Detection of Biomolecules in a Single Cell

Abstract

The present invention relates to a method for determining the concentration of at least one target analyte in a sample, comprising: a. preparing at least a first type of binder and a second type of binder, each containing a label specific to the type of binder, and the first type of binder and the second type of binder specifically binding to a first target analyte; b. determining the binding characteristics of the first type of binder and the second type of binder to the first target analyte; c. preparing a sample containing cells of a known concentration; d. contacting the first type of binder and the second type of binder with the sample, wherein the first type of binder and the second type of binder bind to the first target analyte to form an analyte-binder complex; e. compartmentalizing single cells of the sample into a plurality of first partitions; f. complementing each first partition containing a single cell with a lysis buffer, thereby lysing the single cell within each first partition; g. compartmentalizing a single analyte-binder complex into a plurality of second partitions; h. performing a two-component detection method to thereby determine the number and / or absolute concentration of the complex and the binder in the second partition; and i. determining the absolute concentration of the first target analyte per cell, 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 absolute concentration of the complex and the binder determined in step h.

Description

Technical Field

[0001] The present invention generally relates to the field of molecular biology. More specifically, it relates to methods for detecting targets including proteins, post-translationally modified proteins, and protein interactions in a single cell. The present invention provides a highly sensitive method with a detection limit for targets per cell and preferably less than 100 targets in absolute molecular weight, or preferably less than 10 targets.

Background Art

[0002] Proteomics encompasses proteomic domains that are equally important in the study of proteomic functions, which include expression proteomics regarding changes in protein expression levels under various physiological or pathological cell regimes, protein-protein interactions, enzyme-substrate relationships, and pathway organization determined by post-translational modification of proteins, as well as structural proteomics (complexomics) that predicts the three-dimensional and higher-order structures of proteins and protein complexes. However, to directly relate to the role of proteins in the physiological context of cells, it is also necessary to measure the systematic perturbation or functional inactivation of proteins in their physiological environment to assess all of the above quantitative cellular information in order to address the potential role of target proteins in cellular processes, which is similar to gene disruption for elucidating gene functions. By evaluating the quantitative functions of proteins in parallel at the cellular and molecular levels, it becomes possible to obtain an integrated view necessary to ultimately understand the nature of information processing in the cellular pathways that determine cell function.

[0003] In the case of a single cell, the behavior of individual cells is governed by the principle that the functions, developmental trajectories, and responsiveness of cells are heterogeneous, even if the cells originally had the same ancestor. For example, a cell may be in one transcriptional state or another, and the cell shuttles between states that are only partially governed by known stimuli. When cells interact with the environment, many strange phenotypes should occur purely as a result of these effects. However, observing such cell behavior amidst overwhelming noise and unsatisfactory analytical power, including low measurement sensitivity, is a challenging task, and new single-cell analysis methods are needed.

[0004] Existing single-cell analysis methods are only partially designed to solve this difficult problem and are defined as collecting the comprehensive information processing layer of cells through the relevant chemical information of individual members of large cell populations, which includes genomic methods, transcriptomic methods, and epigenomic methods. Proteomic methods have also been developed, but they have limited scope compared to more developed DNA- and RNA-based single-cell assay methods. Also, there are no methods available for single-cell interactomics, epi-proteomics (for the purposes of this specification, antibody-identifiable modified proteins, including post-translationally modified proteins, are called epi-proteins), and complexomics.

[0005] Regarding the sensitivity of proteomics, interactomics, epi-proteomics, and complexomics single-cell methods, to fully understand the functional significance of the detected amount, the sensitivity of hundreds, or preferably dozens, of proteins in terms of absolute molecular weight is required. Reaching these limits is impossible, especially with modern single-cell assays for interactomics, epi-proteomics, and complexomics.

[0006] The currently used methods are aimed at genome capture, and for example, there are MDA-WGA, isothermal amplification (Non-Patent Document 1), DOP-PCR-WGA, PCR-based ones (Non-Patent Document 2), MALBAC-WGA (Non-Patent Document 3). Single-cell genome sequencing reveals the genetic heterogeneity of single cells, and its applications include the analysis of de novo germline mutations or somatic mutations in normal cells and cancer cells. Since the transcriptome also represents some of these mutations, transcriptome profiling is also used, particularly for elucidating the overall picture of mutations in the B cell-based somatic hypermutation phenotype (Non-Patent Document 4).

[0007] The currently used methods are also aimed at capturing the transcriptome, and for example, there are Smart-seq-WTA with template switching (Non-Patent Document 5), CEL-seq-WTA by in vitro transcription (Non-Patent Document 6), Quartz-seq-WTA with poly(A) tagging (Non-Patent Document 7), C1-CAGE-5'-end RNA-seq (Non-Patent Document 8), lambda-seq-full-length RNA-seq (Non-Patent Document 9), Drop-seq-microdroplet-based (Non-Patent Document 10), microwell-seq-microwell-based (Non-Patent Document 11). Since bulk sample RNA-seq only measures the expression of average transcripts in a cell population, single-cell RNAseq methods provide a more comprehensive description of the cell state, including transitions between states, in order to accurately characterize the transcriptome of single cells in a heterogeneous cell population. scRNAseq transcriptome analysis aims to detect the temporal pattern of the transcriptome and further define the transitions in the pseudo-time space (Non-Patent Document 12).

[0008] The currently used methods also aim to capture other properties of cell physiology (or epigenome), including accessible chromatin, methylation, and histone modifications. For example, scBS-seq-DNA methylation (Non-Patent Document 13), scRRBS-DNA methylation (Non-Patent Document 14), scAba-seq-DNA methylation 5hmC (Non-Patent Document 15), scATAC-seq-chromatin accessibility (Non-Patent Document 16), Drop-ChIP-histone modification chip-seq (Non-Patent Document 17), scChIC-seq-histone modification method (Non-Patent Document 18), CUT&Tag-histone modification method (Non-Patent Document 19), scHi-C-chromatin structure (Non-Patent Document 20).

[0009] The currently used methods also aim to capture combined workflows to measure various properties of cell physiology motivated by the lack of reproducibility of single-cell objects, which include combinations of genomics, transcriptomics, open chromatin, methylation, or even surface protein analysis approaches. For example, G&T-seq-genome and transcriptome: FACS-based MDA / PicoPlex (WGA), SMART-seq2 (Non-Patent Document 21), DR-seq-DNA and RNA sequencing (Non-Patent Document 22), scM&T-seq-DNA methylation and transcriptome (Non-Patent Document 23), scDam&T-seq-chromatin, DamID and CEL-seq-based FACS (384-well plate) transcriptome (Non-Patent Document 24), T-ATAC-seq open chromatin based on scATAC-seq and TCR-seq (Non-Patent Document 25), SNARE-seq-open chromatin and transcriptome (Non-Patent Document 26), FACS-based scCAT-seq-open chromatin and transcriptome (Non-Patent Document 27), CITE-seq-surface protein and transcriptome (Non-Patent Document 28), REAP-seq-surface protein and transcriptome (Non-Patent Document 29).

[0010] Combinations of RNA sequencing (Non-Patent Documents 30 and 31), or proximity ligation assays (PLA and its variations) combined with PCR (Non-Patent Documents 32, 33, 34, 35), or mass spectrometry (Non-Patent Document 36) have been devised for limited-scale simultaneous detection of transcripts and proteins in single cells, including indexed cell sorting. For example, Non-Patent Documents 34 and 37 optimize the PLA reaction only with respect to the optimal concentration of PLA probes for detecting a specific target concentration. In the final quantification of the analyzed proteins in the sample, these approaches only consider the data of the above simple calibration curve / titration curve of the analyzed proteins and / or PLA probes (nucleic acid-binding antibodies). Therefore, the quantification performed in the prior art only considers the potential bias introduced by different concentrations of targets and / or PLA probes in the PLA assay, and further characteristics of the PLA probes are not analyzed or considered, so absolute quantification is impossible with the above methods. Furthermore, these assays are limited in scale, and / or only a very small number of genes and proteins can be profiled in parallel, limited to extracellular proteins that have not been absolutely quantified, unable to handle the interactomics and complexomics of single cells, and significantly restricted in epitroteomics. Conventional techniques such as PLA (proximity ligation assay) and other application methods cannot accurately detect all molecules, often exhibit background noise, and lead to false detection of non-existent molecules. As a result, these techniques cannot obtain the absolute number of molecules essential for absolute quantification without a reference. The method of the present invention addresses these limitations by enabling accurate and precise quantification of molecules without the need for a reference, thereby overcoming the drawbacks of existing technologies.

[0011] Drop-seq, CITE-seq, and REAP-seq perform sequencing-based protein-level readouts by conjugating antibodies to polyA-tagged oligonucleotides (antibody-derived tags - ADTs) that are simultaneously captured by cDNA capture. The antibodies are identified by barcodes included in the labels. The labeled antibodies bind to the targets in a single-cell suspension, the cells are washed to remove unbound antibodies, and then processed for scRNA-seq. Typically, single cells decorated with antibodies are encapsulated in nanoliter-sized reaction droplets to lyse the single cells, extract the cell's mRNA along with the antibody label, and convert it to DNA by the 3' polyA sequence. The transcribed cDNA and ADT are converted into NGS libraries, sequenced, and the quantification of cDNA and ADT is revealed. These assays are widely applied as protein detection performed using FACS (fluorescence-activated cell sorting) (Non-Patent Document 38), which includes a washing procedure, has low specificity, low background, and only provides relative semi-quantitative results, is limited in the same way as FACS, cannot be compared between antibodies, and these methods are classified as not being fully quantitative methods. In the application of ADT, it cannot handle single-cell interactomics and complexomics, and the detection of intracellular proteins is severely limited. As a result, when applied intracellularly, due to the high background and the inevitable washing process with insufficient control, even semi-quantification is lost, so these assays are limited to cell surface proteins.

[0012] Other methods of cell surface protein analysis (Non-Patent Document 39) include magnetic ranking cytometry (MagRC) and flow cytometry (FACS). Methods that can be used for the analysis of methods generally applicable to the detection of proteins include single cell Western blotting, microfluidic proximity ligation assay (PLA), proximity extension assay (PEA), mass cytometry (cytometry by time of flight, CyTOF), mass spectrometry, and single cell barcode chip (SCBC). However, all of these methods have limitations, particularly in terms of providing absolute quantitative detection and high sensitivity, regardless of which targets in the cell compartments of proteomics, interactomics, epi-proteomics, and complexomics they access. The current highest sensitivity has a high background and is limited to thousands of molecules per single cell (Non-Patent Document 34 (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.)).

[0013] Therefore, for quantifying multiple single cell targets in diagnostic and research applications, there is a need for a more efficient composition that is highly sensitive, accesses intracellular targets that reveal proteomic, interactomic, epi-proteomic, and complexomic information, and has universally absolute quantitative and comparable results.

Prior Art Documents

Non-Patent Documents

[0014]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 21

Non-Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Non-Patent Document 25

Non-Patent Document 26

Non-Patent Document 27

Non-Patent Document 28

Non - Patent Document 29

Non - Patent Document 30

Non - Patent Document 31

Non - Patent Document 32

Non - Patent Document 33

Non - Patent Document 34

Summary of the Invention

[0015] In light of the prior art, the technical problem underlying the present invention is to provide an improved method for reliably quantifying at the single-cell level, in an absolute quantitative manner, target analytes such as, for example, proteins, protein modifications, DNA modifications, protein-protein complexes, protein-DNA complexes, or protein-RNA complexes, regardless of the target's cellular localization.

[0016] This problem is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0017] Accordingly, in one aspect, the present invention is a method for determining the absolute 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 comprising a label specific to the 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 the binding characteristics of the first type of binding agent and the second type of binding agent to the first target analyte; c. preparing a sample containing cells of known concentration; d. contacting the sample with the first type of binding agent and the second type of binding agent, wherein the first type of binding agent and the second type of binding agent bind to the first target analyte and preferably form analyte-binding agent complexes under suitable conditions; e. compartmentalizing the (single) cells of the sample into a plurality of first partitions, each compartment containing one or fewer cells (i.e., containing one cell or no cells); f. Complementing each first partition containing a single cell with a lysis buffer, preferably a lysis and / or dispersion buffer, thereby lysing the single cell within each first partition; g. Compartmentalizing a single analyte - binder complex (complex) into a plurality of second partitions; h. Performing a two - component detection method to thereby determine the number and / or concentration of the complex and the binder in the second partition; i. Determining the absolute concentration of the first target analyte per cell in the first partition, 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 the complex and the binder in the second partition determined in step h; relates to a method comprising.

[0018] In embodiments, one or more of the steps of the above - described method may be performed in a different order or a modified order. In some embodiments, certain steps are performed in parallel. In embodiments, for example, depending on the cellular localization of the target analyte, step d. of "contacting the sample with a first type of binder and a second type of binder" may be performed before or after the first compartmentalization step e. (compartmentalizing the sample, preferably a single cell, such that the compartment preferably contains one or fewer cells, i.e., contains one cell or no cells), or may be performed after cell lysis and / or addition of the lysis or dispersion buffer into each first compartment (step f). In such embodiments, prior to the incubation with the binder (step d.), since the nuclear membrane or other membranes have already been lysed in the lysis step f., target factors contained particularly inside the nucleus and / or other organelles of the cell can be detected / bound by the binder.

[0019] Thus, in embodiments, the steps of the above - described method may 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.

[0020] Thus, in an embodiment, the method is a. preparing at least a first type of binder and a second type of binder, each containing a label specific to the type of binder, wherein the first type of binder and the second type of binder specifically bind to a first target analyte; b. determining the binding characteristics of the first type of binder and the second type of binder to the first target analyte; c. preparing a sample containing cells at a known concentration; e. compartmentalizing the sample into a plurality of first partitions, preferably each compartment containing one or fewer cells; f. complementing each first partition containing the sample with a lysis buffer, preferably a lysis and / or dispersion buffer, thereby lysing single cells within each first partition; d. contacting the sample in the first partition with the first type of binder and the second type of binder, wherein the first type of binder and the second type of binder bind to the first target analyte and preferably form an analyte - binder complex under suitable conditions; g. compartmentalizing a single analyte - binder complex into a plurality of second partitions; h. performing a two - component detection method to thereby determine the number and / or concentration of the complex and the binder in the second partition; i. determining the absolute concentration of the first target analyte per cell in the first partition, 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 the complex and the binder in the second partition determined in step i (h?); including (in this order).

[0021] In some embodiments, ligation reaction d. is carried out after the first compartmentalization of the sample components (e.g., single cells) and under lysis and / or dispersion buffer conditions, e.g., after and / or during the addition of the cell lysis and / or dispersion buffer to each first compartment and after and / or during incubation with the buffer.

[0022] In embodiments, the cell lysis / permeabilization treatment step (step f.) and the ligation reaction step (step d.) are carried out in parallel or simultaneously. Thus, in such embodiments, the steps of the aforementioned method can be carried out 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 step f. + d. comprises complementing each first partition containing the sample with a lysis buffer (preferably a lysis and / or dispersion buffer), and a first type of binder and a second type of binder, thereby lysing single cells in each of the first partitions and contacting the first type of binder and the second type of binder with the sample in the first partition, preferably under appropriate conditions, such that the first type of binder and the second type of binder bind to the first target analyte to form an analyte-binder complex. In some of the above embodiments, the binder is added to the first compartment contained within the lysis and / or dispersion buffer (step f. / d.), or the binder is contained within the lysis and / or dispersion buffer when added to the first compartment (step f. / d.).

[0023] In some of the above embodiments where the order of the method steps is changed, it is preferred that the compartmentalization / isolation of single cells is carried out before cell lysis such that only the lysate of single cells (not the bulk lysate) is analyzed.

[0024] Absolute quantification is an important feature of analytical methods that provide accurate measurement of the number of molecules in a sample without relying on an external quantification standard. In the study of biological systems, it is difficult to achieve absolute quantification because of the large variability in protein properties, the wide range of protein abundances, and the complexity of the cellular environment. Existing absolute quantification methods based mainly on mass spectrometry are only partially established, and there is a lack of approaches that do not require external standards.

[0025] The two-component method preferably used in the embodiments herein preferably constitutes an external quantification-free protein assay. To achieve a high signal-to-noise ratio without background noise, it is preferable to use a two-step readout method. In the first step, it is preferable to partition the complex to ensure robust signal generation and mathematically tractable results. Forming the complex under stringent buffer conditions avoids matrix effects and ensures accurate quantification. In the second step, the method employs compartmentalized single-molecule digital PCR for accurate quantification of the complex in the embodiments. To ensure an absolute signal based on the single-molecule sensitivity of the digital PCR measurements, this step is preferably separated from the antibody binding process / step.

[0026] The two-component detection method employed in the present invention offers several advantages compared to existing assays. It provides a reference-free absolute quantification method that does not require an external standard, making it very versatile and applicable to a wide variety of protein analytes. The homogeneous, preferably wash-free workflow of the two-component method of the present invention minimizes signal loss and avoids issues associated with the solid-phase capture step. Since this assay relies on complex formation and compartmentalization in embodiments, it preferably ensures a strict dependence on the binding of the binding agent, prevents dissociation-related problems, and provides reliable quantification. In embodiments, the two-component method used in the present invention enables extensive control of the reaction readout, including determination of binding concentration, parallel normalization, and compensation for dPCR clustering errors, by detecting and quantifying the complexes and individual binding agents.

[0027] Calculations include, in embodiments, determination of the correct concentrations of the binding reactants in the binding reaction, including the binding agent and the complex, and these numerical values may, in embodiments, be corrected by contamination due to the labeling ratio (LR), labeling synthesis error (LE), active fraction (AF) of the binding agent, and / or free label (FL), and may also, in embodiments, be corrected by appropriate mathematical error compensation, such as clustering of individual compartments according to positive or negative evaluations regarding the inclusion of different molecular entities. In embodiments, the absolute concentration is determined by applying chemical equilibrium, and the corrected concentration can be directly used to calculate the absolute concentration of the analyte using, for example, the method described in WO 2020 / 260277, which is incorporated herein by reference.

[0028] In the present invention, one aspect of an embodiment of the absolute quantification method, for example, saturation conditions and the like can be emphasized. This condition preferably enables the direct matching of the concentration of the ternary complex (complex) formed during the assay process with the concentration of the target analyte without the need to determine the dissociation constant (Kd) of the binder (e.g., antibody). In an embodiment, by ensuring the saturation of the assay components, the concentration of the complex can be reliably measured, providing an accurate and simple means for quantifying the target analyte. This approach preferably simplifies the absolute quantification process, eliminates the need to determine the Kd value of the binder (e.g., antibody) used, and enables the direct and accurate quantification of the analyte of interest.

[0029] The step of double compartmentalization (compartmentalization is defined as having a large number of separated and reproducible partitions under specific conditions) and the step of deriving the absolute chemical conditions in the first compartmentalization (the first partition) using the absolute quantification method from the second compartmentalization (the second partition) incorporate the advantages of two different conditions simultaneously, thus presenting a new concept compared to the prior art.

[0030] In the first compartmentalization, the binding reaction preferably has a high local target concentration, which can be of two different types: (1) A suspension of a large number of cells, where the compartmentalization is restricted by diffusion, the cells are in their own concentrated environment, and a high local concentration of the target is achieved, or (2) A small number of cells or a single cell has a high local target concentration.

[0031] In the concept of the present invention, the first compartmentalization and the second compartmentalization are preferably related to each other. (3)Due to the absolute chemical correspondence between the first compartmentalization and the second compartmentalization, it becomes possible to determine the absolute chemical conditions in the first compartmentalization (the first partition) of a single cell from the second compartmentalization (the second partition), and without determining the absolute chemical conditions in the first compartmentalization, the absolute target concentration cannot be derived. (4)Using the determination of the binding characteristics of the binder provides high accuracy in determining the absolute amount within a single cell required for a single cell. Regarding the differences and improvements compared to the standard binding reaction in the bulk solution, since the single cell binding reaction of the present invention is compartmentalized, the separability of the cells is maintained. However, in the reaction with batch-lysed cells, although an absolute chemical correspondence between the compartmentalizations becomes possible, the results cannot be separated for each cell.

[0032] For example, in a preferred embodiment, the step of the second compartmentalization is used to derive / reveal the unknown absolute chemistry (the absolute concentrations of the binder and the complex) present in the first compartmentalization (each in the first partition, each containing the contents of a single cell and potentially the binder).

[0033] In an embodiment, the unit of detection is defined as a complex of two binders bound to a target and is called a complex. When three or more binders are bound, all the unique combinatorial combinations of binder pairs are considered different complexes.

[0034] In a preferred embodiment, the second compartmentalization further provides the exact number of molecules such as the absolute amount / concentration of the target analyte from the determined absolute chemistry, and in order to determine the absolute amount / concentration, the volume of the first compartmentalization (the first partition) and / or the initial concentration of the cells in the initial solution provided for the first compartmentalization, and / or the initial concentration of the binder in the initial solution provided for the first compartmentalization, and most importantly, by taking into account the (apparent) binding characteristics of the pre-determined binder, provides the exact number of molecules.

[0035] Preferably, the two-component detection method determines the number and / or absolute concentration of the binder in addition to the number and / or absolute concentration of the complex in the second partition in step h. Thus, the two-component detection method is also applied to determine the number and / or absolute concentration of the binder in the first partition (which is divided into a plurality of second partitions containing the contents).

[0036] In summary, the present invention surprisingly facilitates a highly reliable and accurate quantitative assessment of the absolute concentrations (amounts) of different types of binder-target (target analyte) in a single cell.

[0037] The present invention achieves a surprising effect preferably by two major improvements to the art: (i) preferably, considering the labeling ratio of the binder, and / or the labeling synthesis error that renders the label undetectable, and / or the active fraction of the binder having binding ability, and contamination by free label, the apparent dissociation constant of the binder (e.g., an antibody) is considered, and the binding characteristics of the binder such as an antibody are determined using a target material of unknown quantity.

[0038] Subsequently, based on these measurements, it is preferable to determine the apparent dissociation constant of the binder, such as an antibody. Thereafter, (ii) double compartmentalization of single cell components follows, enabling an absolute quantitative correspondence between the conditions of the first compartmentalization of the single cell during the binding incubation of the binder and the conditions of the second compartmentalization of the single cell components (e.g., the complex formed by the binder and the target molecule), thereby making it possible to determine the very small amount (up to 10 target analytes per single cell) and the absolute amount of the target analyte in each single cell by considering the previously determined binding characteristics of the binder.

[0039] In an embodiment, the binding characteristics of the active fraction and the contamination by the free label are inherently compensated for by the second compartmentalization method, which represents a surprising finding and feature of the method.

[0040] Thus, in a preferred embodiment of the method according to the invention, determining the binding characteristics in step b. comprises determining, for each of the first and second types of binder, the dissociation constant, the labeling ratio and the labeling synthesis error from the (respective) target analyte.

[0041] In other words, in a preferred embodiment of step b., determining the binding characteristics comprises determining the (apparent) dissociation constant of each of the first and second types of binder, taking into account the labeling ratio and the labeling synthesis error of the binder(s), with the active fraction and the free label being self-compensating.

[0042] In a preferred embodiment, the method determines the apparent dissociation constant of the binder(s) using the labeling ratio and the labeling synthesis error of the binder(s), thereby providing a self-compensation inherently advantageous for contamination by the active fraction and the free label of the binder(s) in order to provide the dissociation constant of the antibody for a bias-free determination of the absolute amount in a single cell.

[0043] In an embodiment, determining the binding characteristics in step b. comprises determining, for each of the first and second types of binder, the dissociation constant from the target analyte required for binding to the target analyte, the labeling ratio, the labeling error rate, the activity ratio and / or the optimal concentration (number of molecules per unit volume).

