phosphatase, respectively. In this way, each bead of a single protein molecule is captured with two enzyme detections. Beads without captured molecules remain unlabeled. The beads are loaded into a 216,000 femtoliter-sized hole array, with each hole accommodating no more than one bead. Beads are added in the presence of substrate, followed by sealing the holes with oil and imaging. If the target analyte has been captured (i.e., an immune complex has been formed), the substrate is converted into a fluorescent product by the captured enzyme label. The reaction is evaluated according to the two-component evaluation concept (Example 6), single-positive and double-positive compartments are read, and all compartments are counted.

[0511] Example 10

[0512] For a large number of target analytes, the readout / analysis of the two-component method can be performed by nucleic acid sequencing such as NGS, in which case the label should be a nucleic acid sequence that contains a sequence that is unique for each type of binder. In an embodiment, a molecule-specific barcode (UMI) is additionally added to the label of each binder during the initial binder labeling reaction or at a later time point in each partition. This example describes the use of the method of the present invention to detect the target using NGS as a readout method. This method advantageously uses the method of the present invention to determine the absolute amount of the target analyte, thereby determining the absolute condition of the first compartment using a next-generation sequencing method.

[0513] NGS reads

[0514] MCF7 ( HTB-22T) cells. In a cell culture incubator (Heracell TM150i CO2 Incubator, 50116048, Thermo Scientific TM ) in a 5% CO2 atmosphere at 37°C until the cells were approximately 90% confluent. The cells were harvested by scraping. The cells were washed twice with DPBS (14040133, Gibco) and counted ( II Automated Cell Counter,Invitrogen TM ), including the use of trypan blue (T10282, Invitrogen TM ) for live / dead staining.

[0515] The antibody was conjugated to a DNA amplifiable marker using a proprietary procedure of Actome GmbH, Germany. The achieved labeling efficiency was between 80-100% and confirmed by SDS-PAGE electrophoresis. The antibody preparation had 3% free labeling, 25% labeling error (see Example 5). Trastuzumab (TTZ) and Pertuzumab (PTZ) are recombinant humanized monoclonal antibodies, both of which target the extracellular region of the HER2 tyrosine kinase receptor and have no overlapping epitopes. The conjugated antibody has two different labels, namely, trastuzumab BL and Pertuzumab P8 labeling incorporating 10bp UMI. The dissociation constant of the antibody was determined according to Example 6, and was TTZ=0.49E-10M and PTZ=0.93E-10, respectively.

[0516] 0.5 μl of LBTW was dispensed into 384-well V-bottom plates (0030623304, Eppendorf) using instant droplet-on-demand technology (I.DOT One; Dispendix, Stuttgart, Germany) [50, 51] with an I.DOT PURE plate 90 μm orifice (Dispendix, Stuttgart, Germany). Prior to dispensing, the I.DOT was calibrated for the applied liquid to ensure reliable dispensing. Diluted crude cell lysate ("cl") or DPBS was dispensed using liquid class "H2O".

[0517] Without lysing the cells, the cell distribution procedure was performed as previously described (T. Groβ, C. Jeney, D. Halm, G. Finkenzeller, GB Stark, R. Zengerle, P. Koltay, S. Zimmermann, Characterization of CRISPR / Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning, BioRxiv. (2020) 1–19. https: / / doi.org / 10.1101 / 2020.08.17.253559.). Briefly, MCF7 and BT-474 cells were adjusted to a concentration of 1 × 10e6 cells / ml and loaded into a dispenser (CYTENA GmbH, Freiburg, Germany) using the F.SIGHT single-cell dispenser (CYTENA GmbH, Freiburg, Germany), a modified version of the single-cell printer (SCP)

[43] . The settings for MCF7 cells were a cell size of 10 to 25 μm (BT-474: 10 to 30 μm) and a circularity of 0.5 to 1 (for BT-474, similarly) ( Figure 1 a, S3a and S3b). F.SIGHT enables reliable single cell dispensing with minimal liquid volumes. Single cell dispensing efficiency (successful single cell isolation events divided by target events) is typically around 90% and is additionally controlled by the cell image that is clearly assigned to each dispensing event. Thus, in addition to single cell dispensing, events such as doublets or empty droplets can be excluded.

[0518] Prepare a master mix as follows: using primers containing cell-specific barcodes, 1X qPCR (Quantabio) and loaded into Crystal Digital PCR System Sapphire chip reaction, and read with Prism3 reader (Stilla Technologies, Villejuif, France). Linked PCR was performed with cycle parameters (PCR conditions of hot start at 95°C for 2 minutes, 240 cycles, denaturation at 95°C for 15 seconds, annealing at 58°C for 30 seconds). Imaging conditions P8 label-FAM green channel, 500ms integration time, gain 6, and BL label-HEX yellow channel, 400ms integration time, gain 6).

[0519] The two-component signal was assessed as described (Example 6). The number of amplified compartments was recorded or calculated using quantitative tracer-linked PCR amplicons.

[0520] After the cycle, the Sapphire chip was removed from the cycler and the blue cover was removed, and the emulsion was recovered using 20% ​​perfluorooctanol (PFO) in chloroform or hydrofluoroether (HFE), the emulsion was destroyed and the cDNA library was purified. In brief, 125 μL of recovery agent (20% PFO in HFE), 55 μL GITC buffer (5M GITC, 25mM EDTA, 50mM Tris-HCl pH 7.4) and 5 μL 1M DTT were added to each individual aliquot of 50 μL emulsion and incubated on ice for 5 minutes. 99 μL of Ampure XP beads were added to the aqueous phase and incubated for 10 minutes. The Ampure beads were precipitated on a Nd magnet and washed twice with 80% EtOH. Elution was performed in 30 μL T8.5-DTT-Tween (10 mM DTT, 0.1% Tween-20 in T8.5 (10 mM Tris-HCl pH 8.5)).

[0521] Linear PCR amplification was performed (40 μL library, 50 μL 2×KAPA HiFi (Roche), 10 μL 10 μM TSO-PCR handle primer). PCR cycles were performed according to (3 min at 95°C, 13 cycles of [20 sec at 98°C, 20 sec at 63°C, 3 min at 72°C], 5 min at 72°C), followed by a final hold at 4°C. 2 μL 1 M DTT was added and 0.6× Ampure XP purification was performed according to the manufacturer's recommendations.

[0522] The library was adapter-ligated using NEBNext Ultra II Adapter Ligation Mix (35 μL fragmented library, 30 μL NEBNext UltraII Ligation Master Mix, 1 μL NEBNext Ligation Enhancer, 2.5 μL NEBNext Adapter for Illumina) at 20°C for 15 minutes, kept at 4°C. 28.5 μL T8.5 was added and 0.8× Ampure purification was performed according to the manufacturer's recommendations and eluted in 30 μL. The eluted library was amplified in a PCR Master Mix (50 μL 2× KAPA HiFi, 10 μL 10 μM Hy-i7 primer, 10 μL 10 μM Hy-i5 primer, 30 μL eluted library) with the following thermal cycle program: 3 minutes at 95°C, 13 cycles of (20 seconds at 98°C, 30 seconds at 64°C, 30 seconds at 72°C), 5 minutes at 72°C, and finally kept at 4°C. Sequencing-ready libraries were purified using 0.8× Ampure purification and eluted in 30 μL T8.5.

[0523] Libraries were sequenced on an Illumina NextSeq2000 system using 50 cycles of read 1, 10 cycles of index 1 (sample index, custom i7 read primers), 10 cycles of index 2 (sample index), and 58 cycles of read 2.

[0524] The library was data processed using a custom pipeline. In brief, the cells, UMIs were deconvoluted, and after describing such library deconvolution, the droplets were deconvoluted as described in WO 2020 / 260277 A1. In brief, during the linked PCR step, deconvolution of the droplets was achieved based on unique pairings between the UMIs of each droplet, such pairings causing the droplets to randomly become local pairs of UMIs, and the network of such pairs was highly unique, clearly defining the droplets, enabling the droplet contents to be reconstructed with reference to the types of antibodies present. Based on the droplet contents, the distribution of antibodies can be inferred, so droplets containing one type of antibody and droplets containing two antibodies can be counted. The total number of droplets is derived by direct counting or using the tracer concept. The obtained droplet information can be evaluated using the methods described for dPCR readout (Example 6).

Claims

1. A method for determining the concentration of at least one target analyte in a sample, comprising: a. providing at least a first type of binding agent and a second type of binding agent, each binding agent comprising a label unique to said type of binding agent, wherein said first type of binding agent and said second type of binding agent specifically bind to a first target analyte, b. determining the binding characteristics of the first type of binding agent and the second type of binding agent to the first target analyte, c. providing a sample containing cells of known concentration, d. contacting the first type of binding agent and the second type of binding agent with the sample, wherein the first type of binding agent and the second type of binding agent bind to the first target analyte, thereby forming an analyte-binding agent complex (conjugate), e. dividing the single cells of the sample into a plurality of first partitions, f. supplementing each first partition containing single cells with a lysis buffer, thereby lysing the single cells in each first partition, g. partitioning a single analyte-binding agent complex (conjugate) into a plurality of second partitions, h. performing a two-component detection method to determine the amount and / or absolute concentration of the conjugate and the binding agent in the second partition, i. determining the absolute concentration of the first target analyte per cell in the first partition by taking into account the initial concentration of the cells in the sample in step a., the binding characteristics determined in step b. and the number and / or concentration of conjugates and binding agents in the second partition in step h.