[0044] In an embodiment, determining the binding characteristics in step b. comprises determining the dissociation constant from the target analyte for each of the first and second types of binder.

[0045] In an embodiment, determining the binding characteristics in step b. comprises determining the labeling ratio for each of the first and second types of binder.

[0046] In an embodiment, determining the binding characteristics in step b. includes determining the labeling error rate for each of the first and second types of binders. In an embodiment, determining the binding characteristics in step b. includes determining the labeling ratio and / or the labeling error (labeling error, labeling synthesis error (LE)) rate for each of the first and second types of binders.

[0047] In an embodiment, determining the binding characteristics in step b. includes determining the contamination by free label (FL) for each of the first and second types of binders.

[0048] In an embodiment, determining the binding characteristics in step b. includes determining the labeling error rate for each of the first and second types of binders.

[0049] In an embodiment, determining the binding characteristics in step b. includes determining the labeling ratio, the labeling error (labeling error; labeling synthesis error (LE)) ratio, and / or the contamination by free label (FL) for each of the first and second types of binders.

[0050] In an embodiment, not only determining the determination efficiency and accuracy of the labeling of the binder (i.e., the "labeling ratio" and the "labeling error" rate), but also in an embodiment, the fact that the binder solution is contaminated with free label (after labeling of the binder) facilitates the correction of labeling-related errors (such as reading of the labeled sequence) from the final result. For example, if the binder does not contain a label (labeling ratio), or if the label contains a sequence error, and / or if the binder contains the wrong label, the binder cannot be detected and / or identified during subsequent data analysis. Contamination of the sample by free label can lead to false positive detection of the complex. Therefore, a complex containing a mislabeled or unlabeled binder cannot be identified in subsequent data analysis. Therefore, by pre-determining the efficiency and accuracy of the labeling of the binder, it becomes easier to correct the above errors later, and the accuracy of determining / calculating the concentration of the target analyte in the sample can be further improved.

[0051] In an embodiment, determining the binding characteristics in step b. includes determining the activity ratio for each of the first and second types of binders.

[0052] In an embodiment, determining the binding characteristics in step b. includes determining the optimal concentration (number of molecules per unit volume) required for binding to the target analyte for each of the first and second types of binders.

[0053] In an embodiment, determining the binding characteristics in step b. includes determining the dissociation constant from the target analyte, the labeling ratio, the labeling error rate, and / or the activity ratio for each of the first and second types of binders.

[0054] In an embodiment, determining the binding characteristics in step b. includes determining the labeling ratio, the labeling error rate, and / or the activity ratio for each of the first and second types of binders.

[0055] In an embodiment, the binding characteristics of the binders preferably include their (re)activity, specificity, concentration, labeling ratio (LR), their labeling synthesis error (LE), the active fraction (AF) of the binder, and / or contamination by free label (FL).

[0056] Thus, the determination of the so-called "binding characteristics" of the binders herein is an improved approach for optimizing the absolute quantification of the target analyte concentration in a sample. By measuring and reducing the "binding characteristics" associated with the binders, the method surprisingly enables the identification of specific conditions where, preferably, the influence of some of these characteristics becomes negligible in absolute quantification. Specifically, in an embodiment, the present invention focuses on establishing saturation conditions characterized by high concentrations of binders such that the influence of reactivity in the determination of absolute quantification can be ignored. Applying the quantification method under these optimized saturation conditions ensures accurate and reliable quantification of the target analyte concentration.

[0057] Therefore, the determination of the so-called "binding characteristics" of the binder herein is an improved approach for optimizing the absolute quantification of the target analyte concentration in a sample. This can identify additional confounding factors in the calculation of the absolute quantification of the target analyte, such as unlabeled or mislabeled binders, and / or the dissociation constant between the binder and its target analyte, which can be considered during the final calculation of the target concentration.

[0058] In a preferred embodiment, step h. of performing the two-component detection method involves thereby determining the number and / or absolute concentration of the complexes and binders within the second compartment.

[0059] The binding characteristics of the binder may also include the reactivity, specificity, and / or optimal concentration (number of molecules per unit volume) required for binding to the target analyte. Reactivity is described by the dissociation constant of the binder, specificity is specifically described by identifying the binding target, and concentration is described by the number of molecules per unit volume. WO 2020 / 260277 describes, for reference herein, a method for determining the dissociation constant of a binder using the described binding characteristics by applying chemical equilibrium.

[0060] The present invention significantly improves the method of WO 2020 / 260277 and extends it to be usable for high-precision and high-sensitivity single-cell measurements, including additional characteristics such as the labeling ratio of antibodies, inability to detect labels due to labeling synthesis errors, and / or the active fraction of antibodies with binding ability, and contamination by free labels. The method then facilitates the determination of the apparent dissociation constant of a binder, such as an antibody, based on these measurements, and provides accurate and sensitive detection of the target analyte (up to about 10 target analytes per single cell) in conjunction with the dual compartmentalization of single-cell components as described.

[0061] In this context, the labeling ratio (LR) of a binder represents the ratio of labeled to unlabeled binder carrying a portion of the binder. The labeling synthesis error (LE) represents, for example, the fraction of undetectable labels due to, for example, sequence errors in the labels. Since binding of the binder to the target analyte cannot be detected if it contains undetectable labels, determining the labeling synthesis error or, for short, the "labeling error" may be relevant to the accuracy of embodiments of the method. The active fraction (AF) of a binder represents the ratio of the reactive fraction to the non-reactive fraction of the binder. Contamination of the binder by free label (FL) accounts for the proportion of labels that did not bind to the binder during its preparation and poses a risk of false positive results during measurement of the target analyte by the method.

[0062] The present invention describes a method for determining a novel correction value for, for example, the apparent dissociation constant of a binder, which is used to determine the absolute amount of a target analyte using the binding properties of the binder, preferably their reactivity, specificity, concentration, labeling ratio (LR), their labeling synthesis error (LE), the active fraction (AF) of the binder, and / or contamination by free label (FL). The determined binding properties may also be referred to as extended binding properties as they extend the molecular binding properties taking into account practical limitations (such as the active fraction (AF) of the binder). In a preferred embodiment, the system involves a discontinuous enrichment of the target analyte, for example, a suspension of single cells.

[0063] In a preferred embodiment, the present invention applies the determination of the (extended) properties of the binder to enable measurement of extremely low and absolute amounts of a target in a single cell, preferably hundreds or, preferably, up to dozens of targets per single cell.

[0064] In an embodiment, the two-component detection method in step h. includes nucleic acid amplification or a nucleic acid amplification step.

[0065] In an embodiment, the first type of binder and the second type of binder each contain a nucleic acid label that is preferably unique to that type of binder, and the two-component detection method in step h. includes amplifying the nucleic acid sequence label of the binder, preferably inside each of the second partitions.

[0066] In an embodiment, the amplification includes ligation of the nucleic acid sequences of the nucleic acid labels of two or more binders that form a complex, and preferably, the ligation can be the synthesis of a nucleic acid sequence complementary to the single-stranded nucleic acid label of the binder. In such an embodiment, the labels themselves are not ligated, but their nucleic acid sequences are ligated in the nucleic acid amplification step. Thus, in an embodiment, the ligation (inside each second partition) of the nucleic acid sequence label of the first type of binder to the nucleic acid sequence label of the second type of binder can be understood to be compatible / synonymous with the ligation / combination of the nucleic acid sequences (sequences of nucleotides) of the above labels. The result of the above ligation and / or amplification step is preferably a nucleic acid sequence that includes the nucleic acid sequences of the labels of at least two binders that form a complex so as to be able to determine the identity of the binder and thus the identity of the target analyte in the complex.

[0067] In an embodiment, the two-component detection method in step h. includes combining (pooling) a plurality of second partitions.

[0068] In an embodiment of the method, the two-component detection method in step h. includes performing nucleic acid sequence analysis.

[0069] In an embodiment of the method according to the invention, the two-component detection method of step h. comprises performing a nucleic acid sequence analysis, such as nucleic acid sequencing or PCR analysis, thereby determining the number and / or identity of the 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 the nucleic acid sequence labels (of the binding agent). In an embodiment, a nucleic acid sequence analysis is performed, thereby determining the number and / or identity of the nucleic acid sequence labels (of the binding agent), and subsequently, preferably from the number and / or identity of the unique nucleic acid sequence labels, the number and / or concentration of the complexes is calculated.

[0070] In an exemplary embodiment, two different binding agents, each containing a unique nucleic acid label that is also characteristic of each type of binding agent, bind to the target analyte (forming a complex) and are compartmentalized (separated) into a second compartment. In this case, the two unique sequences of the nucleic acid labels may be ligated / combined by a nucleic acid amplification step. Since the resulting sequence contains the ligated / combined nucleic acid sequences of the two labels, nucleic acid sequence analysis can be used to determine the identity of the two different binding agents (based on the ligated label sequences), and thus the identity of the target analyte forming the complex. Since the labels preferably contain unique sequences, for example, duplications that occur during amplification and / or sequence analysis can be discarded / ignored during the final analysis. Furthermore, in an embodiment, the identity and / or number of the binding agents forming a complex with one or more target analytes can be normalized by the initial concentration of the binding agent and / or one or more binding characteristics of the binding agent (such as label ratio, label synthesis error, dissociation constant from the target analyte, etc.) in the binding reaction (step d.), and is included in the calculation of the normalization process described in detail herein.

[0071] In an embodiment of the present method, the absolute concentration of the first target analyte per cell in the first partition is calculated from the initial concentration of the cells in the sample (adjusted / determined in step a.), the binding characteristics determined in step b., and the number and / or concentration of the complex and the binding agent in step h. in the second partition, and thus the number and / or concentration of the complex.

[0072] 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 label specific to the type of binding agent, and the two-component detection method in step h. amplifying the nucleic acid sequence label inside each of the second partitions; combining a plurality of second partitions to perform nucleic acid sequence analysis in order to determine the number and identity of the nucleic acid sequence labels, and thereby determine the number and / or concentration of the complex; and includes.

[0073] In an embodiment of the present method, the two-component detection method in step h. includes performing a step of ligating the label of the first type of binding agent and the label of the second type of binding agent for each second partition, and this ligation step provides co-compartmentalization information of the two-component method.

[0074] In an embodiment of the present method, the ligation step in step h. includes ligating the nucleic acid sequence label of the first type of binding agent to the nucleic acid sequence label of the second type of binding agent inside each second partition when both form a complex with the first target analyte.

[0075] In some embodiments, the ligation step involves performing nucleic acid amplification of nucleic acid labels (e.g., PCR, or digital PCR, or ligation - chain reaction). In other embodiments, the ligation step is a classical proximity ligation reaction involving rolling - circle DNA synthesis or rolling - circle amplification. In other embodiments, ligation is achieved using a ligase enzyme. In subsequent nucleic acid sequencing analysis of the label, the presence of the complex within the partition can be demonstrated by a sequence containing both the labeled sequence of the first type of binder and the labeled sequence of the second type of binder, and both the binder specific for the target analyte and the above - mentioned sequence are associated with the co - compartmentalization information.

[0076] In any case, the ligation step varies depending on the type of label used. In some embodiments where detection is achieved using a fluorescent probe or fluorescent label, since the presence of a complex containing two types of binders bound to a target can be detected by the co - localization inside one partition or droplet of two labels containing different fluorescent markers or dyes used to detect the two different types of binders within the complex, the ligation step between nucleotide labels may not be essentially necessary.

[0077] In some embodiments, protein - or peptide - based labels may be cross - linked according to methods known in the art.

[0078] In an embodiment of the method according to the present invention, after contacting the sample in step d., during the first compartmentalization, before contacting the sample with the first type of binder and the second type of binder so that one or more of the first type of binder and the second type of binder enter one or more cells, the cells in the sample are permeabilized with respect to the first type of binder and the second type of binder (preferably to recover the antigen therefrom, in other words, the cells are "antigen - recovered").

[0079] In embodiments where the target analyte is at least partially located on the surface, or membrane, or cell wall of the cell, cell permeabilization may not be essential to contact the binding agent with the target analyte. However, in embodiments, when the target analyte is located inside the cell and the binding agent cannot interact with the target analyte, since the binding agent cannot enter the cell, permeabilization of the cells in the sample and / or permeabilization of the cells suspected of containing the target analyte may be required. Such permeabilization may also include encapsulating the binding agent in a molecular shuttle such as a liposome to enable entry into the cell. In other embodiments, permeabilization may be achieved using other methods known in the art, such as chemicals (e.g., formaldehyde, Triton, Tween, NP-40, leukoperil, digitonin, saponin, acetone, methanol, etc.), electric current or (ultra)sonication. Those skilled in the art know the appropriate means for cell permeabilization depending on the binding agent and / or cells used.

[0080] Thus, in one embodiment, the first compartmentalization preferably provides chemical conditions that facilitate detection of the target on the cell surface within the cell and / or in the nucleus.

[0081] In embodiments 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 label specific to the type of binding agent, and the plurality of first partitions in step f. are optionally further supplemented with PCR reagents and nucleic acid amplification primers optionally containing a barcode specific to each first partition and the single cell therein, and optionally RT-PCR reagents, and an additional nucleic acid amplification step is performed after cell lysis within each second partition to detect additional nucleic acid targets, particularly specific genomic loci or transcripts or specific complexes. In embodiments, the nucleic acid label of the binding agent present in the complex may be ligated and / or barcoded and / or amplified during such amplification.

[0082] After the first partition or droplet is supplemented with the lysis buffer, optionally, it is further supplemented with the first-strand cDNA synthesis reagent and nucleic acid amplification primers that optionally contain a barcode unique to each first partition and the single cell therein, and an additional first-strand cDNA synthesis step is performed after cell lysis within each first compartment.

[0083] In a preferred embodiment, during the above 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, and the unique barcode or UMI is preferably included in the sequence of the amplification primer. According to the above barcode or UMI, a complex single-cell specific analysis can be performed during nucleic acid sequencing analysis.

[0084] In an embodiment of the method according to the present invention, the plurality of first partitions in step f. and / or preferably step e. are further supplemented with a protein cross-linking agent and / or a protease inhibitor and / or a nuclease inhibitor.

[0085] In an embodiment of the method according to the present invention, the first type of binder and the second type of binder each contain a nucleic acid label unique to the type of binder, and each nucleic acid sequence label includes an identifier sequence (barcode) that identifies the reactivity of the binder and its molecular identity and a unique molecular identifier (UMI).

[0086] The determination of the accurate / absolute molecular concentration of the target analyte by this method can be further improved in embodiments when the label of the binder is not only specific to the type of binder but also unique for each individual binder molecule. By using a label unique to each individual binder molecule, when the above unique label is linked to another unique label of the second binder within the complex, the determination of the total number of individual complexes by subsequent nucleic acid sequence analysis becomes easier.

[0087] In an embodiment of the method according to the invention, the first type of binder and the second type of binder each contain a nucleic acid label specific to the type of binder, and the amplification in step h. includes the performance of PCR and / or droplet PCR and / or digital PCR and / or real-time PCR and / or RT-PCR.

[0088] In an embodiment of the method, two or more target analytes and corresponding first and second types of binders are provided, and finally, their absolute concentrations are determined according to the method of the invention.

[0089] In an embodiment of the method according to the invention, at least one target analyte includes at least first and second target analytes, and in step a., for each of at least the first and second target analytes, at least a first type of specific binder and a second type of specific binder are provided.

[0090] In an embodiment, step d. of contacting a sample with a first type of binder and a second type of binder is carried out under suitable conditions, and the first type of binder and the second type of binder bind to a first target analyte to form an analyte-binder complex. The phrase "under suitable conditions" refers to any conditions described herein suitable for contacting the first type of binder and the second type of binder with the sample, and the first type of binder and the second type of binder bind to the first target analyte to form an analyte-binder complex. In an embodiment, the suitable conditions may be conditions that include or enable "permeabilization" of the cells. In an embodiment, the preferred conditions may include specific "antigen recovery" conditions such that one or more antigens are recovered from the cells. In an embodiment, the conditions of step d. are intended to enable the first type of binder and the second type of binder to contact the first target analyte to form an analyte-binder complex. Preferably, the above conditions do not necessarily lyse the cells (release all of their contents), and in an embodiment, for example, the binder(s) are allowed to enter the cells (if present) and interact with the contents (analytes) to permeabilize the cells and / or enable contact between the cell contents and the binder(s). In other embodiments, the permeabilization of the cells prior to addition of the binder(s) may be considered a lysis step that releases the cell contents into respective first compartments and / or enables contact between the cell contents and the binder(s).

[0091] In an embodiment, a lysis and / or dispersion buffer is used or facilitates complete dispersion of the cell material prior to the subsequent compartmentalization step. In other words, in an embodiment, the conditions of step f. are intended to enable lysis of a single cell in each of the first partitions to release (disperse) all of the above cell contents into the above first partitions. In an embodiment, at least two different lysis (or lysis-like) conditions and / or buffers are applied during the method, for example, in step d. and step f. respectively.

[0092] This method also facilitates the detection of two or more target analytes simultaneously. When analyzing multiple target analytes, for each target analyte, two specific binding agents must be selected, one of the first type and the other of the second type. The distinction between different binding agent pairs for different target analytes can be achieved, for example, in some embodiments, using different fluorescent labels, primers, or probes during the final PCR, qPCR, real-time PCR, or dPCR readout. As an example, a probe that specifically binds to the ligation sequence of two binding agent labels of a first target analyte is tagged with one fluorescent color, while a probe specific to the ligated binding agent labels of a second target analyte is tagged / marked with a different fluorescent color. In other embodiments where the labels of the binding agents within the complex are not ligated, each pair of binding agents within the complex is detected by a specific combination of fluorescent colors that can be distinguished during the final PCR readout.

[0093] In embodiments of the method according to the invention, the number of different target analytes is less than 5, and when nucleic acid sequence analysis is performed in step h., the above analysis includes digital PCR and / or real-time PCR.

[0094] In embodiments where the number of target analytes to be analyzed exceeds the number of available fluorescent labels or the number of their combinations, nucleic acid sequencing (e.g., NGS) may be used to specifically analyze the abundance of the complex of the above target analytes.

[0095] In embodiments of the method according to the invention, the number of different target analytes is 5 or more, and when nucleic acid sequence analysis is performed in step h., the above analysis includes next-generation sequencing (NGS).

[0096] In some embodiments where nucleic acid sequence analysis is performed in step h including next-generation sequencing (NGS), the sequencing data is preferably processed using a custom pipeline. In an embodiment, the customized pipeline includes deconvolution of unique sequences contained within nucleic acid labels, such as UMIs. In an embodiment following the description of such deconvolution, sequences initially contained within the first or second partition, such as droplets, may be deconvolved as described in WO 2020 / 260277. Briefly, in an embodiment, deconvolution of the partition is achieved based on unique pairing between UMIs for each partition during a ligation reaction (e.g., PCR) step, and such pairing randomly constitutes UMI pairs within the partition. A unique virtual network can be constructed from the UMI pairs (of preferably only one complex-containing partition) of the ligation reaction partition (droplet) that clearly define the partition (the bound two binder labels), thereby reconstructing the droplet (partition) contents that refer to the type of binder present in the above complex / droplet. Since the distribution of binders can be determined based on the contents of the droplets, droplets (partitions) containing one type of binder and droplets containing both binders can be distinguished and counted separately. The total number of droplets used for sequence analysis (e.g., droplets pooled / combined prior to sequencing) can be directly counted or derived using concepts such as tracers. The obtained droplet information can be evaluated using the method described for dPCR readout.

[0097] Advantageously, the method also facilitates not only the absolute quantification of the target analyte concentration but also the determination of the concentrations of multiple target analytes in parallel in a robust and efficient manner. In one embodiment, the method includes adopting an analysis method based on absolute molecule numbers. Such an analysis method based on absolute molecule numbers preferably refers to applying a digital detection method such as digital PCR or NGS analysis.

[0098] In an embodiment, the method includes employing a digital PCR assay. In one embodiment, the method includes using a target analyte-specific binding agent associated with a unique amplifiable nucleic acid label, and employing a compartmentalized assay, in which nucleic acid amplification is performed for each partition, preferably using a fluorescence-labeled amplification product or a fluorescence-labeled probe that binds to the amplification product of interest.

[0099] In some embodiments, the nucleic acid amplification is PCR and the fluorescence-labeled amplification product is a fluorescence-labeled PCR product. In some embodiments, the nucleic acid amplification is PCR and the amplification product of interest (label of the binding agent forming the complex) is detected by a fluorescence-labeled probe such as a hydrolysis probe or a Taqman probe.

[0100] The analyte-specific binding agent may preferably be labeled with a unique PCR-amplifiable DNA label, and two analyte-specific binding agents, for example, two antibodies, are preferably labeled with single-stranded DNA unique to the binding agent or the type of binding agent (e.g., the first type of antibody). In an embodiment, the binding characteristics of the initially labeled binding agent (e.g., antibody) are determined and then added to the sample or to one or more diluents to enable complex formation. Thereafter, the reactants are highly diluted, for example, with a dilution factor of more than 20,000, preferably more than 50,000, more preferably more than 100,000, and emulsified into droplets using, for example, methods known in the art, so as to achieve single complex separation upon the first compartmentalization into the first partition.

[0101] In an embodiment of the method according to the present invention, the binding agent is selected from the group consisting of an antibody, an artificial protein scaffold, and an aptamer.

[0102] In an embodiment of the method according to the present invention, the target analyte is selected from the group consisting of a peptide, a protein, a chemically modified peptide or protein, a nucleic acid, a chemically modified nucleic acid, and any complex thereof.

[0103] In embodiments, not only proteins or peptides containing specific amino acid sequence motifs or epitopes of antibodies can be analyzed, but also post-translational modifications in proteins or peptides can be analyzed. For example, one type of binder may recognize chemical modifications or post-translational modifications, and a second type of binder may recognize specific epitopes or domains of a protein. This method can also be used to analyze the absolute amount of protein complexes in a single cell, in which case each type of binder recognizes one of the proteins forming the complex. However, nucleic acids or nucleic acid-protein complexes may also be recognized, for example, by antibodies specific for the complex members. For example, one type of binder may recognize a protein within a DNA-protein complex, and another type of binder may recognize, for example, a methylated DNA motif (which may be in the functional context of the interaction). Thus, the term "any complex thereof" can refer to any complex comprising proteins, peptides and / or nucleic acids, or any combination thereof, since the only requirement of the method is that a specific target analyte can be specifically recognized by a pair of two labeled binders to form a complex.

[0104] In embodiments of the method according to the invention, the sample is selected from the group comprising tissue samples, biopsy samples, liquid biopsy samples, blood samples, plasma samples, urine samples, liquor samples, environmental samples, samples derived from cell cultures, and samples derived from microbial cultures.

[0105] In embodiments of the method according to the invention, the sample is prepared in step c. after being processed 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 optically blocked controlled single cell printing.

[0106] In embodiments, the invention provides the following: a. Preparing at least a first type of binder and a second type of binder, each containing a nucleic acid sequence label specific to the type of binder, the first type of binder and the second type of binder specifically binding to a first target analyte, and optionally, the nucleic acid sequence label containing an identifier sequence (barcode) and / or a unique molecular identifier (UMI); b. Determining the binding characteristics of the first type of binder and the second type of binder to the first target analyte; c. Preparing a sample containing cells at a known concentration, optionally where the concentration includes or relates to the number of single cells per unit volume, and / or the concentration is at a known / given / determined single-cell resolution; d. Optionally, performing a permeabilization treatment on the cells in the sample with respect to the first type of binder and the second type of binder; e. Contacting the first type of binder and the second type of binder with the sample, optionally, one or more of the first type of binder and the second type of binder entering the permeabilized cells, the first type of binder and the second type of binder binding to the first target analyte to form an analyte-binder complex; f. Compartmentalizing single cells of the sample into a plurality of first partitions; g. Complementing each partition containing a single cell with a lysis buffer and lysing the single cell in each partition; h. Further compartmentalizing a single analyte-binder complex into a plurality of second partitions; i. Optionally, if both form a complex with the first target analyte, ligating the nucleic acid sequence label of the first type of binder to the nucleic acid sequence label of the second type of binder inside each partition; j. Amplifying nucleic acid sequence labels linked inside each partition and optionally detecting partitions containing both labels of the first type of binder and labels of the second type of binder thereby to determine the number of complexes; k. Combining a plurality of partitions; l. Performing nucleic acid sequence analysis to thereby determine the number and identity of the nucleic acid sequence labels linked in step i; m. Determining the absolute concentration of the initial target analyte per cell, 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 labels determined in step l; relates to a method for determining the absolute concentration of at least one target analyte in a sample comprising.

[0107] Preferably, during step g, incubation between the cell contents and the binder is still possible / still carried out, so that the binder can contact (and form complexes with) the target agent contained within the cell, for example within an organelle such as the nucleus, which was not (or was low) permeable and / or inaccessible to the binder before cell lysis.

[0108] In embodiments, it is particularly advantageous that the sample contains cells at a known concentration, the concentration comprising or relating to the number of single cells per unit volume, and / or that the method can be carried out using a single cell instead of a suspension of a large number of cells or a plurality of cells if the concentration is known / given / determined at single cell resolution.

[0109] In an embodiment of the method according to the invention, the first type of binder and the second type of binder each contain a nucleic acid label specific to the type of binder, complement each partition containing a single cell with a lysis buffer, and lyse the single cell within each partition. A plurality of first partitions in step f. / g. are optionally further complemented with PCR reagents, nucleic acid amplification primers optionally containing a barcode specific to each first partition and the single cell therein, and optionally RT-PCR reagents, and an additional nucleic acid amplification step is further performed after cell lysis within each first partition, enabling the detection of additional nucleic acid targets, particularly specific genomic loci or transcripts or specific complexes. In an embodiment, the nucleic acid label of the binder present in the complex may be ligated and / or barcoded and / or amplified during such amplification. In some embodiments, the sample solution is complemented with PCR reagents and / or reverse transcription reagents and / or RT-PCR reagents prior to the step of compartmentalizing the single cells into the first compartment.

[0110] After complementing the first partition with a lysis buffer, it is optionally further complemented with first-strand cDNA synthesis reagents and nucleic acid amplification primers containing a barcode specific to each first partition and the single cell therein, and an additional first-strand cDNA synthesis step is performed after cell lysis within each first partition.

[0111] In a preferred embodiment, during the above amplification, a barcode or UMI specific to each first partition / droplet (or specific to each encapsulated single cell) is added to each amplified nucleic acid sequence, and the unique barcode or UMI is preferably included in the sequence of the amplification primer. According to the above barcode or UMI, single-cell specific analysis of the complex can be performed during nucleic acid sequencing analysis.

[0112] In an embodiment, step l. of performing nucleic acid sequence analysis further includes determining the RNA composition of the single cell from the sequence information. Preferably, in the above embodiment, the amplification reaction in step g. / j. of amplifying the linked nucleic acid sequence label inside each partition further includes reverse transcription of RNA to 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 label in the first partition. Therefore, it is preferable that the lysis buffer further contains reverse transcription reagents or complements the first partition or the initial sample solution with reverse transcription reagents. The generated cDNA may preferably be labeled with barcodes and / or UMIs during the above reverse transcription step, and the obtained RNA sequence can be assigned to the first partition and thus to a single cell. In an embodiment, it is preferable that the identity / sequence of the RNA present in each single cell can be obtained by determining the nucleic acid sequence of the corresponding cDNA during the final nucleic acid sequence analysis.

[0113] Therefore, in an embodiment of the method of the present invention, the sample contains RNA, the plurality of first partitions are further complemented with reverse transcription reagents in step f. / g., RNA is simultaneously converted to cDNA during any one or all of steps e. / d., the first and second types of binders are contacted with the sample until lysis of the cell in step g., and when nucleic acid sequence analysis is performed in step h. / l., the analysis includes next-generation sequencing (NGS) or qPCR analysis of the cDNA generated in steps e. / d. - g.

[0114] In an embodiment, the method is advantageous for determining the absolute amount of a target analyte using the method of the present invention that forms physically separated first compartments. In an embodiment of determining the absolute amount of a target analyte, the first compartmentalization is performed on a first partition. Here, the compartmentalization is physical, and the binding reaction between the cell and the binding agent is preferably carried out at a high target analyte concentration exerted by a small volume encompassing each single cell. Preferably, the cell is lysed during the first compartmentalization. In an embodiment, each first compartment is compartmentalized a second time, and a single target analyte molecule is counted using a suitable sensitive method (e.g., nucleic acid sequence analysis). Preferably, the concentration of the target in the first compartment (preferably containing a known volume and one cell) is determined using the binding characteristics of the binding agent, and ultimately, as explained in this example, the absolute amount / number of target analyte per single cell will be determined. In an embodiment, the method prepares a single cell sample, incubates the sample with two target-specific binding agents, forms a complex of the target and at least two binding agents, performs compartmentalization into a plurality of first partitions of the single cell sample (each preferably containing one cell or no cells at all), and complements each first partition with a cell lysis buffer. In the "compartmentalized two-component method" (second partition), the composition of the complex contained in the first partition is determined.

[0115] In an embodiment, a binder (e.g., an antibody) is conjugated with a DNA-amplifiable label. The achieved labeling efficiency of the binder can be determined in the embodiment and can be confirmed, for example, by SDS-PAGE electrophoresis in the embodiment. In the embodiment, free (unbound) labels and labeling errors may be determined (see, for example, Example 5). In the embodiment, it is preferable that the binder (e.g., an antibody) does not have overlapping epitopes and the conjugated binder has two different labels. In the embodiment, the dissociation constant of the binder can be determined, for example, as described herein or according to Example 6. In the embodiment, cells are encapsulated using droplet generation technology (e.g., a microfluidic platform) and have a labeled binder in a first partition. The resulting emulsion can be collected and incubated in the embodiment. In the embodiment, droplet dispensing may be performed at an adjusted droplet concentration, for example, delivering a single first compartment to a microplate well. In the embodiment, it is preferable to add dPCR reagents to the above compartment, dispense one droplet into a well, and load it onto another well plate after mixing. It is also preferable to analyze a negative control containing only the labeled binder (binding control). In the embodiment, dPCR may be performed. In the embodiment, the number of complexes and the number of binders can be obtained as described herein (e.g., as in Example 6). In the embodiment, the number of antibodies (copy number) expected on average for each reaction (correcting for antibody labeling ratio and labeling error), and the detected complexes (correcting for antibody labeling ratio and labeling error) can be determined. In an embodiment based on predetermined binding characteristics including the concentration of the binder and the Kd of the binder (see Example 6 for the apparent kd), a calibration curve can be constructed. In the embodiment, the calculated target concentration may be determined (on average) for each cell.

[0116] In an embodiment, the step of determining binding characteristics is for each of a first type and a second type of binder, the dissociation constant from the target analyte, and / or, the specificity of binding to the target analyte, and / or, labeling ratio, and / or, labeling synthesis error, and / or, active fraction, and / or, contamination by free label, and / or, the optimal concentration (number of molecules per unit volume) required for binding to the target analyte, including determining.

[0117] As described herein, the number of complexes can be determined by analyzing the co-localization of the label of the first type of binder and the label of the second type of binder within one partition or emulsion droplet using nucleic acid amplification, such as PCR or dPCR, such as a probe or primer labeled with a fluorescent dye, and / or alternatively next-generation sequencing (NGS).

[0118] In one embodiment, the present invention relates to a method for processing a sample, the method comprising: A. preparing an antibody having a uniquely identifiable and amplifiable label, the antibody being two for each target analyte (i.e., the first type and the second type of binder), the label comprising an identifier sequence (barcode) and optionally a unique molecular identifier (UMI); B. determining the binding characteristics of the antibody, including determining the labeling ratio, labeling synthesis error, and also the active fraction of the antibody, and contamination by free label, and determining an apparent dissociation constant based on these measurements; C. preparing a sample containing cells at a certain concentration containing a target analyte such as DNA, RNA, protein, epitope protein, or interacting protein; D. optionally, permeabilizing the cells to make the cells permeable to macromolecules (especially antibodies); E. combining an antibody having the determined binding characteristics with the sample to form a binding reaction between the antibody and the cells containing the target analyte; F. performing a first compartmentalization of a plurality of first partitions of single cells; G. Combining the compartmentalized single cells with a buffer having lysis conditions; H. Further compartmentalizing the single target analyte of the target analyte into a plurality of second partitions of the formed antibody-target analyte complexes (complexes) of the individual single cells; I. Optionally, simultaneously converting RNA to cDNA between steps E and H; J. Amplifying the antibody labels and thereby ligating the antibody labels of two antibodies forming a complex to the target analyte and optionally to the DNA and the cDNA; K. Determining nucleic acid sequence information; L. Identifying the barcode of the antibody label 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 complex; Optionally, determining the composition of the first partition by identifying their sequence information based on the UMI of the ligated antibody labels; M. Optionally, determining the RNA composition of the single cell from the sequence information; N. Determining the absolute amount of the target analyte in a single cell taking into account the determined binding properties of the antibody in step B; Optionally, a step of determining the chemical composition of the first partition based on the ligated antibody labels identified in the second partition in step L, wherein the determined chemical composition preferably includes the amount, concentration, or presence of the target analyte in the first partition; Including.

[0119] Preferably, during step G, incubation between the cell contents and the binding agent is still possible / still carried out, so that the binding agent can contact (and form a complex with) the target agent contained in the cell, such as an organelle such as the nucleus, which was not permeable (or had low permeability) and / or inaccessible to the binding agent before cell lysis.

[0120] In some embodiments, step K of identifying array information involves collecting array information of ligated antibody labels using a compartmentalized two-component assay.

[0121] In one embodiment, the present invention relates to a method as described herein, wherein the step of combining the compartmentalized single cell with a buffer having lysis conditions exposes the single cell to lysis conditions together with a cell-specifically distinguishable barcoded amplification primer to amplify the ligated label, and optionally DNA and cDNA, during a second compartmentalization such that a distinguishable barcode of the single cell is included. In other words, the single cell is exposed to lysis conditions together with a cell-specifically distinguishable barcoded amplification primer in step G (as disclosed above), and the ligated label, and optionally DNA and cDNA, are amplified during a second compartmentalization such that a single cell distinguishable barcode is included in step J (as disclosed above).

[0122] In one embodiment, the present invention relates to a method of applying a chemical treatment comprising an inhibitor that prevents protein cross-linking to stabilize protein interactions, sample or degradation of the labeled antibody prior to the lysis conditions applied to the single cell in the step of combining the compartmentalized single cell with a buffer having lysis conditions.

[0123] In one embodiment, the present invention is applied in the step of combining an antibody with a sample, the number of antibodies applied being advantageously less than 10, and the method for determining the sequence information of the nucleic acid label of the binder is the same compartmentalized PCR two-component method described in International Publication No. 2016 / 083793, and this method is applied from the step of second compartmentalization to the step of arbitrarily determining the RNA composition of the single cell from the sequence information. In other words, prior to the application of the lysis conditions to the single cell as disclosed above in step G., a chemical treatment is applied that includes an inhibitor that prevents protein cross-linking stabilization of protein interactions, degradation of the sample or the labeled antibody.

[0124] In one embodiment, the present invention relates to a method in which the number of the antibodies in step E is advantageously more than 10, and the method for determining the sequence information of the nucleic acid label of the binder is a compartmentalized NGS two-component method similar to that described in International Publication No. 2016 / 083793, and is applied up to steps H to M (or L). In other words, it is advantageous that the number of antibodies applied in step E (above) is less than 10, and the method for determining the sequence information of the nucleic acid label of the binder is the same compartmentalized PCR two-component method described in International Publication No. 2016 / 083793 applied from step H to step M (above).

[0125] In one embodiment, the present invention relates to a method in which a plurality of the single cells are subjected to the above method, and for the plurality of single cells, information obtained from the single biomolecule, the identity and the absolute amount of the biomolecule recognizable by the antibody are arbitrarily determined from the information in step M (including determining the RNA composition of the single cell from the sequence information as described above) in combination with the information on the RNA content of the single cell.

[0126] In some embodiments, the first compartmentalization preferably provides chemical conditions that enable the implementation of other detection methods including the detection of a plurality of RNA molecules. Such RNA detection methods for single cells are known in the art.

[0127] In embodiments, the target or target analyte can comprise a number of epitopes and can bind to a plurality of binders. In a preferred embodiment, all binders are detected pairwise using the methods of WO 2016 / 083793 or WO 2020 / 260277.

[0128] The method of the invention is preferably carried out under physiological conditions so that the target can be detected in its native context, i.e., under the same conditions as it exists in living cells. This provides information about the naturally occurring target.

[0129] The method preferably applies a first compartmentalization. The chemical composition of the first compartmentalization is partially unknown but can be inferred from the second compartmentalization. It is a preferred embodiment of the invention to maintain an absolute quantitative correspondence between the first compartmentalization and the second compartmentalization. The first compartmentalization is preferably a binding reaction between the binder and the target in a single cell. In embodiments of the first compartmentalization, the concentration of the target is preferably unknown. Further, in embodiments, either the concentration of the binder is unknown, or the concentration of the single cell is unknown, or the volume of the first compartmentalization is unknown. During the second compartmentalization in embodiments, the method determines the number of binders and the number of complexes corresponding to the first compartmentalization. From the numerical conditions of the method and the determined number of the first compartmentalization, the concentration of the binder and the concentration of the complex can be derived to determine the chemical composition of the first compartmentalization.

[0130] The first compartmentalization preferably provides chemical conditions, the concentration of the target in a single cell, and a binder that promotes the binding reaction. Such conditions can be provided, for example, by a larger number of many single cells or a smaller number of few single cells.

[0131] The step of bringing the binding agent into targeted contact is usually carried out in a known buffer system, for example, a buffer system that has already been used in the study of the target of the binding agent. The reaction can be carried out at room temperature or 4 °C, or other appropriate conditions. In order to obtain a reproducible signal, the optimal time, optimal temperature, and other assay conditions are determined, including the steps of binding and detection. The optimal conditions can be determined by those skilled in the art, but are optimized especially with respect to fixation, permeabilization treatment, and incubation time.

[0132] The present invention enables the detection of the absolute amount of a single-cell target at levels that were previously undetectable, down to 100 copies, 10 copies, or 0 copies of the target per single cell.

[0133] Using binding agents and targets at various concentrations (various numbers of single cells), quantitative parameters of targets such as protein-protein interactions, proteins, epitope proteins (modified / PTM proteins), etc. can be calculated. Thus, in a preferred embodiment, the method is carried out using binding agents and / or targets at various concentrations.

[0134] In another preferred embodiment, complexes are formed during the first compartmentalization.

[0135] In relation to the second compartmentalization, it is necessary to separate, i.e., isolate, the complex from other complexes before ligating the complex to a labeling sequence, i.e., a related unique nucleotide sequence. The separation is carried out by methods known in the art. For example, it can be carried out by dilution, specific binding, or separation based on physical and / or chemical properties. Preferably, the above dilution is limiting dilution. The complex is separated into a second compartment such as an emulsion droplet, a microcavity, etc., and preferably into a diffusion-limited compartment or a separation compartment as described in WO 2016 / 083793.

[0136] In a preferred embodiment, the binder and the target analyte are not immobilized and are present in solution, so that the complex is also formed in solution.

[0137] Preferably, the two analyte-specific binding components and the analyte are not immobilized and are present in solution, so that the two-component / analyte complex is also formed in solution. In some embodiments, the method includes the step of preparing a sample containing an analyte at a known concentration. In some embodiments, the method is an in-vitro method, and preferably the step of preparing the sample does not include a surgical or invasive procedure on a living human or animal body.

[0138] Preferably, the separation includes, inter alia, any one or more of solid surface binding, dilution, or phase separation, or more preferably includes preparing diffusion limitation or separated compartments / partitions. The separation limits the number of unbound binders per compartment. For example, the average number of unbound binders in the compartment is 1 or less, or the average number of unbound binders in the compartment is 1 to 3. Compartmentalization (e.g., effective separation or isolation of multiple reactions) follows a Poisson distribution. Sufficiently separating the complex before further analysis provides a situation where (optionally linked) pairs of nucleic acid labels based on co-localization of the binders are generated. By separating, the number of non-specific co-localizations of nucleic acid identity labels is reduced, enabling the identification of specific binding partners, especially when examining particularly complex protein mixtures.

[0139] As a result of the separation, it is preferred that, on average, there is one complex per compartment, providing only the linked nucleotide sequences of the binders by ligation, thereby reducing the likelihood of random ligation between other members of the binders. Such separation is suitable for counting binder / target complexes within a compartment, and their absolute amounts can be determined based on the Poisson distribution, but there are mathematical concepts to fully complement other situations regarding other final states of the separation.

[0140] On average, separation results in a single unlinked binder per compartment, and ligation provides only self-ligated nucleotide sequences, reducing the likelihood of random ligation between other members of the binder. Such separation enables the counting of binders within a compartment and the determination of their absolute amounts based on the Poisson distribution, which is well known in the art.

[0141] In embodiments where ligated nucleotide sequences are generated in compartments (partitions), amplification methods can be used. Compartmentalized amplification methods, such as emulsion polymerase chain reaction (Schuetze et al., Anal. Biochem. 2011 March 1; 410(1):155-7), are well known to those skilled in the art.

[0142] In embodiments where ligated nucleotide sequences are generated within a compartment, amplification is used to create covalent bonds between labels and / or to determine co-localization-based ligation of non-covalent labels. In embodiments, generating ligated nucleotide sequences in a compartment or partition by amplification can include ligating the UMIs of labels to create uniquely ligated sequences, or simply unique sequences (including unique combinations of two label sequences).

[0143] As demonstrated herein, in preferred embodiments, an absolute quantification method for single cell analysis is provided that can be applied, for example, as a novel single cell method in proteomics and / or interactomics and related fields, and furthermore as nucleic acid analysis (basically any target that may be recognized by a binding analyte according to the present invention), and the scope of the invention is generally known to those skilled in the art. The method can be used for single cells after various separation methods, including, for example, fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), laser capture microdissection (LCM), manual cell picking / micromanipulation, various microfluidic platforms, and on-demand optically blocked controlled single cell printing (A. Gross, J. Schoendube, 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), enabling highly sensitive, absolute quantitative, and parallel proteomics analysis of single cell targets. The methods of the present invention can also be combined to detect cellular mRNA. The methods described herein enable a broader detection of different molecular species within single cells, thus allowing for a better understanding of cell-to-cell heterogeneity.

[0144] The method is also useful for generating functional quantitative information regarding cell function, which may be useful for determining disease states or species between different cell types or any cellular objects having some functional differences.

[0145] The novelty of the present invention, in a preferred embodiment, enables the characterization of a binder and then applying the determined characteristics of the binder to analyze single cells previously contacted with the binder in a suspension to detect the target of the binder. In a cell suspension, both the advective transport rate and the diffusive transport rate affect binding in the dispersion solution. In a cell suspension, the effector concentration of the target can be increased to the level of saturated binder binding, demonstrating the saturation from the characterization of the binder, and the absolute amount can be derived. Preferably, the binding of the binder that is less than the saturation level can also be used together with the characterization of the binder to derive the absolute amount. Thus, the method described herein is also suitable for generating quantitative proteomics and / or interactomics, or even genomic and / or transcriptomics data corresponding to any binder-recognizable target of interest, and is not limited to very abundant target analytes such as highly expressed proteins or RNAs.

[0146] The novelty of the present invention lies, for example, in a novel quantitative assessment of antibody targets in single cells. In a preferred embodiment, the present invention provides two major improvements over the art, namely, (i) preferably, using cell target material of unknown quantity, determining the binding characteristics of a labeled binder (e.g., an antibody) including the labeling ratio of the binder, labeling synthesis errors that render the label undetectable, and the active fraction of the binder having binding ability, and contamination by free label, and based on these measurements, determining the apparent dissociation constant of the binder to provide accurate and sensitive zero-target measurements of the target in single cells, and (ii) performing dual compartmentalization of single cells, the first compartmentalization of single cells during binding incubation and the second compartmentalization of single cells using the determined binding characteristics of the binder to determine the chemical composition (e.g., the presence or absence of the target) of the first compartmentalization to enable an absolute quantitative correspondence between the conditions for determining the absolute amount of the target of the binder, which is very low, on the order of hundreds or dozens of copies of the target per single cell.

[0147] Compartmentalization enables a large number of separated and uniform conditions to be achieved.

[0148] A preferred embodiment of the first compartmentalization (to the first partition) includes a suspension of single cells, the compartmentalization is diffusion-limited, and the cells are each in their own concentrated environment.

[0149] Also, the compartmentalization preferably achieves an overall high concentration (enrichment) of the target by isolating single cells such that individual single cells exhibit locally different concentrations.

[0150] In another preferred embodiment of the first compartmentalization (to the first partition), the compartmentalization is physically performed, advantageously containing a small number of several cells or single cells. Preferably, the compartmentalization can increase the overall concentration of the target by preferably encapsulating single cells in small amounts, and such volume is preferably within the nanoliter range or picoliter range. Methods for encapsulating cells in such low-volume compartments are known in the art, including, for example, droplet microfluidics, microwells, and hydrodynamic trapping (Murphy, T. W., Zhang, Q., Naler, L. B., Ma, S. & Lu, C. Recent advances in the use of microfluidic technologies for single cell analysis. Analyst 143, 60 (2018)).

[0151] In a preferred embodiment, after exposing single cells to a labeling binder (e.g., an antibody or an aptamer), the separation of single cells is maintained during the first compartmentalization (to the first partition) such that the first compartmentalized single cells can be analyzed separately.

[0152] The second compartmentalization (to the second partition) facilitates the determination of the chemical composition of each single cell (preferably the concentration of the binding agent and the complex herein) partitioned during the first compartmentalization by applying the (apparent) binding characteristics of the binding agent thereafter so that the absolute amount / concentration of the target(s) can be derived.

[0153] For example, as described in Example 1, using such a method, targets of less than 100 copies or less than 10 copies per cell can be detected. The method is also useful for generating functional quantitative information regarding the function of cells, which may be useful for determining the disease state or species between emerging functional differences between cell types or any cell objects having some functional differences.

[0154] The reactivity of a binding agent such as an antibody can be expressed by the on-rate and off-rate of the binding agent and its derivative, and is also known as the "dissociation constant" in the art. Reactivity is generally regarded as a constant related to the chemical properties of the binding agent (e.g., antibody), but 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. Also, the structure of the epitope and conformational changes also change the dissociation constant of the binding agent (e.g., antibody), and since the epitope is also part of a macromolecule in most cases, the epitope is also affected by the same chemical environment. There are methods to compensate for these effects by determining, for example, the dissociation constant of an antibody in a given chemical environment, including NMR spectroscopy, equilibrium dialysis, dynamic light scattering, analytical ultracentrifugation, ultrafiltration, electrophoresis, differential scanning calorimetry, homogeneous time-resolved fluorescence, fluorescence correlation spectroscopy / fluorescence cross-correlation spectroscopy, spectroscopic assays, affinity capillary electrophoresis, biolayer interferometry, dual-polarization interferometry, static light scattering, and microscale thermophoresis (Wilkinson KD. 2004 Quantitative analysis of protein-protein interactions. Methods Mol. Biol. 261, 15 - 32. doi:10.1385 / 1 - 59259 - 762 - 9:015).