2. The method according to claim 1, wherein determining the binding characteristics in step b. includes determining the following binding characteristics of each binding agent of the first type and the second type: dissociation constant with the target analyte, specificity of binding to the target analyte, labeling ratio, labeling synthesis error and / or optimal concentration (number of molecules per unit volume) required for binding to the target analyte.

3. The method according to any one of the preceding claims, wherein the first type of binding agent and the second type of binding agent each comprise a nucleic acid tag unique to said type of binding agent, and wherein the two-component detection method in step h. comprises the following steps: - amplifying said nucleic acid sequence marker in each of said second partitions, and / or - combining the plurality of second partitions and subjecting them to nucleic acid sequence analysis in order to determine the number and identity of the nucleic acid sequence tags, and thereby determine the number and / or concentration of the conjugate.

4. A method according to any of the preceding claims, wherein the two-component detection method in step h. includes a connection step between the labels of the first type of binding agent and the second type of binding agent in each second partition.

5. A method according to any of the preceding claims, wherein if the nucleic acid sequence tag of the first type of binding agent and the nucleic acid sequence tag of the second type of binding agent both form a conjugate with the first target analyte, the connection step in step h. includes connecting the nucleic acid sequence tag of the first type of binding agent to the nucleic acid sequence tag of the second type of binding agent within each second partition.

6. A method according to any of the preceding claims, wherein before contacting the first type of binding agent and the second type of binding agent with the sample, the cells in the sample are permeabilized for the first type of binding agent and the second type of binding agent, so that one or more of the first type of binding agent and the second type of binding agent enter one or more of the cells after contacting the sample in step d.

7. A method according to any one of the preceding claims, wherein the first type of binding agent and the second type of binding agent each comprise a nucleic acid marker unique to that type of binding agent, and wherein the plurality of first partitions in step f. are further supplemented with PCR reagents and nucleic acid amplification primers containing a barcode unique to each first partition and the single cell therein, and optionally supplemented with reverse transcription reagents, and wherein a further nucleic acid amplification step is performed after cell lysis in each first partition, and Optionally wherein the plurality of first partitions in step f. are further supplemented with a protein cross-linking agent and / or a protease inhibitor and / or a nuclease inhibitor.

8. A method according to any of the preceding claims, wherein the first type of binder and the second type of binder each comprises a nucleic acid tag unique to that type of binder, and wherein each nucleic acid sequence tag comprises an identifier sequence (barcode) and a unique molecular identifier (UMI) unique to each tagged molecule.

9. A method according to any of the preceding claims, wherein the sample comprises RNA, and wherein the multiple first partitions in step f. are further supplemented with a reverse transcription reagent, and during any one or all of steps dg, the RNA is simultaneously converted into cDNA, and wherein if nucleic acid sequence analysis is performed in step h., the analysis comprises next generation sequencing (NGS) or qPCR analysis of the cDNA produced in step dg.

10. A method according to any one of claims 3-9, wherein the first type of binder and the second type of binder each contain a nucleic acid marker unique to that type of binder, and wherein the amplification in step h. comprises performing PCR and / or droplet PCR and / or digital PCR and / or real-time PCR and / or RT-PCR.

11. A method according to any of the preceding claims, wherein the at least one target analyte comprises at least a first target analyte and a second target analyte, and wherein in step a., for each of the at least first target analyte and the second target analyte, at least a first type of specific binding agent and a second type of specific binding agent are provided.

12. A method according to the preceding claim, wherein If the number of different target analytes is less than 5, and if nucleic acid sequence analysis is performed in step h., said analysis comprises digital PCR and / or real-time PCR, or If the number of different target analytes is greater than or equal to 5, and if nucleic acid sequence analysis is performed in step h., the analysis comprises next generation sequencing (NGS).

13. The method according to any one of the preceding claims, wherein the binding agent is selected from the group comprising: antibodies, engineered protein scaffolds and aptamers.

14. The method according to any one of the preceding claims, wherein the target analyte is selected from the group comprising a peptide, a protein, a chemically modified peptide or protein, a nucleic acid, a chemically modified nucleic acid and any complex thereof.

15. A method according to any of the preceding claims, wherein the sample is provided in step c. after being processed by a method selected from the group consisting of: fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), laser capture microdissection (LCM), manual cell picking / micromanipulation, microfluidic separation, and on-demand optical interception and controlled single-cell printing.

Images