[0155] In embodiments for determining the dissociation constant of a binding agent such as an antibody, the compartmentalized two-component method is advantageously applied to derive the absolute amount of the formed complex (or ternary complex), facilitating the target external standard-free determination of the dissociation constant of the antibody in a complex chemical environment and also in a native environment. One embodiment is described in Example 3. The method for determining the dissociation constant of the binding agent(s) is affected by additional factors including, for example, as shown in Example 4, the labeling ratio of the binding agent(s), labeling synthesis errors, the active fraction of the binding agent(s), and contamination by free label. In such embodiments, in order to control and compensate for the accuracy of the above-described compartmentalized two-component method for determining the dissociation constant of the binding agent(s), it is necessary to separately determine only the labeling ratio and / or labeling synthesis errors of the binding agent(s). Those skilled in the art can devise methods for determining, for example, as described in Example 5, the labeling ratio of the binding agent(s) and the labeling synthesis errors of the binding agent(s).

[0156] However, as shown in Example 4, in the determination of the dissociation constant of an antibody, advantageously, the active fraction of the binding agent(s) and contamination by free label are confused. Example 6 shows the determination of the dissociation constant of an antibody with varying labeling ratio, labeling synthesis errors, and active fraction of the binding agent(s). Preferably, the compartmentalized two-component method using the labeling ratio and labeling synthesis errors of the binding agent(s) determines the apparent dissociation constant of the above binding agent(s), thereby providing an inherently advantageous self-compensation against the active fraction of the binding agent(s) and contamination by free label and providing the dissociation constant of the antibody for a bias-free determination of the absolute amount in a single cell.

[0157] In one embodiment, preferably, both the determination of the dissociation constant of the antibody (binding agent) and the determination of the absolute amount of the target analyte in the single cell are determined using the same compartmentalized / partitioned two-component method.

[0158] In a preferred embodiment, the two analyte-specific binding components and the analyte of the two-component method are not immobilized and are present in solution, so the two-component / analyte complex is also formed in solution.

[0159] In one embodiment, the determination of the absolute amount of the target analyte is performed in a first compartmentalization where the compartmentalization is diffusion-limited, and the binding reaction between the cell and the antibody is performed at a diffusion-limited concentration, as shown in Example 1 for example. The concentration of the target protein (target analyte) of the binding reaction is preferably determined by the volume of the cell suspension containing the cells and the total target protein content of that volume. As described in Example 1, under appropriate conditions and assumptions where the volume of the cells is negligible or the cell volume is included in the entire sample volume by making the antibody permeable or the target protein / analyte permeable, the concentration of the target protein / analyte can be treated as if it were in solution. Thus, in such an embodiment, their concentration per cell can be inferred from the concentration calculated for the entire sample volume and the number of cells and binding agents present in the sample volume. By separating each single cell and using an appropriate highly sensitive method, the number of targets per cell can be determined from the value obtained by dividing the extrapolated concentration of the target in the suspension volume by the number of cells in the suspension. Preferably, the suitable sensitive method is a second compartmentalization method that can be used for counting single molecules. Preferably, the cells are lysed before the second compartmentalization. Preferably, the extrapolated concentration of the target in the suspension volume is determined using binding characteristics, and ultimately, as described in Example 1, the absolute amount of the target analyte per single cell is determined.

[0160] The present invention discloses a method for determining the absolute amount of a target analyte per single cell using a compartmentalization approach. In one embodiment, the binding reaction between cells and antibodies is carried out in a first compartmentalization. The concentration of the target protein (target analyte) in the binding reaction is determined by the volume of the compartment and the total content of the target protein within that volume. By considering a negligible amount of cells or making them permeable to antibodies or target protein / analyte by lysis, the concentration of the target protein / analyte can be treated as if it were in solution. By combining single-cell isolation with a highly sensitive second compartmentalization method capable of counting single molecules, the number of targets per cell can be determined by extrapolating the target concentration in the first partitioning volume. The method utilizes lysed cells in the first compartmentalization, exploits binding characteristics to extrapolate the target concentration, thereby ultimately enabling the absolute quantification of the amount of target analyte per single cell as described in Example 2.

[0161] In one embodiment, the determination of the absolute amount of a target analyte (e.g., a biomolecule recognizable by an antibody) is carried out in a first compartmentalization, the compartmentalization is physical, and the binding reaction between cells and the binding agent is preferably carried out at a high target analyte concentration exerted by a small volume encompassing each single cell. Preferably, the cells are lysed during the first compartmentalization. Each first compartment is re-compartmentalized and a suitable highly sensitive method is used to count single target analyte molecules. Preferably, the concentration of the target in the first compartment is determined using binding characteristics, and ultimately the absolute amount / number of target analyte per single cell is determined, for example, as described in Example 8.

[0162] In one embodiment, a suitable highly sensitive method is a homogeneously compartmentalized two-component method without a washing step for maintaining binding conditions, similar to that described in WO 2016 / 083793.

[0163] In another embodiment, the two-component method further has the ability to perform absolute quantification with a predetermined binding property of the binder having a predetermined linked array information (of the linked label array).

[0164] In one embodiment, a suitable sensitive method is a compartmentalized two-component method similar to that described in WO 1999 / 043855. Such a method uses the sequence information of nucleic acid labels of two binders of the complex (herein referred to as (two) components) to detect the absolute amount of protein per individual single cell. Such a 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 complex can be determined for each single cell from the solution-based partitioning data of the second compartmentalization.

[0165] In one embodiment, a suitable sensitive method is a compartmentalized two-component method similar to that described in WO 2016 / 083793, and for the readout, a dPCR-based method using ligation information based on co-compartmentalization is applied.

[0166] In one embodiment, a suitable sensitive method is a compartmentalized two-component method similar to that described in WO 2016 / 083793, and for the readout, an NGS sequencing-based method is applied. In such a method, the binding information based on the co-compartmentalization of the nucleic acid label of the binder is based on the determination of the linked unique molecular identifier (UMI), see also Example 10 of the present invention.

[0167] In one embodiment, a suitable high-sensitivity method is a compartmentalized two-component method that applies unique molecular identifiers (UMIs), where the UMI sequences include 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. The UMI sequences are described in the art, such as 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 molecule incorporating the UMI can be encoded and there is information content in the encoded information. The UMI sequences are random sequences that can be added to other sequences.

[0168] In a preferred embodiment, determining the absolute amount of the antibody-recognizable biomolecule of the single cell is combined with a method of measuring multiple RNA molecules of the single cell as in Example 7.

[0169] In one embodiment, a suitable high-sensitivity method for detecting the absolute amount of protein per individual single cell is combined with RNA detection. Methods of RNA analysis are known in the art and preferably include using nucleotide sequences that can hybridize to nucleic acids, i.e., nucleotide sequences designed to hybridize to the poly-A tail of mRNA, such as a poly-T sequence. In some embodiments, the first-strand synthesis is primed with an (anchor) oligo-dT primer (or optionally a randomer or a combination of the two) to which a UMI, an amplification primer binding site, and optionally a template-switching primer sequence are added.

[0170] In embodiments of the present invention, preferably, nucleic acid sequence barcodes can be used to identify labels, molecules, or binding agents derived from a single cell. The barcode can be identified during analysis of nuclear sequence information. The unique barcode sequence can also be used to associate data with individual cells.

[0171] In addition to, or instead of, this, the processed RNA may also contain UMI. Thus, the sequence information of the nucleic acid label of the binding agent may include UMI that may be ligated, and / or the processed RNA may contain at least one of a barcode and UMI. Thus, in some embodiments, UMI is added to oligo dT or random hexamer priming. Also, in some embodiments, the template switching oligo also contains UMI.

[0172] In one embodiment of the present invention, the sample is preferably a single cell or an isolated nucleus. The present invention is not limited to a specific cell type. The cell is preferably a nucleated cell. The cell is preferably a mammalian cell, preferably a human cell.

[0173] There are many variations of single cell cDNA synthesis in the literature and are well known to those skilled in the art and can be applied in the present invention.

[0174] In embodiments where the detection of a protein, protein interaction and / or post-translationally modified protein according to the present invention is combined with RNA detection, the readout / analysis is provided by Roche, Illumina, and Applied Biosystems, or is also referred to as third generation sequencing as described by David J Munroe and Timothy J R Harris in Nature Biotechnology 28, 426-428 (2010) in the art, or is preferably performed by next generation sequencing (NSG) such as that provided by Pacific Biosciences and Oxford Nanopore Technologies.

[0175] In one embodiment, the method of the present invention further comprises depleting ribosomal RNA (rRNA).

[0176] One of ordinary skill in the art will readily appreciate that there may be additional or alternative methods for amplifying the resulting cDNA, and that the methods applicable here are not limited to polymerase chain reaction (PCR).

[0177] In different steps of the method embodiments described herein, one or more adapters linked to the sequence may be present. In addition to, or instead of, this, additional nucleotide sequences may be added during the reverse transcription and / or amplification steps by incorporating these sequences into the primers used for each of the reverse transcription and / or amplification.

[0178] In one embodiment, the method of the present invention further comprises the step of adding a single cell-specific or sequencing method-specific adapter or barcode.

[0179] In one embodiment, one or more universal sequences flank the sequences obtained during the read by the method of the present invention, for example, the ligated sequences and / or cDNA molecules obtained by the method of the present invention. Universal sequences that may be present in different members of a plurality of nucleic acid molecules may enable the replication or amplification of a plurality of different sequences using a single universal primer complementary to the universal sequence. In embodiments, the universal sequence may comprise a binding site for a sequencing primer, preferably a binding site for an NGS or deep sequencing primer.

[0180] For any embodiment of the method according to the present invention, the embodiments and features of the present invention described in connection therewith are considered to be disclosed with respect to any other aspect of the disclosure such that features characterizing one embodiment of the method may be employed to characterize other suitable embodiments of the method. The various aspects of the present invention are unified by, and benefit from, are based on, and / or are related to, the general and surprising discovery that the method enables absolute quantification of a target analyte at the single cell level.

[0181] Detailed Description The present invention provides a method for quantitative assessment of binder targets in single cells. The present invention preferably incorporates two major improvements over the art: (i) determining the binding characteristics of a binder (e.g., an antibody) using a large amount of solubilized target material, which includes considering the labeling ratio of the binder, the inability to detect the label due to labeling synthesis errors, as well as the active fraction of the binder with binding ability and / or contamination by free label, and determining the apparent dissociation constant of the binder based on these measurements, thereby enabling accurate and sensitive (up to dozens of targets per single cell) measurement of the target analyte, and (ii) applying double compartmentalization of 1. single cells and then 2. their contents, which allows for an absolute quantitative correspondence between the conditions of the first compartment (first partition) of the single cell during the binding incubation of the binder and the conditions of the second compartment (second partition) of the single cell contents, and at the same time determining the chemical composition of the binder-binding reactant and using the determined binding characteristics of the binder to determine the absolute amount of the target analyte even in very small amounts.

[0182] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One of ordinary skill in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. Indeed, the present invention is not limited to the methods and materials described.

[0183] The practice of conventional techniques in cell culture, biochemistry, molecular biology, recombinant DNA, computational biology, bioinformatics, genomics, proteomics, interactomics, sequencing, and related fields is well known to those of ordinary skill in the art and is described, for example, in the following references: (Sambrook et al. Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates, and the series Methods in Enzymology, Academic Press, San Diego.).

[0184] The use of singular terms includes plural references unless the context specifically dictates otherwise. Thus, for example, reference to "a single cell" includes, as appropriate, cases of one, two or more cells, etc.

[0185] As used herein, the term "ligating" is used to describe and explain any useful ligation method and provides ligated co-compartmentalized information obtained for the labeling of a binder in the above two-component method and for the ligation of the unique nucleotide sequence of the binder. The term "ligating" is used broadly and describes physical and non-physical ligation methods and is the same as the terms "ligation information" or "ligated sequence information".

[0186] As used herein, the term "about" is used to account for small variations. For example, it may refer to less than or equal to 10 (such as less than or equal to 1 in appropriate cases), or it may refer to greater than or equal to 10 (such as greater than or equal to 100 in appropriate cases). It should be understood that the range format is used for simplicity and conciseness, and should be flexibly understood to include each numerical value and sub-range, including the numerical values explicitly described as the boundaries of the range.

[0187] As used herein, "taking into account" preferably refers to including one or more parameters in a calculation. In other words, depending on each value, the coefficient or parameter to be taken into account may refer to including a specific parameter (or parameters) in each or the corresponding calculation used to determine the above value, coefficient, or parameter. In embodiments, taking into account may include an offset between parameters or values. Examples of parameters taken into account when calculating a specific value or constant are described herein and in the examples. For example, in embodiments, when calculating / determining the absolute amount / concentration of a target analyte in a sample or cell, the volume of the first compartmentalization (the first partition) and / or the initial concentration of cells in the initial solution are taken into account (used in the calculation). Or, for example, in embodiments where the labeling efficiency of a binding agent, such as an antibody, is determined, as described herein, the concentrations [ng / μl] of the light chain, heavy chain, and labeled heavy chain are taken into account (used in the calculation).

[0188] In an embodiment, the pooled droplets / second compartment can be analyzed using nucleic acid sequencing such as next-generation sequencing (NGS). The results of the NGS can be analyzed according to the procedure described in international application PCT / EP2020 / 067493, and briefly, in an embodiment, prior to sequencing (bevor), "compartmentalized" dPCR is used to amplify a binder label containing a unique molecular identifier (UMI), thereby obtaining, in the case of a complex, a nucleic acid sequence containing two linked label sequences. During NGS data analysis, in an embodiment, labeled sequences containing two or more UMIs, such as "dimerized UMIs", can be deconvoluted to reconstruct the content of each droplet (ddPCR amplification of the label is performed, preferably containing only one complex). That is, in an embodiment, the evaluation of the binding reaction may be based on the NGS reads of binder-specific "dimerized" UMI labels generated according to the standard protocol for emulsion coupling (ligating two or more binder labels within the complex on a dPCR basis). In an embodiment, the number of (labeled) binders, such as antibodies, in each droplet (preferably forming a complex) can be determined by counting all the unique UMI labels of a given binder, such as an antibody (counts limited to a given binder-specific sequence). In an embodiment, possible PCR or sequencing duplicates may be discarded during analysis using overlapping "dimerized" label sequences. In an embodiment, the complexes may be counted based on their dimerized labels (containing two or more UMIs).

[0189] In an embodiment, once the droplet content is specified, Poisson-based calculations (as described in European Patent No. 22161450.6) may be performed to determine the number of detected complexes. Briefly, since the distribution of the binder during compartmentalization is governed by the Poisson distribution, the number of occurrences of each binder is counted (by determining the relative abundance of the binder by NGS after droplet-based PCR amplification), and if the number of droplets is known, preferably the background detection of the complex can be calculated. A large number of specific binding events are used to identify the exact target agent of the binder. For example, in a multi-protein complex, the information contained in the linked labels of the binders is based on the co-localization of each binder on the complex and indicates the direct interaction between the detected target analytes. The resulting results may be further normalized in an embodiment using the background signal and / or the expected Poisson distribution of single binders and their labels.

[0190] In other words, in an embodiment, the ligation reaction of the labels of the binders contained within a single complex, for example by amplification, is itself preferably isolated within a second compartment (droplet), resulting in a ligated nucleic acid label containing the nucleic acid sequences to which the respective labels are bound. For example, if two binders bind to a target agent to form a complex, the ligated label contains the nucleic acid sequences to which both binder labels are bound. Thus, the ligated label sequence preferably includes at least two binder type-specific sequences (one from each binder) and at least two UMIs, each being unique to the binder molecule and unique among the plurality of binders used in the experiment. In an embodiment, the nucleic acid sequence of the ligated ( "dimerized") label is identified using nucleic acid sequence analysis (e.g., using NGS). The data of the sequence analysis can then be analyzed for such sequences containing the ligated label sequence.

[0191] Thus, in embodiments using nucleic acid sequence analysis, ligated label sequences can be identified, and each unique combination of at least two labels (within one sequence including a unique combination of at least two different UMIs) indicates the formed complex. Thus, in embodiments during sequencing analysis, each unique sequence containing at least two label sequences can be counted, and it is preferable that the total number (of the unique ligated label sequences) is equal to the total number of detected / analyzed complexes. In embodiments using the binding properties described herein, the absolute concentration of the target analyte can be calculated from the number of the above complexes. In embodiments, determining the absolute concentration of the first target analyte per cell in the first partition is carried out (taking into account) by offsetting against each other the initial concentration of cells in the sample in step a., the binding properties determined in step b., and the number and / or concentration of complexes and binders in the second partition in step h.

[0192] In embodiments, sample dilution can be measured in the same sequencing reaction using sample-specific DNA barcodes (e.g., barcoded primers), and since different types of binders have distinguishable labels, in embodiments, many measurements (using different antibody pairs for different antigens) can be performed in parallel (multiplexed).

[0193] The terms used to conjoin the descriptions refer to a situation where one or more of the recited instances may occur alone or in combination with at least one of the recited instances of all the recited instances, and include terms such as "including" which are inclusive, open-ended, and not exclusive.

[0194] Amplification refers to polymerase chain reaction (PCR and its many variations - 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, R.K. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-91.), particularly fluorescence readout (Holland, P.M. 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 including isothermal amplification via loop (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 No. 6,410,278, Notomi T, Hase T, 'Process for synthesizing nucleic acid', published on June 25, 2002, assigned to Glorious Research & Development Co., Ltd.), whole genome amplification technology (Non-Patent Document 3 (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), Klein CA, Schmidt-Kittler O, Schardt JA, Pantel K, Speicher MR, Riethmueller G. Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. Proc Natl Acad Sci U S 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 multiple displacement amplification. Proc Natl Acad Sci U S A. 2002;99: 5261-5266. Pmid:11959976, Langmore JP. Rubicon Genomics, Inc. 2002;3: 557-560., Non-Patent Document 2 (Telenius H, Carter NP, Bebb CE, Nordenskjoeld 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 U S A. 1992;89: 5847-51. pmid:1631067), Strand displacement amplification (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. 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, H.M.; Abd El Wahed, A.; Shalaby, M.A.; Almajhdi, F.N.; Hufert, F.T.; 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 amplification based on nucleic acid sequences (Deiman, Birgit; van Aarle, Pierre; Sillekens, Peter (2002).'Characteristics and Applications of Nucleic Acid Sequence-Based Amplification (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 chemical biology, materials science and medicine'. Chemical Society Reviews. 43 (10): 3324-41. doi:10.1039 / C3CS60439J. PMID 24643375), and the like, and may refer to various amplification reactions, including but not limited to these.

[0195] In the context of the present invention, the PCR reagent and / or reverse transcription reagent and / or RT-PCR reagent comprises one or more reagents selected from the group consisting of RNA and / or buffer reagent and / or nuclease inhibitor(s) and / or DNA polymerase enzyme for reverse transcription of further agent or enzyme and / or reverse transcriptase (RT-) polymerase enzyme and / or dNTP and / or amplification primer and / or poly A primer. Those skilled in the art know the components of the PCR and reverse transcription reagents necessary to successfully amplify and / or reverse transcribe nucleic acids.

[0196] Digital polymerase chain reaction (dPCR) is a PCR technique for absolute quantification of target nucleic acids that utilizes the enhanced amplification effect of a small amount of PCR reaction. The quantification principle is based on Poisson distribution-based correction of the amplified single molecule number and single molecule sensitivity of the partitioning reaction in dPCR (Sykes P.J., Neoh S.H., Brisco M.J., Hughes E., Condon J., Morley A.A. Quantitation of Targets for PCR by Use of Limiting Dilution. Biotechniques. 1992;13:444-449.). The main design parameters of the dPCR platform include the number of partitions, the volume of each partition, the total reaction volume, and the variation in partition volume. The statistical accuracy of dPCR is further affected by sample preparation variability and the rates of molecular dropout and false positives. The dPCR platform currently provides inherent assay multiplexing capabilities while qPCR does not have sample multiplexing capabilities (Quan PL, Sauzade M, Brouzes E. dPCR: A Technology Review. Sensors (Basel). 2018;18(4):1271. Published 2018 Apr 20. doi:10.3390 / s18041271).

[0197] There are many forms of real-time PCR or qPCR that mainly differ in the detection chemicals used for monitoring the reaction products (e.g., Gelfand et al., U.S. Patent No. 5,210,015 (“Taqman”), Wittwer et al., U.S. Patents Nos. 6,174,670 and 6,569,627 (intercalating dyes), Tyagi et al., U.S. Patent No. 5,925,517 (molecular beacons). The detection chemicals for real-time PCR are reviewed (Mackay et al., Nucleic Acids Research, 30: 1292-1305, 2002).

[0198] Regarding the "absolute quantification" of this method, a unique molecular identifier-based approach for obtaining information from the quantitative composition of single-molecule compartments is described (International Publication No. WO 2020 / 260277). The terms "absolute concentration", "absolute number", or "absolute amount" of a target analyte or biomolecule refer to the total or exact concentration, number, or amount of the analyte. In other words, absolute quantification determines the number or concentration of an analyte in terms of the absolute number of molecules or copies. In contrast, relative quantification determines the fold change in the number, amount, or concentration between two samples. Conversely, unlike the method described in the present invention where the absolute number of molecules in a volume defines the molar concentration of a target without comparison to an external (relative) quantification standard material, an external quantification standard is required to determine the amount.

[0199] "Sequencing" refers to determining the order of nucleotides (base sequences) in a nucleic acid sample. Next-generation sequencing, as exemplified by methods commercialized by Roche, Illumina, Applied Biosystems, Oxford Nanopores, Pacific Bioscience, etc., is described (David J Munroe & Timothy J R Harris in Nature Biotechnology 28, 426-428, 2010). A common feature of these technologies is determining the sequences of multiple single molecules of DNA, thereby effectively and quantitatively revealing the composition of a DNA sample that extends from hundreds to billions of individual reads in a single run. In a sequencing reaction, it is necessary to introduce specific DNA sequences (generally called adapters) that vary depending on the technology used. This process is called library preparation and includes methods such as ligation (e.g.), PCR extension, etc. Adapters may contain an identifier sequence for distinguishing samples. As used herein, "next-generation sequencing (NGS)" encompasses recently developed technologies for sequencing nucleic acids and typically enables much higher throughput than the conventional Sanger approach (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 January; 11(1):31-46). These platforms enable sequencing of single-molecule nucleic acid fragments that are either clonally expanded or not amplified. Specific platforms include, for example, sequencing by ligation of dye-modified probes (including cyclic ligation and cleavage), pyrosequencing, and single-molecule sequencing. Nucleotide sequence species, amplified nucleic acid species, and detectable products resulting therefrom can be analyzed on such sequence analysis platforms.In the method of the present invention, for example, next-generation sequencing can be used to quantify a unique PCR-amplifiable DNA label in order to assess the formation of a two-component / analyte complex as follows.

[0200] In embodiments, the methods described herein use a compartmentalized two-component detection method. As used herein, the term "compartmentalized two-component detection method" preferably refers to a two-component detection method in which an analyte-specific binding component is contacted with an analyte to quantify complex formation and then the sample is compartmentalized. Generally, compartmentalized two-component detection methods are preferably based on compartmentalizing a solution into small compartments such that, in the absence of a complex formation method, the likelihood of both binding components being present within a compartment is low and follows a Poisson distribution. For compartmentalization, various assays can be envisioned. For example, the emulsion droplet method can be used to form droplets (e.g., water-in-oil) in an emulsion, with each droplet representing a separate compartment. Compartmentalization can include a location or physical compartment, or an environment with restricted diffusion.

[0201] As used herein, the term "droplet" preferably refers to an isolated 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 dilutions to which a two-component detection system or other reagent has been added. The second fluid is preferably a hydrophobic fluid such as a hydrocarbon, silicone oil, mineral oil, or organic solvent. Emulsion techniques for compartmentalizing a sample solution are well known in the art. In a preferred embodiment, the compartmentalized two-component detection method is emulsion coupling, which refers to the concept of a digital assay based on the detection of individual ternary molecular complexes of double labels (two components) in an emulsion and can be identified, for example, by droplet digital PCR (ddPCR) or next-generation sequencing (NGS).

[0202] Preferably, the two analyte-specific binding agents and the analyte are not immobilized, i.e., they are present in solution, so that the complex is also formed in solution. The term two-component detection method as used herein thus preferably targets the liquid-phase formation of the complex and is different from the common sandwich immunoassay involving a solid-phase immobilized (primary) capture binding agent and a (secondary) detection binding agent. Thus, the term "non-immobilized" preferably refers to components such as analyte-specific binding components that can freely diffuse within a (liquid) solution, whereby the binding kinetics to the analyte that equally freely diffuses in the above 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.

[0203] In a preferred embodiment, determining the binding characteristics includes, for each of the first and second types of binding agents, determining the dissociation constant of the target analyte, the specificity of binding to the target analyte, the labeling ratio, the labeling synthesis error, the active fraction, the contamination by free label, and / or the optimal concentration (number of molecules per unit volume) required for binding to the target analyte. Thus, in other words, in an embodiment, the binding characteristics of a binding agent may include the activity (reactivity), specificity, and / or concentration of the binding agent. Reactivity can be described by the dissociation constant of the binding agent, specificity is described by identifying the binding target, and concentration is described by the number of molecules per unit volume.

[0204] Generally, the dissociation constant (KD) is an equilibrium constant that indicates the tendency of a binder to dissociate (separate) reversibly from a target analyte. In other words, the dissociation constant may indicate the "reliability" and / or strength of a binder that binds to a target analyte to facilitate its detection. In embodiments, a two-component method using the labeling ratio and / or labeling synthesis error of the binder(s) is used to determine the apparent dissociation constant of the binder(s). Thus, in embodiments, the consideration of the dissociation constant for calculating the absolute concentration of the target agent herein is more complex than the consideration of a simple titration curve in the prior art (e.g., methods for determining the concentration of a target agent in a sample or cell), and in the context of the present method, it is preferred that the labeling ratio and / or labeling error of the binder is additionally (independently) determined to control and compensate (normalize) the accuracy of the method for determining the dissociation constant of the binder, thereby achieving a higher accuracy of absolute quantification of the target agent than was possible with any method of the prior art.

[0205] In embodiments, the concentration calibration curve of the two-component detection method or the dissociation constant relationship of the two-component detection method may be provided in the form of reference data. In embodiments, the reference data of the concentration calibration curve of the two-component detection method preferably relates to any data that can provide a mathematical function reflecting the dependence of the signal reflecting the formation of the two-component / analyte complex formed in the above solution on the concentration of the analyte. In embodiments, the reference data of the dissociation constant relationship of the two-component detection method preferably relates to any data that enables the provision of a mathematical function of the relationship between the dissociation constants of the analyte-specific binding components (kd1 and kd2 respectively) and the analyte according to the concentration of the two-component / analyte complex and the signal reflecting the concentration of the analyte and / or the analyte-specific binding component.

[0206] The compartmentalized generation of "linked array information" in this embodiment may refer to the physical linkage of two binder labels, preferably nucleic acid labels, which, when they bind synchronously to a target analyte, co-localize in a complex within a partition, thereby forming a complex. In other words, the ligation process may form random multimeric nucleic acid products based on the co-localization of these nucleic acid identity labels under appropriate assay conditions. In embodiments, the ligation reaction may be amplification-based or may include other techniques. Amplification-based ligation can utilize two or more pairs of amplification primers having the same binding ability, but the use of complementary 5' tags or dimer linker sequences results in the formation of a polymerase-extendable nucleic acid duplex. The tag or dimer linker sequence means that the sequence amplified by one pair of primers hybridizes to the sequence amplified by a second pair of primers. Thereby, the identification label representing the type of binder is ligated. By physically ligating the nucleotide labels of the binders, when a complex is formed with the target analyte, it becomes easier to "translate" the information regarding the co-localization of the binders and their targets within the partition into a nucleotide sequence (physically linked nucleotide labels). In embodiments, the identity of the binder and thereby the presence of their targets can be determined from the nucleotide sequence, which in embodiments can be achieved by nucleic acid sequencing or PCR, such as digital (droplet) or real-time qPCR. One embodiment of this process is described in International Publication No. WO 2016 / 083793. Optionally, before identifying the ligated labels, the ligated nucleotide sequences from two or more complexes are combined. The identity of the binder(s) and / or target(s) can be determined, for example, by sequencing the ligated nucleotide label sequences. This can be performed using a high-throughput parallel system. Combining the ligated sequences allows for performing one reaction to identify all the ligated sequences. For example, all the ligated nucleotides can be sequenced in one reaction. The ligated sequences can be quantitatively determined to measure the relative abundance of the ligated sequences.In some embodiments, the labels of the complex's co-localized binding agents are not physically linked, but their co-localization within the second partition, and thus within the complex, is detected by PCR, preferably dPCR and / or real-time qPCR using fluorescence detection methods. This method of detecting co-localized nucleic acid labels, which reliably determines the presence of the complex and thus the target analyte within the second partition, is facilitated because the distribution of the components of a single cell in the first partition to the second partition follows a Poisson distribution. Thus, when forming a complex without binding to the target analyte, the probability of having two binding agents in the same second partition is lower than the probability of co-localization because the two binding agents are physically connected by binding to the target analyte to form a complex. Thus, in embodiments, those nucleic acid labels can be detected by PCR methods using, for example, two different labeled (e.g., two different fluorescent colors) probes in droplet PCR without physically linking the labels beforehand.

[0207] Therefore, in this embodiment, the presence of two different fluorescent colors within the second partition, for example an emulsion droplet, during detection indicates the presence of a complex within the second partition. In embodiments where multiple target analytes are analyzed, for each target analyte, two specific binding agents, one of the first type and the other of the second type, should be selected. The discrimination between different binding agent pairs for different target analytes can be achieved, for example, in some embodiments, using different fluorescent labels, primers or probes during the final PCR, qPCR, real-time PCR or dPCR readout. As an example, a probe that specifically binds to the ligation sequence of two binding agent labels of a first target analyte is tagged with one fluorescent color, while a probe specific for the ligated binding agent label of a second target analyte is tagged / marked with a different fluorescent color. In other embodiments where the labels of the binding agents within the complex are not ligated, each pair of binding agents within the complex is detected by a specific combination of fluorescent colors that can be distinguished during the final PCR readout. In embodiments, the readout of the labeled sequences can be performed by nucleic acid sequencing. In such embodiments, the nucleic acid labels of the binding agents within the complex are preferably ligated, prior to the sequencing-based readout, within the second partition, for example, by a ligase reaction or PCR or other means known in the art, followed by a pooling step of all second partitions and a batch sequencing analysis of all labels contained within the partitions. During the analysis of the acquired sequence information, the ligated labeled sequences can be determined and assigned to a single complex, i.e., a single target analyte molecule.

[0208] Examples of linked sequence information for the detection of proteins, epitope proteins (PTM- / modified proteins), and protein-protein interactions, in particular for the labeling, analysis, detection, and measurement of protein-protein interactions by fewer than 10 antibodies, are described in WO 2016 / 083793 and Karakus et al. (Karakus U, Thamamongood T, Ciminski K, Ran W, Guenther SC, Pohl MO, Eletto D, Jeney C, Hoffmann D, Reiche S, Schinkoethe J, Ulrich R, Wiener J, Hayes MGB, Chang MW, Hunziker A, Yangueez E, Aydillo T, Krammer F, Oderbolz J, Meier M, Oxenius A, Halenius A, Zimmer G, Benner C, Hale BG, Garcia-Sastre A, Beer M, Schwemmle M, Stertz S. MHC class II proteins mediate cross-species entry of bat influenza viruses. Nature. 2019 Mar;567(7746):109-112. doi: 10.1038 / s41586-019-0955-3. Epub 2019 Feb 20. PMID: 30787439., WO 2016 / 083793).

[0209] As used herein, the "ligation" reaction may also be referred to as "ligation of the label", or "linkage of the label", or "linkage of the binder label", or "linkage of the antibody label". In some embodiments, the ligation reaction referred to herein is a classical proximity ligation reaction that includes rolling circle DNA synthesis or rolling circle amplification. In other embodiments, ligation is achieved using a ligase enzyme. Non-limiting examples of proximity-based two-component methods include 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-label 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 regarding FRET or BRET, see, for example, (Pfleger, Seeber, & Eidne, 2006), for protein complementation assays see Morel!, Ventura, & Aviles, 2009, for AlphaScreen see Taouji, Dahan, Bosse, & Chevet, 2009, and for DNA-label proximity methods such as PLA (proximity ligation assay) or PEA (proximity extension assay) see Soderberg et al., 2006.

[0210] As used herein, "barcode" refers to a nucleic acid sequence preferably used to identify a single cell when obtaining sequence-based linked information. With a unique barcode sequence, data can be associated with individual cells. As used herein, "barcode" may also refer to a nucleic acid sequence preferably used to identify a single cell when obtaining sequence-based linked information. With a unique barcode sequence, data can be associated with individual cells.

[0211] The processed RNA may contain UMIs. Thus, in embodiments, the ligated sequence information may include UMIs that can be ligated, and / or the processed RNA may include at least one of a barcode and a UMI. Thus, in some embodiments, UMIs are added to oligo dT or random hexamer priming. Also, in some embodiments, the template switching oligo also includes a UMI. There are many variations of single-cell cDNA synthesis in the literature and are well known to those skilled in the art and thus can be applied in the present invention.

[0212] Specific embodiments of the compartmentalized generation of "linked sequence information" using unique molecular identifiers (UMIs) are described in International Publication No. WO 2020 / 260277, and the UMIs are described in Kivioja et al. (Teemu Kivioja, Anna Vaehaerautio, 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.). To improve the quantitative accuracy of various polynucleotides in a sample using next-generation sequencing technology, various methods have been developed, including methods such as competitive polymerase chain reaction (PCR) described in U.S. Patent No. 5,213,961, and deep barcode sequencing using unique molecular identifiers (UMIs) described by Smith, A.M., Heisler, L.E., Mellor, J., Kaper, F., Thompson, M.J., Chee, M., Nislow, C. (2009). Quantitative phenotyping via deep barcode sequencing. Genome Research, 79(10), 1836-1842. https: / / doi.org / 10.1101 / gr.093955.109).

[0213] "Nested PCR" means PCR in which the product of the first amplification serves as the template for the second amplification PCR reaction. As used herein, the inner primer with respect to the nested amplification reaction means the primer used to generate the first amplicon, and the outer primer means the primer used to generate the second, or nested amplicon.

[0214] Multiplexing of PCR means that multiple target sequences are provided and PCR is 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, with low multiplexing within compartments and a large multiplicity of PCR in the overall volume of dPCR (Alexandra S. Whale, Jim F. Huggett, Svilen Tzonev. Fundamentals of multiplexing with digital PCR. Biomolecular Detection and Quantification. Volume 10, 2016, Pages 15-23, ISSN 2214-7535, https: / / doi.org / 10.1016 / j.bdq.2016.05.002).

[0215] As used herein, the term "target analyte" or "target" may refer to a "biomolecule" or a biomolecule recognizable by an antibody, or simply a "target". These terms can be used interchangeably herein. Thus, in this specification, these terms can be used interchangeably. The target used herein includes artificial or natural modified proteins, non-protein molecules (e.g., nucleic acids or modified nucleic acids) recognizable by a binder, including phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and proteolysis, as well as protein-protein interactions, and non-exhaustively includes proteins, modified or post-translationally modified (PTM) proteins (epitope proteins), and a single molecule or 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) that forms a complex with a binder (which may be plural). The complex is usually formed under conditions that maintain the conformation of the target in the subject organism. In this specification, a target or target analyte can be selected from the group including peptides, proteins, chemically modified peptides or proteins, nucleic acids, chemically modified nucleic acids, substances, and any complex thereof (any complex including one or more of those listed).

[0216] As used herein, "complex" refers to a complex formed by two binders (a pair of binders) or an analyte-specific binder and the target analyte itself to which the two binders specifically bind. The complex may preferably be referred to as a "two-component / analyte complex" or a "ternary complex" in the context of a two-component detection method as described in International Publication No. WO 2020 / 260277. The complex includes a first type of binder and a second type of binder, and each preferably binds to a different epitope on the same target analyte molecule. In embodiments, the first type of binder and the second type of binder are two different antibodies that recognize different isotopes of the target analyte and can bind to the target analyte simultaneously. In embodiments, the binding and detection of two different binders (of different types) to the same target analyte can have more advantages than detection by a single binder. For example, in embodiments, one type of binder may recognize a chemical modification or a post-translational modification, and the second type of binder may recognize a specific protein. In another embodiment, one type of binder may recognize a conserved epitope of a protein, while the second type recognizes a specific mutation or variant of the target protein. In another embodiment, the target analyte may be a protein complex or a DNA-protein complex, one type of binder recognizes a protein within the complex, and the other type of binder recognizes, for example, a methylated DNA motif, or in the case of a protein-protein complex, each binder type recognizes one of the proteins forming the complex. The second compartmentalization step of the method facilitates the separation of single molecules or molecular complexes contained within lysed cells according to a Poisson distribution. Thus, the presence of two different types of binders in one droplet, bead, or partition indicates that the two types of binders bind together to the target analyte and form a complex with the target analyte. The probability (impossibility) that the two binders are accidentally present in the same partition but not in the complex can be easily calculated.

[0217] In embodiments, the method can be used to identify multiple binders that bind to a single target. For example, in embodiments where the target is a protein, the method can identify antibodies that bind to different epitopes on the protein. Alternatively, the target may be a protein complex, and the method can identify multiple binders that bind to different proteins within the complex. For example, in embodiments, a nucleic acid label associated with one binder within the complex can be ligated to a nucleotide sequence associated with a second binder within the complex. If the identity of the binder is known (from the ligated nucleic acid label sequence), it may be possible to identify the components of the target and, thus, for example, the proteins within the target that naturally interact. For example, in embodiments where the binder is an antibody with known binding properties, the protein bound by the antibody may be identified. In this way, the identity of the proteins within the target can be determined. Thereby, protein-protein interactions within a sample can be detected and identified. Further, if the protein-protein interaction is identified, the method can be used to monitor the effect of a compound on the interaction. Alternatively, in embodiments, a nucleotide sequence associated with the binder in the binder / target complex can be ligated to a nucleotide sequence associated with the target in the binder / target complex. In embodiments, the method can be used to identify which binder interacts with which target. For example, it can be used to identify which members of a binder library can form a complex with a known target. This information can be used to characterize the members of the binder library and obtain binding property information. Preferably, in embodiments, the generation of the randomly paired and ligated nucleic acid products involves using at least two pairs of PCR primers to amplify the same or non-identical amplicons, and optionally, the 5'-terminal PCR primer has a sequence tag, and when amplified using the tagged primer, randomly paired and ligated nucleic acid products are generated. More preferably, the amplification is emulsion PCR amplification or digital PCR (dPCR), and the generation of the amplicons and the randomly paired and ligated nucleic acid products is a parallel process.Preferably, the sequencing of the ligation amplification product is a highly parallelized sequencing method (e.g., NGS).

[0218] The "binding agent" is preferably an antibody, an aptamer, or based on an artificial protein scaffold. Preferably, the term "antibody" means any binding agent. Alternatively, the binding agent may be a compound. In the present specification, binding agents can be distinguished into various "types", and a specific type of binding agent preferably recognizes a specific epitope or target binding motif on the target analyte. Thus, different types of binding agents recognize different motifs or epitopes of the target analyte or on the target analyte. Thus, it is preferred to use two analyte-specific binding agents, both of which exhibit binding ability to the target analyte. Preferably, the two analyte-specific binding agents do not compete with each other for binding to the analyte and are selected and designed such that the components can bind simultaneously to form a complex. Thus, in the present specification, two types of binding agents specific for one target analyte can bind to the target analyte simultaneously at different positions or sites of the analyte. Thereby, one type of binding agent can specifically bind to a domain, chemical modification (e.g., PTM) or motif of the target analyte, and the other type of binding agent can recognize or specifically bind to another domain, epitope, or motif of the target analyte. When the molecular complex is the target analyte, different types of binding agents may recognize different members or parts of the complex, thereby facilitating the detection of a specific complex rather than an individual member or part of the complex. Such a complex may be a protein-protein, protein-peptide, peptide-peptide, protein-nucleic acid, compound-nucleic acid, compound-protein, compound-peptide or compound-compound complex, or any complex that may be specifically recognized by a binding agent.

[0219] The binder may be a member of an antibody display library or an antibody library in which each antibody is labeled with a unique nucleotide sequence. The method may use an antibody agent as the binder, and the binding characteristics, such as the target to which the binder binds, the chemical nature of the binding (including numerical representations of the binding affinity, the on / off rates of binding, and further including numerical consideration of some or all of the following labeling ratios, labeling synthesis errors, active fractions, contamination by free label), are known, the unique nucleotide sequence associated with the plurality of displayed antibody agents is determined, and the binding characteristics and the unique nucleotide sequence are correlated with each other. In an embodiment, when two binders, preferably one first type of binder and one second type of binder, bind to the same target agent molecule, thereby forming a complex, the complex (including the two binders and the target analyte) may be referred to as an "analyte-two-component complex". The two binders may also be referred to as "analyte-specific binding components" or simply "binding components", especially in the context of two-component detection methods.

[0220] The binder used in the present invention may 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 that contains an antigen-binding site that specifically binds to an antigen, whether natural or produced by partial or complete simulation. The term "antibody" includes antibody fragments, derivatives, functionally equivalent and homologous of antibodies, humanized antibodies (whether natural or wholly or partially simulated) including any polypeptide containing an immunoglobulin binding domain, and any polypeptide or protein having a binding domain that is an antibody binding domain or homologous to an antibody binding domain. Thus, included are chimeric molecules containing an immunoglobulin binding domain or equivalent fused to another polypeptide. Cloning and expression of chimeric antibodies are described in European Patent Application Publication Nos. 0120694 and 0125023. Examples of antibodies include immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD, IgA) and their isotype subclasses, fragments containing antigen-binding domains such as Fab, scFv, Fv, dAb, Fd, and diabodies. The antibody may be polyclonal or monoclonal. Furthermore, fragments of whole antibodies can perform the function of binding antigens.Examples of antibody fragments include: (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains; (ii) an Fd fragment consisting of a VH domain and a CH1 domain; (iii) an Fv fragment consisting of the VL domain and the VH domain of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward, E.S. et al., Nature 341:544-546 (1989)); (v) an isolated CDR region; (vi) an F(ab')2 fragment, which is a bivalent fragment containing two linked Fab fragments; (vii) a single-chain Fv molecule (scFv), in which the VH domain and the VL domain are linked by a peptide linker that enables these 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) a bispecific single-chain Fv dimer (International Application PCT / US92 / 09965); and (ix) a "diabody", i.e., a multivalent or multispecific fragment constructed by gene fusion (International Publication No. WO 94 / 13804; P. Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)).

[0221] An "antigen-binding domain" is a part of an antibody that specifically binds to a part or all of an antigen and includes a region that is complementary to a part or all of the antigen. When the antigen is large, the antibody binds only to a specific part 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 the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH).

[0222] Alternatively, the binder may be based on an artificial protein scaffold. The protein scaffold is derived from a stable and soluble native protein structure and is modified to provide a binding site for the target molecule of interest. Examples of artificial protein scaffolds include, but are not limited to, affibodies based on the Z domain of staphylococcal protein A that provide a binding interface for two of its α-helices (Nygren, P. A. (2008). FEBS J 275(11): 2668-76), anticalins derived from lipocalins that incorporate a binding site for a small ligand at the open end of the β-barrel fold (Skerra, A. (2008) FEBS J 275(11): 2677-83), nanobodies, and DARPins. Artificial protein scaffolds typically target binding to the same antigenic protein as an antibody. Also, short peptides may be used to bind to the target protein. A phylomer is a natural structured peptide derived from a bacterial genome. Such peptides represent the folding of diverse protein structures and can be used to inhibit / disrupt protein-protein interactions in vivo (Watt, P. M. (2006). Nat Biotechnol 24(2): 177-83).

[0223] Alternatively, the binder 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 very easily obtainable in vitro and can bind to simple ions (e.g., Pb 2+, can be developed to recognize various molecular classes from nucleotides, small molecules, proteins, viruses, cells, and even whole organisms (Menger et al., 2006. Handbook of Experimental Pharmacology, 359 - 373). High - affinity aptamers have been selected by the well - known SELEX method (Ellington & Szostak, 1990. Nature, 346, 818 - 822) not only for 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), 17β - estradiol (Kim et al., 2007. Biosens. Bioelectron., 22, 2525 - 2531), but also for the detection of 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 (for reviews, see Tombelli et al., 2007, Biomolec Eng., 24, 191 - 200). Some of the above - mentioned aptamers have been used in ELISA - like assays on the surface of microplates or biosensor transducers (QCM, SPR). An aptamer - modified AuNP colorimetric system has also been developed for determining the protein PDGF in a sandwich - based assay (Huang et al., 2005, 77, 5735 - 5741). Recently, modified nucleotide - based aptamer libraries have been applied for highly parallel measurement of proteins.The aptamer sequence can be tagged with a unique sequence or can itself function as a unique nucleotide label for the binder.

[0224] The binder may be part of a library such as a display binder library, for example, a library of bacterial display, mRNA display, bacteriophage display, aptamers, ribosome display, or yeast display. Each member of the library preferably has a detectable nucleic acid identity label that is unique to one member of the library. Preferably, the unique nucleic acid identity label is ligatable. "Ligatable" means that based on the co-localization / compartmentalization of these nucleic acid identity labels under appropriate assay conditions, the ligation process may form random nucleic acid binding products. The product preferably has a dimer or pair, or a higher-order co-compartmentalized label without covalent bonds.

[0225] Preferably, the binder can preferably detect two or more targets with different apparent affinities. Alternatively, the binder can detect a single target using different epitopes or binding sites, preferably with different apparent affinities.

[0226] Preferably, any binder that binds to any target and is associated with a unique nucleotide sequence is applicable to the present invention.

[0227] One binder can recognize a specific target such as the corresponding protein, etc. This is called specificity. The assay of International Publication No. WO 2016 / 083793 applied in this specification enhances these specificities using two antibodies per detection. Alternatively, multiple binders may recognize one target, for example, a specific target such as the corresponding protein, etc. This is called redundancy. Similarly, one binder can recognize two or more targets such as protein species based on, for example, the similarity of the target conformation due to the conformation of the protein or the protein sequence. This phenomenon is called cross-reactivity. Furthermore, the recognition of the target protein by the binder is based on the conformation of the protein or its protein sequence. This is called reactivity and its numerical representation, and its affinity is the dissociation constant of the binder. The protein-binding affinity of the binder can be calculated, for example, from the quantitative information according to International Publication No. WO 2020 / 260277.

[0228] The predetermined binding properties of the binder can include reactivity, the active fraction of the binder, specificity and cross-reactivity with redundancy, and the calculated affinity, for example, the apparent dissociation constant of the binder. Furthermore, the binder is preferably associated with a unique nucleotide sequence, and as described in the present invention, the associated unique nucleotide sequence modifies the apparent measurable dissociation constant of the binder, and these further include, as influencing factors, contamination by free label, the labeling ratio of the binder, and labeling error.

[0229] In an embodiment where one or more binders are antibodies (or fragments thereof), the step of determining the labeling efficiency includes calculating the labeling fraction (LE) of the binder using the concentrations in [ng / μl] of the light chain (lc), heavy chain (hc), and labeled heavy chain (L) of the antibody as follows:

Equation

[0230] In an embodiment, determining the concentration of the free label (label not bound to the binder) after the labeling reaction of the binder(s) involves, for example, performing nucleic acid amplification by dPCR, thereby determining the labeling reaction amount or the concentration of the free label (label not bound to the binder) in the other (stock solution) containing the binder (e.g., in units of cp / μl) and the concentration of the labeled binder (e.g., in units of cp / μl), and calculating the ratio of the free label therefrom.

[0231] In an embodiment, 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). It is preferred that dPCR can determine the exact amplifiable / detectable amount (concentration or percentage) of the binder label. In an embodiment, then, the difference between the two amounts / concentrations (binder versus control) determined in the above measurement can be used for the calculation of the labeling synthesis error.

[0232] In an embodiment, the apparent dissociation constant of the binder(s) is determined using a two-component method that uses the labeling ratio and / or labeling synthesis error of the binder(s). Thereby, it is preferred to provide an advantageous and native self-compensation against the active fraction of the binder(s) and contamination by the free label in order to determine the absolute amount in a single cell in an unbiased manner and to provide the dissociation constant of the antibody.

[0233] In an embodiment, the calibration curve can be calculated for each sample as described herein. The calibration curve can be calculated as described in WO 2020 / 260277 by adjusting the binding characteristics. Using the individually determined labeling ratio and / or labeling error of the antibody, for example, the concentrations of the antibody and the complex are corrected using a calibration curve corrected with the corrected values of the single and double positive values of dPCR. This represents a significant improvement compared to the prior art, which can generally only determine the concentration of the target analyte in a sample using an uncorrected calibration curve. However, the prior art calibration curves do not account for errors or biases due to errors in the unlabeled binder and / or label synthesis, etc. This drawback of the prior art is surprisingly overcome by the present method, and in an embodiment, labeling errors or biases due to incorrectly labeled binders are taken into account during the quantification of the target analyte, i.e., used, for example, to normalize the data obtained from the calibration curve and / or sequence analysis.

[0234] In an embodiment, assuming that all samples contain the target analyte at the same concentration, the apparent dissociation constant of the binder can be recursively calculated by minimizing the standard deviation of the target analyte concentrations recalculated for all measurements.

[0235] In chemistry, biochemistry, medical technology, and pharmacology, the dissociation constant (Kd) is a specific type of equilibrium constant that measures the tendency of a large complex to reversibly dissociate into smaller components (Friguet, Chaffotte, Djavadi-Ohaniance, & Goldberg, 1985). The dissociation constant is the reciprocal of the binding constant. In the special case of an embodiment of a two-component assay, the dissociation constant is the dissociation constant between two components (binders) and the target analyte. Since the components can be antibodies, the determination of the dissociation constant has received wide interest. Several approaches for determining the dissociation constant are known in the prior art.

[0236] Accordingly, the terms "two-component detection method" or "two-molecule detection method" refer to a method that uses two analyte-specific binding components and determines a signal that reflects the formation of a two-component / analyte complex when the two analyte-specific binding components are contacted with a solution containing the analyte. The term "two-component detection system" or "two-molecule detection system" preferably refers to the components or reagents necessary to perform the two-component detection method. This preferably includes two analyte-specific binding components (binders) provided in a single or separate solution of known concentration, and a sample solution containing any analyte to be analyzed.

[0237] Preferably, the two-component detection method is compartmentalized. The compartmentalization has restricted diffusion and is physical. Preferably, the compartmentalized two-component detection method is in solution or preferably contains surface-bound molecular components.

[0238] In the two-component detection method, preferably, two analyte-specific binding components that both exhibit binding ability to the analyte are used. Preferably, the two analyte-specific binding components are selected and designed such that they do not compete with each other for binding to the analysis and can bind simultaneously to form a two-component / analyte complex. As described above, the analyte may refer to an aggregate or complex formed from a plurality of entities, such as a protein-protein complex. One of the two analyte-specific binding components can bind to one entity of the analyte complex (e.g., the first protein), and the other can bind to the second entity of the analyte (e.g., the second protein). Accordingly, the two-component / analyte complex is formed only when both proteins interact and is then labeled with the binding component.

[0239] In one embodiment, determining the "dissociation constant" of a binding agent may include preparing a sample or one or more dilutions of the binding agent having a known dilution factor and applying a two-component detection method to the sample and the one or more dilutions, and applying the 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-component binding agent / target analyte complex (complex or two-component / analyte complex) formed in the solution. Dilution changes the concentration of the analyte-specific binding agent or the sample, providing dilution with a known dilution factor. The signals detected in the sample and the one or more dilutions, and the concentrations of the target analyte and / or the target analyte-specific binding agent can be used as constraining inputs for mathematical approximation. The signal can take various forms and depends on the two-component detection method used. In the case of a compartmentalized two-component method such as coupling described in WO 2020 / 260277, the signal may refer to a signal resulting from digital droplet PCR (dPCR) and / or NGS sequencing indicating the presence of a two-component (two binding agents) / target analyte complex (complex) based on a deviation from the expected Poisson distribution within the compartmentalized droplet.

[0240] As used herein, "emulsion" refers to a water-in-oil emulsion used to compartmentalize a single cell or molecule into a single emulsion droplet or compartment containing a very small amount. Those skilled in the art are familiar with various methods for generating water-in-oil emulsions, such as in microfluidic devices in some embodiments. Droplet-based microfluidics manipulates separate volumes of fluid in immiscible phases to generate (micro) droplets with small volumes (from μl to fl). Alternatively, in other embodiments, it may be a gel bead in an emulsion generated using a partition, such as the Gel Beads in Emulsion (GEM) technology according to 10×Genomics.

[0241] As used herein, "partition" refers to the above emulsion and physically separated partitions. Those skilled in the art are familiar with various methods for generating partitions, such as in microfluidic devices in some embodiments. Alternatively, in other embodiments, the partition may be a nanowell, such as a QIAcuity nanowell, Qiagen.

[0242] In the context of the present invention, a "first partition" is a partition, compartment, or emulsion droplet that contains one single cell or no cells. In this method, it is preferred to dilute and compartmentalize the sample in a manner that allows a single first partition or droplet to contain one single cell or no cells, preferably no more than one cell. Separation and compartmentalization in such first partitions are essential for facilitating analysis at the single cell level. In some embodiments of the method, each single cell is lysed inside its first partition to release the components of the cell into the volume of the first partition or droplet, thereby facilitating further separation of the single molecules of each cell into a second partition. Thus, in the context of the present method, a "second partition" preferably contains only one target analyte, preferably bound to binding agents of the first and second types to form a complex. Thus, in the context of the present invention, it is preferred to dilute the compounds of the single cell inside the first partition and separate them into a second partition such that each complex formed is contained within a separate second partition or droplet. This separation allows for an accurate determination of the number of individual complexes formed in the method. In the context of the present invention, since a "first partition" may sometimes be referred to as a "first compartment" and a "second partition" may sometimes be referred to as a "second compartment", the terms "partition" and "compartment" may be used interchangeably herein. The same applies to "compartmentalization" and "partitioning".

[0243] "Emulsion coupling" or "coupling" or "protein coupling" or "PICO" is the concept of a digital assay based on the detection of individual molecular complexes (e.g., complexes) of dual labels (ternary) in an emulsion, which is identified, for example, by dPCR or next-generation sequencing (NGS).

[0244] Preferably, by using specific nucleotide-binding agents, especially for RNA and DNA targets, the target can be evaluated simultaneously with other targets having different properties unsuitable for the assay. In such cases, other methods, especially methods for detecting single-cell RNA expression, can be advantageously combined with the present invention.

[0245] The target preferably contains a protein. More preferably, the target is part of a protein sample. Optionally, the target may be cross-linked to a plurality of targets, for example, other targets within the protein sample. For example, the proteins in the sample may be cross-linked to one or more other proteins in the sample and may form a ternary or higher-order complex target as a new cross-linked entity.

[0246] The target can be associated with a specific nucleotide sequence. "Associated" means that the presence of the target in the binder / target complex can be detected by the presence of the nucleic acid sequence within the linked sequence generated by the present method. The nucleotide sequence may be attached to the target as a label, or may be present within the nucleic acid within the target, for example, within a phage, or the nucleotide sequence is associated using a cross-linking process. For example, a DNA-binding protein is DNA, and for example, DNA is cross-linked to a histone protein. Alternatively, the nucleotide sequence may be part of an aptamer known to bind to the target. By contacting the binder / target complex with the aptamer and ligating the specific nucleotide sequence containing the aptamer, the existing target can be identified. However, many alternative methods for associating labels are known in the art.

[0247] The present invention can be applied to any single-cell-derived protein sample. The single cell can be derived from any biological specimen, including but not limited to tissues, cytological specimens, body fluids, cell cultures, or other single-cell-containing substances. Examples of body fluid samples include blood, saliva, urine, cerebrospinal fluid, and buffy coat. Preparation of single cells from specimens can be carried out 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 various microfluidic platforms are more frequently applied. Recently, on-demand optically addressable valve-controlled single cell printing (A. Gross, J. Schoendube, 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.).

[0248] As used herein, the term single cell refers to one cell. Single cells useful in the methods described herein can be obtained from a subject's tissue, or from a biopsy sample, blood sample, or cell culture. Additionally, cells can be obtained from a specific organ, tissue, diseased tissue, etc. and used in the methods described herein. Further, generally, cells from any population such as a population of single-celled organisms, prokaryotic or eukaryotic, including bacteria, fungi or yeast can be used in this method. Thus, in this specification, it is preferred to select a sample from the group including tissue samples, biopsy samples, liquid biopsy samples, blood samples, plasma samples, urine samples, liquor samples, environmental samples, samples derived from cell cultures, and samples derived from microbial cultures.

[0249] In some embodiments of the methods described herein, obtaining a sample can include first obtaining single cells and then lysing the cells.

[0250] A single cell suspension can be obtained using standard methods known in the art, including, for example, enzymatically using an enzyme on a tissue sample or mechanically releasing adherent cells in a culture. Single cells can be placed in any suitable reaction vessel in which the single cells can be individually processed. For example, a 96-well plate, where each single cell is placed in a single well, which is also referred to as compartmentalization. Individual cells can be individually selected based on measurable parameters such as location, morphology, or gene expression, or based on detectable features by various analytical methods.

[0251] Methods for lysing cells are well known in the art. Lysis can preferably be achieved, for example, by using a detergent or other chemical method, or by a combination thereof.

[0252] A single cell specimen can be chemically treated prior to the second compartmentalization. For example, various fixing chemicals or crosslinking agents (such as BS3 - (bis(sulfosuccinimidyl)suberate)) can be used, or formaldehyde and glutaraldehyde can create bonds between lysine residues to generate crosslinked proteins. Formaldehyde and glutaraldehyde are usually used at a concentration of 0.5% - 4% in PBS depending on the sample. The single cell sample may or may not be crosslinked. Depending on the experimental purpose and the type of target being investigated, the single cell target can be analyzed in either a denatured or non - denatured form, and / or in either a crosslinked or non - crosslinked form. The single cell protein sample can be analyzed under multiple conditions to collect information regarding the quantitative characteristics of multiple targets. For example, the dissociation constant and other kinetic parameters can be determined by varying the concentration or amount of the binding agent.

[0253] Another permeabilization treatment step can be applied to the single cell to access intracellular targets. Thereby, the binding agent can access the intracellular structure without damaging the cell morphology. Triton, digitonin, and saponin are examples of permeabilization reagents that act by disrupting the cell membrane. The level of permeabilization is important because epitope access may require different levels of permeabilization (e.g., cytoplasmic epitopes vs. nuclear epitopes). Such techniques are commonly used in fluorescence - activated cell sorting studies and are well known in the art. Many commercially available kits that provide reagents for both fixation and permeabilization are also currently available. However, the preferred method described in International Publication No. WO 2016 / 083793 is a homogeneous assay and does not require a washing step, so it is preferred that the present invention does not need to remove unbound antibodies.

[0254] In an embodiment, the present invention relates to a method for performing a homogeneous assay using the binding agent within a first partition exposed to the lysed cell material. In some embodiments where step d. (binding reaction) is carried out after step e. and step f. (compartmentalization and lysis), efficient and reliable analysis by additional binding of the binding agent under lysis conditions becomes possible. Thereby, it is preferable to prevent interference of binding from cell components. By adopting such an approach, the assay preferably achieves an improvement in sensitivity and accuracy in detecting and quantifying analytes present in the lysed cell material.

[0255] Single cells can be preselected. For example, single cells may be enriched in a particular protein present, such as a protein from a particular cellular location, a protein from a particular cell type, a protein having a similar size or electrostatic charge, a protein having similar binding properties, similar sequence properties, or a similar function (e.g., an enzyme) (Current Protocols in Molecular Biology (2006) 20.0.1 - 20.0.6 CHAPTER 20 Analysis of Protein Interactions.). The particular protein preferably includes a phosphorylated protein, a membrane protein, or a protein artificially modified after natural translation.

[0256] Single cells may present protein display either extracellularly or intracellularly. Examples are well known in the art and are described in [Galan, A., Comor, L., Horvatic, A., Kules, 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].

[0257] The target can include a number of epitopes and can bind to multiple binding agents. In a preferred embodiment, the methods of International Publication No. WO 2016 / 083793 or International Publication No. WO 2020 / 260277 are used to detect all bound binding agents in pairs.

[0258] The unit of detection is defined as a complex of two binding agents bound to a target and is referred to as a complex. When three or more binding agents are bound, all the unique combinatorial combinations of binding agent pairs are considered different complexes.

[0259] The "label" or unique label herein includes or consists of a unique DNA, RNA, and / or protein sequence. Any suitable label known in the art can be used as the label for the binding agent herein. The label can be attached, linked, or bound to the primer, probe, molecule, or binding agent covalently or non-covalently. In some embodiments, the target may include or consist of a fluorophore and / or a quencher.

[0260] In next-generation sequencing detection in embodiments, the binding agent can preferably be labeled with a unique PCR-amplifiable nucleic acid, preferably a DNA label, specific for the type of binding agent (e.g., antibody), a label individual to each binding agent molecule, i.e., a unique molecular barcode or unique molecular identifier - UMI (see Parekh et al., 2017). The labeled binding agent can be added to the sample or a dilution thereof in a preferred embodiment.

[0261] In embodiments involving PCR, qPCR, or dPCR detection, the PCR reagent may be added before emulsification of the sample, or may be added individually to all emulsion droplets (partitions), for example, by droplet fusion or microinjection. PCR or dPCR may be performed using standard dPCR protocols known in the art. Evaluation of the reaction may be based on partitioning of the label in a dPCR reaction using a fluorescently tagged PCR product (e.g., using a real-time PCR probe labeled with FAM or VIC). According to dPCR, standard evaluation of a cluster of droplets can be determined according to the fluorescence signal of the droplets. Here, the number of label binders (e.g., antibodies) in each reaction is determined (counting all label-positive droplets for a given label and using the same defined droplet clusters for all reactions). Furthermore, the number of two colors (e.g., having two different binder labels of different types of binders or binders for different target analytes) is also determined. In the absence of a ternary complex, the labeled antibodies are partitioned according to a Poisson distribution, generating a countable number of two-color droplets (accidentally having two binding components in one compartment but not binding to the present target). In the case of a ternary complex present during the reaction, the number of detected, e.g., two-color droplets (having additional ternary complexes) is greater than the number expected by the Poisson distribution. Based on this measurement, the number of complexes can be calculated (see, for further details, European Patent Application Publication No. 20 3224360 or Karakus et al., 2019, etc.). This provides absolute (molecular number) quantification of the ternary analyte complex.

[0262] In embodiments where multiple target analytes are to be detected, a library can be provided that includes binding agents specific to different analytes, and each binding agent is equipped with a unique label not only for the type of binding agent but also for the specific analyte to be detected. When the number of different target analytes to be detected is small, this uniqueness can be encoded by different fluorescent labels having different colors. In the case of a large number of different target analytes, readout / analysis can be performed by nucleic acid sequencing such as NGS, in which case the label must be a nucleic acid sequence, and the nucleic acid sequence contains a unique sequence for each target analyte and for each type of binding agent. In embodiments, a droplet-specific or molecule-specific barcode is added to the label of each binding agent during the first binding agent labeling reaction or at a later time to each emulsion droplet (partition).

[0263] For each labeled molecule, one or more labels may be used. All labels are molecularly unique as described in WO 2012 / 042374, but are preferably read using WO 2020 / 260277. One labeled molecule preferably has more labels, and all of them are preferably molecularly unique.

[0264] The unique label can also be an identity label for clearly identifying all members of the binding agents and targets of the present invention, i.e., a related unique sequence used in the binding agent and / or target, and this identity is not the identity of molecularly different labels. The label is preferably both an identity label and a molecule-unique label.

[0265] In embodiments, since the unique label may have different biological backgrounds, the amplification and physical ligation processes based on different biologically specific primer pairs (e.g., one or more primer pairs) amplify the target sequence. By ligating different labels, specific information of the binding agent can be linked to the target information.

[0266] To perform nucleic acid amplification for each compartment or partition to generate linked nucleic acid barcodes can be achieved by highly diluting the sample prior to nucleic acid amplification, for example, with a dilution factor of more than 20,000, preferably more than 50,000, more preferably more than 100,000. In a preferred embodiment, the nucleic acid amplification is PCR. In other preferred embodiments, the nucleic acid amplification is any suitable method known in the art. Addition of PCR reagents can achieve separation of a single complex and nucleic acid amplification for each emulsion droplet or partition. For this purpose, a digital PCR (dPCR) standard protocol may be particularly suitable. International Publication No. WO 2016 / 083793 describes methods of simultaneously using ligation PCR or methods only for generating linkage information. Thereafter, compartments, such as emulsion droplets, may be recombined into a common pool, and antibody-specific dimerization labels (e.g., UMI) can be evaluated using parallel nucleic acid sequencing techniques. As described herein, the number of labeled binders, e.g., antibodies, can be determined in each reaction by counting all unique labels (e.g., UMI) of a given binder, e.g., an antibody. Complexes can be counted based on dimerized dual (UMI-) labeled PCR products containing two different binder-specific labels. In the absence of ternary complex (complex) formation, the labeled binders are partitioned according to a Poisson distribution, and a countable number of complex droplets are obtained. In the case of complexes (target analytes present in the sample) present during the reaction, the number of detected complexes will be more than the number predicted by a pure Poisson distribution.

[0267] Methods for attaching nucleotides to binding components such as antibodies or compounds are known in the art. Alternatively, where the binding component library is a phage display library, the unique nucleotide sequence may be a sequence encoding one or more CDR regions, or it may be the displayed binding domain. For example, a display library can be generated by inserting a sequence encoding the amino acid sequence to be displayed into a phage at a known location. The binding sequence can then be identified using universal primers that amplify the inserted sequence.

[0268] Alternatively, in an embodiment, the binder is an aptamer, and the aptamer itself can be a unique nucleotide sequence. The nucleotide sequence may be an oligonucleotide and may include single-stranded or double-stranded RNA or DNA. The nucleotides used to label the binder or target are preferably 5 to 150 bases in length, for example, 10 to 40 bases or 40 to 80 bases in length. Nucleotides forming the nucleic acid can be chemically modified to enhance the stability of the molecule, improve bioavailability, or impart additional activity to the molecule. For example, pyrimidine bases can be modified at the 6 or 8 position, and purine bases can be modified at the 5' position with a halogen such as CH3 or I, Br, or Cl. Modified or pyrimidine bases also include 2-NH3, 0-CH3, N-CH3, and N2-CH3. Modifications at the 2' position are sugar modifications and typically include an NH2 group, an F group, or an OCH3 group. Modifications 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 also be used. Morpholino oligonucleotides are assembled from various morpholino subunits, each subunit containing one of four genetic bases (adenine, cytosine, guanine, and thymine) linked to a 6-membered morpholine ring. The subunits are linked by non-ionic phosphorodiamidate and the bonds between the subunits to produce a morpholino oligonucleotide. LNA monomers are characterized in that the conformation of the furanose ring is restricted by a methylene linker connecting the 2'-O position and the 4'-C position. PNA is an analog of DNA in which the backbone is a pseudopeptide rather than a sugar.

Brief Description of the Drawings

[0269]

Figure 1

Figure 2

Figure 3-1

Figure 3-2

Figure 4-1

Figure 4-2

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0270] Example 1 This example shows an example of the initial compartmentalization of the binding reaction between cells and antibodies, and this compartmentalization can preferably be carried out at a high cell diffusion rate-limiting concentration. The concentration of the target protein (target analyte) of the binding reaction is preferably determined by the volume of the cell suspension and the total content of the target protein in that volume. As shown in this example, under appropriate conditions and assumptions, the concentration of the target protein / analyte can be treated as if it were in solution. Thus, in such an embodiment, their concentration per cell can be inferred from the concentration calculated for the entire sample volume and the number of cells in the above sample volume. By separating each single cell and using an appropriate high-sensitivity method, the number of targets per cell can be determined from the value obtained by multiplying (``divided'') the extrapolated concentration of the target in the suspension volume by the number of cells in the suspension. This method preferably includes a second compartmentalization that can be used for the counting of single molecules containing both the complex and the binding agent. Preferably, the cells are lysed prior to the second compartmentalization. Preferably, the extrapolated concentration of the target in the suspension volume is determined using binding characteristics, and ultimately, as described in this example, the absolute amount of the target analyte per single cell is determined. This example further demonstrates that the method, by the double compartmentalization described herein, surprisingly facilitates the detection of target analyte concentrations of less than 1000 copies per cell, or less than 100 copies or less than 10 copies.

[0271] The method employed in this example MCF7 (ATCC® HTB-22) and BT-474 (ATCC® HTB-20) cells were obtained from BIOSS Centre for Biological Signaling Studies (Freiburg, Germany). MCF7 cells were cultured in DMEM, GlutaMAX Supplement (31966021, Gibco), and BT-474 cells were cultured in DMEM / F12, GlutaMAX Supplement (31331028, Gibco) in Nunc EasYFlask cell culture flasks (156340, Thermo Scientific®). Both media were supplemented with 10% FBS (10270106, Gibco) and 1% Pen / Strep (15140122, Gibco). Cells were cultured in a cell culture incubator (Heracell® 150i CO2 incubator, 50116048, Thermo Scientific®) at 37 °C in a 5% CO2 atmosphere until they reached approximately 90% confluence. Cells were harvested by scraping. Cells were washed twice with DPBS (14040133, Gibco) and counted (Countess® II Automated Cell Counter, Invitrogen®) including live / dead staining with trypan blue (T10282, Invitrogen®).

[0272] Using an immediate drop-on-demand technology (I.DOT One; Dispendix, Stuttgart, Germany) equipped with an I.DOT PURE plate 90 μm orifice (Dispendix, Stuttgart, Germany) [50, 51], 0.5 μl of LBTW was dispensed into a 384-well V-bottom plate (0030623304, Eppendorf). Prior to dispensing, the I.DOT was calibrated according to the applied liquid to ensure reliable dispensing. Diluted crude cell lysate ("cl") or DPBS was dispensed using the liquid class "H2O".

[0273] The binder was an antibody conjugated with a DNA label using a unique procedure of Actome GmbH of Germany in this example. The achieved labeling efficiency was 80% - 100%, which was confirmed by SDS-PAGE electrophoresis. The antibody preparation had 3% free label and 25% labeling error (see Example 5). Trastuzumab (TTZ) and pertuzumab (PTZ) are recombinant humanized monoclonal antibodies, both targeting the extracellular region of the HER2 tyrosine kinase receptor with non-overlapping epitopes. The conjugate antibody has two different labels, trastuzumab BL label and pertuzumab P8 label. The apparent dissociation constants of the antibodies were determined according to Example 6, being TTZ = 0.49E-10 M and PTZ = 0.93E-10, respectively.

[0274] The two-component assay used was similar to that described in WO 2016 / 083793 and is included herein by reference. Briefly, the cell concentrations of MCF7 and BT-474 were adjusted to 1×10 7 cells / ml, and 100 μL of the cell suspension was incubated overnight with the conjugate antibody (a mixture of ABX-antibodies), or the cell suspension was lysed using a unique lysis buffer supplied by Actome GmbH of Germany, and the lysate was incubated overnight with the antibody (ABX was added).

[0275] For non-dissolved cells, the cell dispensing procedure was performed as described above (Gross, 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, an F.SIGHT™ single cell dispenser (CYTENA GmbH, Freiburg, Germany), an improved version of the single cell printer (SCP), was used (A. Gross, J. Schoendube, 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.). The cell concentrations of both MCF7 and BT-474 were adjusted to 1×10e6 cells / ml and loaded into a dispensing cartridge (CYTENA GmbH, Freiburg, Germany). The settings for MCF7 cells were a cell size of 10 μm to 25 μm (BT-474: 10 μm to 30 μm) and a circularity of 0.5 to 1 (the same as BT-474), respectively. F.SIGHT can reliably dispense single cells with a minimal amount of liquid. The single cell dispensing efficiency (excluding single cell isolation success events from the target events) is typically about 90% and is further controlled by cell images clearly assigned to each dispensing event.Therefore, events other than single-cell dispensing events such as doublets or empty droplets can be excluded.

[0276] QIAGEN's QIAcuity probe master mix (41 μL) was added, and the sample (0.5 μL for both the lysed bulk sample and the single-cell sample) was loaded onto a QIAcuity nanoplate 26k 24-well plate. Digital PCR was performed using cycling parameters according to the PCR conditions of 40 cycles of denaturation at 95°C for 15 seconds and annealing at 58°C for 30 seconds with a hot start at 95°C for 2 minutes using a QIAGEN QIAcuity digital PCR system. Imaging conditions: P8-labeled - FAM green channel, integration time 500 milliseconds, gain 6, and BL-labeled - HEX yellow channel, 400 milliseconds integration time, gain 6.

[0277] The cells were processed according to the following steps. (a) Single cells were each labeled with PTZ and TTZ and incubated at 1×10e6 cells / 50 μl. (b) After overnight incubation, the cells were printed into 0.5 μL (I-DOT dispensing) of lysis buffer. (c) QIAcuity probe master mix (41 μL) was added, and the sample (0.5 μL for both the lysed bulk sample and the single-cell sample) was loaded onto QIAcuity. A negative control (antibody binding control) containing only the labeled trastuzumab antibody and pertuzumab antibody was also set up with either a cell-free buffer or one derived from an ABX antibody mixture (antibody binding control).

[0278] The calibration curves referred to herein are derived in the same manner as those described in International Publication No. WO 2020 / 260277, but the binding characteristics described herein are applied.

[0279] In the case of bulk lysates, the calibration curve indicates the molar concentration of the target analyte (HER2 protein in this example). The number of complexes in the dPCR reaction of the dispensed single-cell equivalent lysates was directly read out and multiplied by the dilution factor of the binding reaction to obtain the molar concentration of the complexes in the binding reaction (the dilution factor of the binding reaction is determined by the volume fraction of the binding reaction measured in the dPCR reaction). The molar concentration of the antibody was also measured in the same way, and the row counts were obtained by directly reading the dPCR reaction and applying the dilution factor of the binding reaction in the same way. The apparent dissociation constant of the antibody was determined previously. Using the value compensated for the binding characteristics, the absolute concentration of the analyte in the bulk binding reaction was determined.

[0280] In the case of single cells, the calibration curve indicates the number of target analytes (HER2 protein) determined for each single cell. The molar concentrations of the antibody and the complexes in the binding reaction were determined by applying the dilution factor of the binding reaction in the same way. However, the calculation method of the dilution factor is different. Since a single cell represents a single-cell fraction of the target protein (analyte), the number of complexes of the measured single cell was multiplied by the number of cells in the suspension volume, and as a result, the number of cells in the suspension volume was treated as the dilution factor of the binding reaction. The apparent dissociation constant of the antibody was determined previously. Using the molar input compensated for the binding characteristics and the apparent dissociation constant of the antibody, the absolute number of target analytes in the suspension (sample) was determined. Briefly, considering all the cells in the suspension, the molar concentrations of the complexes and the binder were calculated, and in the suspension, solution-based conditions were assumed during the calculation. Stoichiometry was performed in the same way as described in International Publication No. WO 2020 / 260277 to determine the absolute molar concentration. Considering that the molar concentrations of the complexes and the binder are derived from individual single cells, this is a mathematical compensation for the target forming non-complexes that are not bound, and the absolute concentration is obtained. The absolute (compensated) molar concentration of the target was obtained, and considering the number of cells in the suspension, the absolute number of target analytes per single cell was determined. The number of target proteins per single cell was derived from the absolute concentration of the target by dividing by the number of cells in the suspension.

[0281] Regarding the effectiveness of this method, in order not to significantly reduce the free antibody concentration, it was assumed that the target concentration for single cells was lower than the antibody concentration in the cell suspension. However, at the physiological concentration of proteins in cells, this assumption usually holds, and it is known to those skilled in the art that recursive calculations compensate for it.

[0282] It should be noted that the detection of single cells without permeabilization is limited to the cell surface only, so the cell bulk lysate contains more detectable HER2 molecules than single cells.

[0283] In the case of MCF7 cells, the average number of HER2 proteins per single cell is 4500 ± 1060 molecules, while the expected number under bulk conditions is 10150 ± 1260 molecules. Similarly, BT474 cells show approximately 208000 ± 1260 HER2 proteins per single cell compared to the result of 2.3E+6 ± 8.E+5 molecules under bulk conditions. Such a difference in the amount of HER2 protein is due to the essential difference between the protein pool accessible on the surface and the protein pool accessible in bulk. The method disclosed herein provides a reliable means for quantifying HER2 proteins in single cells and comparing them with bulk conditions, thereby facilitating a comprehensive understanding of protein distribution at both the cell level and the population level.

[0284] Example 2 The plate-based "scPICO" method according to the present invention Reagents and buffers On the day of the experiment, the reagents and buffers were prepared as follows.

[0285] Additive C (5× stock solution) 500 μl PBS

[0286] BSA (5× stock solution) 20 mg BSA 400 μl PBS

[0287] EDTA-free protease inhibitor cocktail (PIC), (25× stock solution) Complete Protease (trademark) inhibitor cocktail, 1 tablet Make up to 2 ml with PBS.

[0288] Cell lysis buffer (LBT), (2× stock solution) 200 μl of additive T (10×) 400 μl of dissolved additive C (5×) 80 μl of PIC (25×) 200 μl of additive L 120 μl of PBS

[0289] Cell lysis buffer (LBTW), (1×) 300 μl of LBT buffer (2× stock solution) 300 μl of PBS Antibody binding control (ABC) buffer, (1×) 250 μl of LBT buffer (2× stock solution) 100 μl of BSA (5×) 150 μl of PBS

[0290] Oligonucleotide

[0291]

Table A

[0292] Antibody mix For the detection of HER2 in single BT474 cells, two anti-HER2 humanized monoclonal antibodies, pertuzumab (PTZ) and trastuzumab (TTZ), were used. Both antibodies were modified by covalent bonding with a DNA oligonucleotide label using the PICOglue technology. The label contains a 10-base pair random nucleotide sequence (UMI) and TaqMan hydrolysis probe annealing sites for binding to P8 and NOS-6 (PTZ-P8 and TTZ-NOS6), respectively (see below). The antibody mix was prepared by diluting the labeled antibody stock solution with LBTW so that the molar concentration of each antibody was 5.00×10-10 It was prepared to be M. Subsequently, the antibody mix was diluted 1:2 with LBTW and stained with fluorescein until the final concentration reached 1 μM. The applied concentration was aimed at providing saturation conditions.

[0293] Cell culture Mammary duct carcinoma BT474 cells were grown in T175 flasks in DMEM F12 medium supplemented with GlutaMax + 10% FCS + 1% P / S and split 1:2 to 1:3 at 80% - 90% confluence every 5 - 7 days. After washing with 10 ml of PBS, 5 ml of Versene non - enzymatic cell dissociation reagent (Thermo) was incubated at room temperature for 3 - 5 minutes to recover the cells. When the cells began to detach, the reaction was stopped by diluting 1:2 with PBS, and then the cell suspension was centrifuged at 400 g for 5 minutes. Subsequently, the cells were resuspended in 40 ml of fresh medium per T175 flask and seeded.

[0294] In the single - cell analysis according to the present invention (the "single - cell PICO approach"), T175 flasks of BT474 cell culture were harvested as described above, resuspended in 1000 μl of PBS, and subsequently filtered through a 30 - μm cell strainer. The cells were counted, centrifuged at 400 g for 2 minutes, washed again with 1 ml of PBS, and resuspended in PBS to a density of approximately 0.5×10 -6 cells / ml and stained with 100 μM of fluorescein. The cells were stored on ice until use.

[0295] Preparation of 384 - well plates and single - cell isolation The wells of a 384-well Eppendorf microplate 384 / V-PP were pre-filled with 4 μl of vapor lock PCR overlay (Qiagen) and centrifuged briefly at 1000 g. Using an I.DOT liquid handler (Dispendix), 12 nl of antibody mix (1:2 dilution in LBTW, 1 μM fluorescein) was dispensed into each well. 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 dispensing and positioning were microscopically controlled with a Dino-Light fluorescence microscope (AM4115T-GFBW).

[0296] Single BT474 cells were isolated with an f.sight single cell dispenser (Cytena) with automatic offset correction (AOC) enabled. Single cells with a size of 15 μm to 25 μm and a circularity setting of 0.6 to 1.0 were selected, and only reactions receiving one cell were valid for downstream processing.

[0297] The fusion of antibody mix droplets and droplets containing single cells was considered successful if an increase in the fluorescence signal was observed compared to reactions not receiving single cells. Additional reactions not receiving single cells also functioned as in-plate antibody binding controls (ip ABC) (see below).

[0298] After isolating single cells, the plate was sealed with a PCR foil (4titude) and incubated at 4 °C for approximately 24 hours, resulting in both cell lysis and a binding reaction (complex formation) between HER2 and the two antibodies during incubation.

[0299]

Table B

[0300] Preparation of control binding reactants and sample diluent Before use as input for dPCR, 12 nl of single-cell binding reactants were diluted with PBS as follows. To achieve a 1:5000 pre-dilution, 60 μl of PBS was added directly to the single-cell reactants, followed by repeated pipetting and centrifugation at 2000 g for 2 minutes. 5 μl of the pre-diluent was transferred to 42.5 μl of PBS (1:9.5), and pipetting was repeated to mix. 1 μl and 1.32 μl of this diluent were used as input for the dPCR master mixes of Stilla and QIAcuity, respectively. With partition sizes of 0.78 nl and 0.59 nl for the Stilla Sapphire Chip and QIAcuity system, respectively, the input amounts were correct and a lambda value of 0.15 was achieved.

[0301] This corresponds to total dilution factors of approximately 1.19×10 6 and 1.52×10 6 respectively.

[0302] dPCR master mix

[0303]

Table C

[0304] The partitioning reaction and dPCR reaction were performed using the Naica Geode system with a Sapphire Chip (Stilla) for coupling PCR and the QIAcuity system (Qiagen) with a nanoplate 24 26k.

[0305] Naica Sapphire system - dPCR parameters: 1. Partitioning at 40 °C with Sapphire V1 2. 10 minutes at 95 °C 3. Start 40 cycles 30 seconds at 95 °C 15 seconds at 60 °C 4. Start 20 cycles 30 seconds at 95 °C 15 seconds at 50 °C 5. End cycle 6. Release pressure, Sapphire V1

[0306] QIAcuity system - nanoplate 24 26k - dPCR parameters: 1. QIAcuity standard priming 2. 2 minutes at 95 °C 3. Start 40 cycles 30 seconds at 95 °C 15 seconds at 60 °C 4. Start 20 cycles 30 seconds at 95 °C 15 seconds at 50 °C 5. End cycle

[0307] dPCR reaction Emulsification, ddPCR, and readout were performed using the Stilla Naica system according to the standard dPCR protocol. As a negative control, the antibody was mixed without antigen and processed in the same way as the samples (antibody binding control, ABC).

[0308] The amount of protein HER2 was measured by detecting the difference in partitioning induced by the simultaneous binding of the antibody to HER2. The measurements were normalized against an ABC control that defines zero-level detection by the ABC signal. Figure 1 shows the results of dPCR for single-cell analysis using appropriate controls.

[0309] The NTC and ABC readings were zero, indicating a contamination-free dPCR reaction and a balanced dPCR reading. The BT474sc sample showed an average of approximately 987,000 (±7.6%) HER2 molecules per cell, considering the number of complexes detected using the applied dilution method and other factors affecting the number of complexes (as described in detail in Karakus et al., European Patent No. EP 22161450.6, which is hereby incorporated by reference). In contrast, the rHER2 sample showed approximately 876,000 (±9.8%) HER2 molecules considering the same procedure.

[0310] Droplet Recovery and DNA Extraction from Sapphire Chip First, the inlets and outlets of the sapphire chip were completely emptied with a pipette, and the collected oil was transferred to a collection tube. Next, an Eppendorf Combitip Advance (1.0 mL, yellow) was firmly inserted into the outlet. Then, 50 μl of Novec 7500 was filled into each inlet. After that, the Combitip piston was slowly pulled to recover the emulsion in the chip until the liquid level in the inlet dropped to the bottom. This process was repeated once, 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 using a pipette. Demulsification was performed by adding 60 μl of 20% PFO (volume / volume) to Novec 7500, vortexing for 10 seconds, and centrifuging at 1000 g for 2 minutes. The separation of the aqueous and oil phases was achieved by slowly inverting the tube and recovering the aqueous phase from the bottom of the tube.

[0311] Preparation of NGS Library For the preparation of the NGS library, modifications were made to the manufacturer's protocol with the following settings. All reaction volumes were reduced by 50%, and approximately 5 ng of unpurified amplicon DNA was used as the input amount. The NEB adapters were diluted 25-fold (1:25) to 0.6 μM with 10 mM Tris-HCl (pH 8.0) containing 10 mM NaCl, and size selection was not performed prior to PCR. The library DNA was purified with 22.5 μl (0.9×) of resuspension beads (NEB) according to the protocol.

[0312] NGS on the Illumina MiSeq Platform The libraries were pooled equimolarly, and NGS was performed using the MiSeq platform equipped with the MiSeq Reagent Kit v2 in 300-cycle paired-end according to the manufacturer's protocol. After denaturation, the library pool was diluted to 5 pM, and 10% PhiX control was spiked in.

[0313] The NGS results were calculated according to the procedure described in (International Application PCT / EP2020 / 067493), briefly, compartmentalized dPCR was performed using molecularly unique labels (UMIs), and dimers were deconvoluted to reconstruct the droplet content (here, ddPCR was performed, preferably containing only one complex). That is, the evaluation of the reaction may be based on the NGS readings of binders generated according to the standard protocol of emulsion coupling, such as antibodies, specific dimerizing UMI labels. The number of labeled binders, such as antibodies, can be determined in each reaction by counting all the unique UMI labels of a given binder, such as an antibody (counting limited to a given binder). Multiple labels of the same binder, such as an antibody, can be excluded by using preferentially dimerized sequences (if there are multiple labels for each binder, they will always form a double UMI-labeled dimer with a given antibody-specific label context because they co-localize in the same droplet). Complexes are counted based on dimerized double UMI-labeled PCR products (in relation to two different binder-type specific labels, such as an antibody against a specific HER2 antigen).

[0314] Once the contents of the droplet were obtained, Poisson-based calculations (described in European Patent No. 22161450.6) were performed to confirm the number of detected complexes. Briefly, since the distribution of the binder during compartmentalization is governed by the Poisson distribution, the number of occurrences of each binder can be counted directly by dPCR (or measurement of the abundance of the binder by NGS after droplet-based PCR amplification), and the background detection of the complex can be calculated if the number of droplets is known. Using a large number of specific binding events, the exact target agent of the binder can be identified, which can also be performed in the same way. For example, in a multi-protein complex, the information contained in the linked labels of the binder is based on the co-localization of each binder on the complex and indicates a direct interaction between the detected target analytes. For each protein / protein complex, when calculating the background detection, any variation in the detected protein / protein complex is due to different interactions, and it can be calculated by subtracting the calculated background detection of the protein / protein complex (or Poisson correction subtraction because the protein / protein complex bound to the binder changes the total number of binders). The value compensated by ABC is the BT474sc sample, indicating that approximately 943,000 (±14.8%) HER2 molecules were detected per cell.

[0315] Example 3 This example shows an example of a method for determining the binding characteristics of a binder under various conditions of dissociation constant (Kd), or labeling ratio, labeling synthesis error, active fraction, and contamination by free label, and then applying it to the method according to the present invention to derive the absolute amount of the formed complex (or ternary complex), facilitating the determination of the target standard free dissociation constant of the antibody in a complex chemical environment and a native environment.

[0316] [Table 1] Name Complex

[0317] Table 1: Simulated equimolar titration data. Column headings are "Name" - sample name, "Complex" - two antibody-target complexes, "ableft" - concentration of antibody x1, "right" - concentration of antibody x2, all concentrations in M units. ABX means "antibody mix" and contains equimolar concentrations of the two antibodies shown.

[0318] Figure 2 shows a graphical representation of the simulated equimolar titration data (see Table 1). The preset concentration of the target is 1.0E-11 M (isoCC), the preset Kd values are 5.0E-11 (Kdx1) and 1.0E-10 (Kdx2), and the data values are such that the labeled fraction value is 1, the active fraction value is 1, and there is no labeled error and free label. Antibody concentrations are shown on the curve and are set to 4.0E-11, 1.0E-11, 4.0E-10, 1.1E-9, and 5.0E-9. The concentration of the complex is derived by solving the equation described in WO 2020 / 260277. The recursively minimized standard deviation of the antigen concentration was 0.18% (STDp) or 1.81E-14 (STD).

[0319] [Table 2] Name Complex

[0320] Table 2: Simulated equimolar titration data for determining new complex concentrations using the preset Kd values of the antibodies, where the active concentration of antibody x1 is changed to 0.5 of the active fraction x1. Column headings are "Name" - sample name, "Complex" - complex, "ableft" - concentration of antibody x1, "right" - concentration of antibody x2, all concentrations in M units. ABX means "antibody mix" and contains the concentrations of the two antibodies shown. From the data, it can be seen that when the amount of "ableft" is half of its original value, the corresponding complex value also becomes smaller (the amount of the complex was determined by simulation).

[0321] [Table 3] Name Complex

[0322] Table 3 Equimolar titration data simulated using the complex concentration at a value of 0.5 of the active fraction of antibody x1 in Table 2 to determine the new apparent Kd of the antibody. The concentration of the complex is the same as in Table 2. The active fraction is 0.5, but the active fraction is considered unknown and "ableft" was not compensated as in Table 2. Column headings are "Name" - sample name, "Complex" - two antibody - target complexes, "ableft" - concentration of antibody x1, "right" - concentration of antibody x2, all concentrations are in M units. ABX means "antibody mix" and includes the concentrations of the two antibodies shown. When the active fraction is 1, the "ableft" value is adjusted, but the complex concentration follows the value of 0.5 of the active fraction.

[0323] Figure 3 shows a graphical representation of the equimolar titration data simulated at an active fraction of 0.5 (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 values are 1.0E - 10 (Kdx1) and 1.0E - 10 (Kdx2), respectively. The recursively minimized standard deviation of the antigen concentration was 0.12% (STDp) or 1.16E - 14 (STD). B. Data from Table 2. The recursively minimized standard deviation of the antigen concentration was 2.28% (STDp) or 2.33E - 13 (STD), and the (apparent) Kd values are 5.0E - 11 (Kdx1) and 1.0E - 10 (Kdx2), respectively. These values are the same as those in Figure 2 and reproduce the preset values. (A) describes the case where the active fraction is unknown and the active concentration of the antibody is incorrectly determined based on the labeled antibody (the actual concentration decreases because the inactive antibody is non - reactive). After recursively minimizing the standard deviation of the antigen concentration, it is shown that isoCC is the same as the original isoCC, but the apparent Kd is not. (B) Control simulation. The antibody concentrations are adjusted according to the active fraction to reproduce the original Kd along with the original isoCC.

[0324]

Table 4

[0325] Table 4 Simulated equimolar titration data using the corrected antibody x1 and complex concentration at a value of 0.5 of the labeling fraction to determine the apparent Kd of the antibody. Column headings are "Name" - sample name, "Complex" - complex, "ableft" - concentration of antibody x2, "right" - concentration of antibody x1, all concentrations are in M units. ABX means "antibody mix" and contains the concentrations of the two antibodies shown. The values of "ableft" and "Complex" are adjusted so that the labeling fraction is 0.5 compared to Table 1.

[0326] Figure 4 graphically represents the simulated equimolar titration data at a labeling fraction of 0.5 (see Table 4). The preset concentration of the target was 1.0E-11 M (isoCC). Figure 4A shows that the apparent antibody concentration and complex concentration were adjusted based on the simulation, and the indicated antibody concentrations were 2.0E-11, 5.0E-11, 2.0E-10, 5.5E-10, and 2.50E-9 (half if only the labeling fraction was detected). Subfigure A does not show recursive minimization. The Kd values were 5.0E-11 (Kdx1) and 1.0E-10 (Kdx2), respectively. The standard deviation of the antigen concentration was 17.11% (STDp) or 1.04E-12 (STD). Figure 4B shows that the standard deviation of the antigen concentration after recursive minimization was 0.37% (STDp) or 1.84E-14 (STD), the apparent Kd values were 5.0E-11 (Kdx1) and 1.0E-10 (Kdx2), respectively, and the antigen concentration (isoCC) was 5.0E-12. Since the labeling fraction is 0.5, it is necessary to adjust the antigen concentration to the same 1.0E-11 M as the original antigen concentration.

[0327] The simulation shows that the labeling ratio of the antibody, i.e., the active fraction of the antibody, affects the absolute quantitative determination of the analyte concentration, and the effects are different for them. The active fraction has a self-compensating effect, the apparent Kd confounds the effect of the active fraction, the labeling ratio needs to be determined individually, and numerical compensation is required in the absolute quantification result. Numerical adjustment based on the labeling ratio and active fraction of the antibody improves the accuracy of the absolute quantitative measurement of the analyte concentration using this method.

[0328] Example 4 This example provides an example where the determination of the dissociation constant of the binder(s) is affected by additional factors including the labeling ratio of the binder(s), labeling synthesis error, the active fraction of the binder(s), and contamination by free label. However, as shown in this example, in the determination of the dissociation constant of an antibody, advantageously, the active fraction of the binder(s) and contamination by free label are confounded. As shown herein, preferably, only the labeling ratio and labeling error of the binder need to be determined individually to control and compensate for the accuracy of the method (e.g., the compartmentalized two-component method) for determining the binding characteristics of the binder in the method of the present invention. This method is advantageous for determining the binding characteristics of the binder in the method of the present invention.

[0329] Labeling efficiency and labeling error The "labeling fraction" or "labeling efficiency" or "labeling fraction" of a binder such as an antibody is an independent parameter for determining the absolute quantitative concentration of the target analyte using this method. To mathematically prove b10 (absolute concentration of antibody 1), b20 (absolute concentration of antibody 2), and c12 (absolute concentration of the complex formed in the reaction), the following equations can be expanded:

Equation

[0330] In the formula, lf1 and lf2 are the labeling fractions of two antibodies (the first type of binder and the second type of binder), respectively. lf1 and lf2 are independent variables and are related to other parameters. Note that if the exact values of lf1 and lf2 are determined, it is easy to reconstruct the absolute concentration of the antibody by reversing this conversion.

[0331] Labeling errors can be regarded as binders, such as antibodies, that are unlabeled or contain defective labels, but have the same analytical effect on the absolute quantification of analytes and can be derived in the same mathematical form.

[0332] Active fraction and free label The active fraction of a binder, such as an antibody, is a dependent parameter for the absolute quantitative determination of the concentration of the target analyte. To mathematically prove that b1 (the free absolute concentration of antibody 1), b2 (the free absolute concentration of antibody 2), and c12 (the absolute concentration of the complex formed in the reaction), a (the free absolute concentration of the analyte), c1 and c2 are the absolute concentrations of the bimolecular complexes (complexes) of the analyte with the respective antibodies b1 and b2, the following formula can be expanded.

Equation

[0333] In the formula, af1 and af2 are the active fractions of two antibodies. With some rearrangement, the equation can be converted into an equation with the original chemical equilibrium terms on the right side and the modified terms of constants on the left side. Thus, the change in the active fraction can be deleted because it is self-compensated with respect to the change in the measured apparent dissociation constant:

Equation

[0334] Note that these derived dissociation constants no longer conform to the original definition of the dissociation constant, but represent the apparent effect of the antibody in the reaction. Since the original chemical constants explaining the chemical reaction cannot be used, the correct numerical values can be obtained.

[0335] The presence of free labels that can be regarded as inactive antibodies has the same analytical effect on the absolute quantification of analytes because it can be derived in the same mathematical form.

[0336] Example 5 In a preferred embodiment, in order to control and compensate for the accuracy of the compartmentalized two-component method for determining the dissociation constant of the binder(s), it is necessary to individually determine the labeling ratio and / or labeling synthesis error of the binder(s). This example shows an example of a method for determining the labeling ratio of the binder(s) and the labeling synthesis error of the binder(s). This method is advantageous for determining the binding characteristics of the binder for the method of the present invention for determining the labeling fraction of the binder.

[0337] The labeling efficiency of the antibody is analyzed using an Agilent Protein 230 Chip installed on an Agilent 2100 Bioanalyzer. For this purpose, 4 μl of a solution of labeled antibody and unlabeled (as a control) antibody was loaded according to the manufacturer's instructions. 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) are recorded. The labeling fraction (LE) is calculated using the following concentration values.

Equation

[0338] Figure 5 shows an Agilent Protein 230 Chip on the 2100 Bioanalyzer electrophoresis diagram of a labeled antibody against GAPDH. The labeling fraction was determined according to the text and was 97.6%.

[0339] The labeled antibody was purified using an agarose A / G affinity matrix with several washing steps. The concentration of the labeled antibody was determined by digital PCR (dPCR) using a QIAcuity dPCR device. The method is described elsewhere (see Example 1). Both the appropriately diluted final wash sample and the antibody were measured. The free fraction of the label was calculated by dividing the concentration of the free label by the concentration of the labeled antibody. 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 was 14.4%.

[0340] Labeling error For both the BL label and the P8 label, the labels were synthesized according to the proprietary DNA sequences of Actome GmbH (see Example 1), and these labels were amplified using the dPCR protocol (described in Example 1). dPCR enables determination of the exact amount of amplification / detection of the label.

[0341] For all reactions, 100 μL aliquots of the labeled samples and standards were pipetted into microplate wells, and 100 μL of the OliGreen working solution was added to each well. The OliGreen working solution was freshly prepared by diluting the OliGreen concentrated reagent 1:200 with TE according to the manufacturer's instructions. The samples were incubated at room temperature for approximately 5 minutes, and fluorescence was measured using a Bio-Tek Instruments FL600 fluorescence plate reader equipped with an excitation filter of 485 nm, 20 nm bandwidth and an emission filter of 530 nm, 25 nm bandwidth. The sensitivity settings were changed as needed, and the data were collected using static sampling with a 0.35 second delay with a 3 mm probe from the top, and 50 readings were taken per well.

[0342] The difference in the measured values of the two methods determines the labeling synthesis error.

[0343] Example 6 This example shows the determination of the dissociation constant (Kd) of an antibody considering the labeling ratio, labeling synthesis error, and active fraction of the binder(s). (See Example 3.) Preferably, by using a compartmentalized two-component method using the labeling ratio and labeling synthesis error of the binder(s) to determine the apparent dissociation constant of the binder(s), an advantageous and inherent self-compensation against the active fraction of the binder(s) and contamination by free label is provided, and obtaining the dissociation constant of the antibody results in a bias-free determination of the absolute amount within a single cell. This method is advantageous for determining the binding characteristics of the binder for the method of the present invention for determining the labeled fraction of the binder.

[0344] The antibody was conjugated with a DNA-amplifiable label using the proprietary procedure of Actome GmbH, Germany. The achieved labeling efficiency was 80% - 100%, confirmed by SDS-PAGE electrophoresis (Example 5). The antibody preparation has 3% free label and 25% labeling error (see Example 5). Trastuzumab (TTZ) and pertuzumab (PTZ) are recombinant humanized monoclonal antibodies, both targeting the extracellular region of the HER2 tyrosine kinase receptor with non-overlapping epitopes. The conjugated antibody has two different labels, trastuzumab BL label and pertuzumab P8 label.

[0345] The two-component assay used was similar to that described in International Publication No. WO 2016 / 083793, which is incorporated herein by reference. Briefly, BT-474 cells were randomly crosslinked with 5 mM BS3 in PBS at 4°C for 1 hour. The BT-474 cell concentration was adjusted to 1×10e7 cells / ml and lysed with 100 μL of a proprietary lysis buffer supplied by Actome GmbH, Germany, and the lysate was incubated overnight with the antibody at different concentrations of both antibodies targeting concentrations of 1.00E-09 M, 2.00E-10 M, 4.00E-11 M, 8.00E-12 M, and 1.60E-12 M.

[0346] The incubated lysate was diluted with PBS, and when the concentration of the antibody used was high, a 0.15 lambda concentration was achieved in the final dPCR. Usually, the antibody concentration is the same in ABX. A negative control containing only the labeled trastuzumab antibody and pertuzumab antibody was also set using an antibody concentration of 4.00E-11 M (antibody binding control).

[0347] QIAGEN's QIAcuity probe master mix (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 cycling parameters according to the PCR conditions of 40 cycles of denaturation at 95°C for 15 seconds and annealing at 58°C for 30 seconds with a hot start at 95°C for 2 minutes using the QIAGEN QIAcuity digital PCR system. Imaging conditions: P8 label - FAM green channel, integration time 500 milliseconds, gain 6, and BL label - HEX yellow channel, 400 milliseconds integration time, gain 6.

[0348] From dPCR, the measured raw dPCR parameters (different positive rates) are obtained as the concentration of the antibody (single-color positive rate) and the dual-color positive rate (a two-component complex called misaggregated statistical duplicates and complexes). From these three parameters of individual measurements, the concentration of the complex is obtained using the two-component assay theory (see International Publication No. WO 2020 / 260277). Briefly, the fluorescence readout of the obtained compartments is thresholded and clustered into empty groups, single-positive groups, and dual-positive groups. The dPCR evaluation provides a determination based on the number of positive compartments (antibodies A, B), the number of dual-color compartments, and the number of empty compartments on a single color based on general dPCR theory (Basu, A. S. (2017) Digital Assays Part 15 I: Partitioning Statistics and Digital PCR. doi: 10.1177 / 2472630317705680). The evaluation of the reaction is based on the partitioning of the labels in the dPCR reaction. Normal dPCR evaluation determines the absolute number of labels included in each reaction and then uses this to calculate the number of dual-positive droplets (spontaneous duplicates of the labels) based on the statistical probability of co-compartmentalization (Poisson background). The Poisson background was compared with the number of detected dual-positive droplets (including complexes), and a statistical model was developed to calculate the number of molecular complexes that account for the number of dual-positive droplets above the Poisson background. The concentration of the complex was determined and multiplied by the previously obtained dilution factor to obtain the molar concentration of the complex in the binding reaction. The molar concentration of the antibody was determined similarly, and the concentration was obtained by directly reading the dPCR reaction and applying the dilution factor of the binding reaction as well. Four parallel experiments were performed for a given antibody concentration. The ABC control had a predicted zero complex value, so the deviation from zero indicated evaluation (clustering) and other biases and was used to normalize all measurements when measuring non-zero complexes of ABC.

[0349] Figure 6 shows the calculated calibration curves for each sample. The calibration curves were derived using a method similar to that described in International Publication No. 2020 / 260277, but with adjusted binding characteristics. Using the individually determined labeling ratios and labeling errors of the antibodies, calibration curves corrected with the corrected values of the single-positive and double-positive values of dPCR were created to obtain the corrected concentrations of the antibodies and complexes.

[0350] The apparent dissociation constant of the antibody was calculated by recursively minimizing the standard deviation of the HER2 concentrations recalculated for all measurements, assuming that all samples contained the same concentration of HER2.

[0351] Figure 6 is a graphical representation of the determination of the apparent dissociation constants of trastuzumab and pertuzumab. The samples had equimolar concentrations, but the exact concentration of HER2 was unknown (the samples were obtained from lysed HER2 cells), and the antibody concentrations varied (for the antibodies, 5.5E-9, 1.50E-9, 4.0E-10, 1.2E-10, 3.5E-11, and 1.0E-11). The lines represent the calibration curves of a HER2-specific two-component assay similar to the method described in the assay of International Publication No. 2020 / 260277 at these antibody concentrations and the apparent Kd. The latter was determined by minimizing the SD of the HER2 concentrations recalculated for all measurements, assuming that all samples contained the same concentration of HER2. The molar HER2 concentration is shown as the average HER2, and STD and pSTD are the standard deviations of the measured values, respectively, as a percentage of the average. The dotted line shows the average of the HER2 measurements at various antibody concentrations. Finally, the dissociation of the antibodies (Kd1TTZ and Kd2PTZ) was recursively determined to be 2.9E-11 and 9.3E-11, respectively.

[0352] Example 7 This method is advantageous for determining the RNA content of a sample simultaneously with the determination of the absolute amount of the target analyte using the method of the present invention.

[0353] Simultaneous Detection of RNA To perform RNA detection simultaneously, the DNA-dependent DNA polymerase activity of MMLV reverse transcriptase is utilized, which is described as the SMART library preparation protocol (a mechanism for switching the 5' end of a Clontech-RNA template). Briefly, the MMLV reverse transcriptase switches to a TSO (template-switching DNA oligonucleotide) at the end of RNA-based DNA synthesis initiated from the 3' end of the mRNA (by which a 5' PCR primer site (commonly called a PCR handle) can be introduced), introduces a similar PCR handle at the 3' end at the start of first-strand synthesis, and the handles form an amplicon that can be amplified universally. Briefly, the MMLV reverse transcriptase terminal transferase activity adds several nucleotides (mainly deoxycytidine) to the 3' end of the newly synthesized cDNA strand. These deoxycytidine bases form an annealing site for the template-switching (TS) oligo (TSO). The reverse transcriptase recognizes this new template strand formed against the TSO by cellular RNA and continues the synthesis of the second strand incorporating the universal sequence, i.e., the PCR handle. Single-cell RNA-seq protocols have applied this concept [Non-Patent Document 5 (Ramskoeld, D., Luo, S., Wang, Y.C., Li, R., Deng, Q., Faridani, O.R., Daniels, G.A., Khrebtukova, I., Loring, J.F., Laurent, L.C., et al. (2012). Full-length mRNA-seq from single-cell levels of RNA and individual circulating tumor cells. Nat. Biotechnol. 30, 777-782)].

[0354] Template Switching Oligo (TS) Structure. The simplest version of TSO is a DNA oligo sequence with three riboguanosines (rGrGrG) at the 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.]. 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.]. Approaches have been reported to reduce library background and thereby improve cDNA yield by incorporating isomeric nucleotides into the TS oligo [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 added to the 5' end of the TS oligo and formed hydrogen bonds with each other, but the naturally occurring C and G nucleotides did not effectively lock the 5' end of the TSO, which typically demonstrates the effectiveness in minimizing the concentration of the TS oligo resulting from the cycle 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.].

[0355] MCF7 (ATCC™ HTB-22) cells were cultured in DMEM, GlutaMAX Supplement (31966021, Gibco) supplemented with 10% FBS (10270106, Gibco) and 1% Pen / Strep (15140122, Gibco). Cells were cultured in a cell culture incubator (Heracell™ 150i CO2 Incubator, 50116048, Thermo Scientific™) at 37 °C in a 5% CO2 atmosphere until they reached approximately 90% confluence. Cells were harvested by scraping. Cells were washed twice with DPBS (14040133, Gibco) and counted (Countess™ II Automated Cell Counter, Invitrogen™) including live / dead staining with trypan blue (T10282, Invitrogen™).

[0356] The antibody was conjugated with a DNA-amplifiable label using Actome GmbH's proprietary procedure in Germany. The achieved labeling efficiency was 80% - 100%, which was confirmed by SDS-PAGE electrophoresis. The antibody preparation has 3% free label and 25% labeling error (see Example 5). Trastuzumab (TTZ) and pertuzumab (PTZ) are recombinant humanized monoclonal antibodies, both targeting the extracellular region of the HER2 tyrosine kinase receptor with non-overlapping epitopes. The conjugated antibody has two different labels, trastuzumab BL label and pertuzumab P8 label. The dissociation constants of the antibodies were determined according to Example 6 and were TTZ = 0.49E-10 M and PTZ = 0.93E-10, respectively.

[0357] Using the instant drop-on-demand technology (I.DOT One; Dispendix, Stuttgart, Germany) equipped with an I.DOT PURE plate 90 μm orifice (Dispendix, Stuttgart, Germany), 0.5 μl of LBTW was dispensed into a 384-well V-bottom plate (0030623304, Eppendorf). Before dispensing, the I.DOT was calibrated according to the applied liquid to ensure accurate dispensing. Diluted crude cell lysate ("cl") or DPBS was dispensed using the liquid class "H2O".

[0358] For non-dissolved cells, the cell dispensing procedure was carried out as described above (T. Gross, 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, an F.SIGHT single cell dispenser (CYTENA GmbH, Freiburg, Germany), an improved version of the single cell printer (SCP), was used (A. Gross, J. Schoendube, 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). The cell concentrations of both MCF7 and BT-474 were 1×10 6Adjusted to cells / ml and loaded into a dispensing cartridge (CYTENA GmbH, Freiburg, Germany). The settings of MCF7 cells were cell sizes of 10 μm to 25 μm (BT-474: 10 μm to 30 μm) and roundness of 0.5 to 1 (the same as BT-474) respectively (Figure 1a, Figure S3a and Figure S3b). F.SIGHT can reliably dispense single cells with a minimum amount of liquid. The single-cell dispensing efficiency (excluding single-cell isolation success events from target events) is usually about 90% and is further controlled by cell images clearly assigned to each dispensing event. Therefore, events other than single-cell dispensing events such as doublets or empty droplets can be excluded.

[0359] Add the sample (0.5 μL of single-cell sample) and prepare the master mix 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, random hexamer with PCR handle tag), load it into 4 naica (trademark) Crystal Digital PCR system Sapphire Chip reactions, and read it with a Prism3 reader (Stilla Technologies, Virjust, France). Digital PCR was performed with cycling parameters of (5 cycles of incubating at 42°C for 10 minutes and 25°C for 15 seconds, followed by a 2-minute hot start at 95°C, 40 cycles of denaturation at 95°C for 15 seconds and annealing at 58°C for 30 seconds). Imaging conditions: P8 label - FAM green channel, 500 milliseconds integration time, gain 6, and BL label - HEX yellow channel, 400 milliseconds integration time, gain 6.

[0360] Evaluate the two-component assay signal according to the instructions (Example 6).

[0361] After cycling, remove the Sapphire chip from the cycler, remove the blue cap, recover the emulsion, and use 20% perfluorooctanol (PFO) in chloroform or hydrofluoroether (HFE) to break the emulsion and purify the cDNA library. Briefly, add 125 μL of the recovery agent (20% PFO in HFE), 55 μL of GITC buffer (5 M GITC, 25 mM EDTA, 50 mM Tris-HCl pH 7.4), and 5 μL of 1 M DTT to separate aliquots of 50 μL of the emulsion and incubate on ice for 5 minutes. Add 99 μL of Ampure XP beads to the aqueous phase and incubate for 10 minutes. Pellet the Ampure beads with an Nd magnet and wash twice with 80% EtOH. Elute with 30 μL of T8.5-DTT-Tween (10 mM DTT, 0.1% Tween-20 in T8.5 (10 mM Tris-HCl, pH 8.5)).

[0362] Perform linear PCR amplification (40 μL library, 50 μL 2×KAPA HiFi (Roche), 10 μL of 10 μM TSO-PCR handle primer). Conduct PCR cycling according to (3 minutes at 95°C, [20 seconds at 98°C, 20 seconds at 63°C, 3 minutes at 72°C] for 13 cycles, 5 minutes at 72°C), followed by holding at 4°C finally. Add 2 μL of 1 M DTT and perform 0.6×Ampure XP purification according to the manufacturer's recommendation.

[0363] The final sequencing library was prepared according to the following customized NEB Ultra II FS protocol (NEB E7805S). 80 ng of amplified cDNA was fragmented with the Ultra II Fragmentation Mix (26 μL of amplified cDNA, 7 μL of NEBNext Ultra II FS Reaction Buffer, 2 μL of NEBNext Ultra II FS Enzyme Mix) using a thermocycling protocol of holding at 37 °C for 10 minutes, 65 °C for 30 minutes, and finally at 4 °C. 15 μL of T8.5 was added, and 0.8× Ampure purification was performed according to the manufacturer's recommendation and eluted in 50 μL. To the fragmented library, adapter ligation was performed at 20 °C for 15 minutes using the NEBNext Ultra II Adapter Ligation Mix (35 μL of fragmented library, 30 μL of NEBNext Ultra II Ligation Master Mix, 1 μL of NEBNext Ligation Enhancer, 2.5 μL of NEBNext Adapter for Illumina) and held at 4 °C. 28.5 μL of T8.5 was added, and 0.8× Ampure purification was performed according to the manufacturer's recommendation and eluted in 30 μL. The eluted library was amplified with the PCR Master Mix (50 μL 2× KAPA HiFi, 10 μL 10 μM Hy-i7 primer, 10 μL of 10 μM Hy-i5 primer, 30 μL eluted library) using the following thermocycling program: 3 minutes at 95 °C, (20 seconds at 98 °C, 30 seconds at 64 °C, 30 seconds at 72 °C) for 13 cycles, 5 minutes at 72 °C, and finally held at 4 °C. The sequencing-compatible library was purified using 0.8× Ampure purification and eluted in 30 μL of T8.5.

[0364] The library was sequenced on the Illumina NextSeq2000 system using 50 cycles for Read 1 (3' end of cDNA), 10 cycles for Index 1 (sample index, custom i7 read primer), 10 cycles for Index 2 (sample index), and 58 cycles for Read 2 (5' end of cDNA).

[0365] The library was processed using a custom pipeline but following the main guidelines of scRANseq. Briefly, reads were mapped using STARsolo (Kaminow B, Yunusov D, Dobin A. 2021. GenomicSuperSignature: 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), cells were demultiplexed, and cells were filtered based on the expression of up to 4000 genes. The filtered expression matrix was scaled to total counts and log-normalized.

[0366] Example 8 This method is advantageous for determining the absolute amount of a target analyte using the method of the invention that forms a physically separated first compartment.

[0367] When determining the absolute amount of the target analyte, the first compartmentalization is performed in a first partition, the compartmentalization is physical, and the binding reaction between the cell and the binding agent is preferably carried out at a high target analyte concentration exerted by a small volume encompassing each single cell. Preferably, the cell is lysed during the first compartmentalization. A second compartmentalization is performed on each first compartment, and an appropriate sensitive method is used to count single target analyte molecules. Preferably, the concentration of the target in the first compartment is determined using binding properties, and ultimately, as described in this example, the absolute amount / number of target analyte per single cell is determined.

[0368] Preparing a single-cell sample includes incubating the sample with two target-specific antibodies, and while compartmentalizing the single-cell sample into a plurality of first partitions, the target and the antibody form a complex while each first partition containing a single cell is supplemented with a lysis buffer. During the compartmentalized two-component method (second partitioning), the state of the complex within the first partition is determined.

[0369] MCF7 (ATCC™ HTB-22) cells were cultured in DMEM, GlutaMAX Supplement (31966021, Gibco) supplemented with 10% FBS (10270106, Gibco) and 1% Pen / Strep (15140122, Gibco). Cells were cultured in a cell culture incubator (Heracell™ 150i CO2 incubator, 50116048, Thermo Scientific™) at 37 °C in a 5% CO2 atmosphere until reaching approximately 90% confluence. Cells were harvested by scraping. The cells were washed twice with DPBS (14040133, Gibco) and counted (Countess™ II Automated Cell Counter, Invitrogen™) including live / dead staining with trypan blue (T10282, Invitrogen™).

[0370] Antibodies were conjugated with DNA-amplifiable labels using a proprietary procedure of Actome GmbH, Germany. The achieved labeling efficiency was 80% - 100%, confirmed by SDS-PAGE electrophoresis. The antibody preparation had 3% free label and 25% labeling error (see Example 5). Trastuzumab (TTZ) and pertuzumab (PTZ) are recombinant humanized monoclonal antibodies, both targeting the extracellular region of the HER2 tyrosine kinase receptor with non-overlapping epitopes. The conjugated antibody has two different labels, trastuzumab BL label and pertuzumab P8 label. The dissociation constants of the antibodies were determined according to Example 6 and were TTZ = 0.49E-10 M and PTZ = 0.93E-10, respectively.

[0371] (In PBS, 25 mM DTT, 15% Optiprep, 1.3 U / μL RNase inhibitor, 1× cOmplete Protease inhibitor cocktail tablet, 4.4% PEG-8000), cells were encapsulated in HFE-7500 Novac oil containing EA-008 surfactant (RAN Biotech) using a droplet generation chip (LabSmith) on an Onyx microfluidic platform (Droplet Genomics). Picoinjection was performed using the Onyx system, and 1× LBTW containing trastuzumab (TTZ) and pertuzumab (PTZ) (both at a concentration of 1.e-11 M) labeled in the first partition was achieved. The resulting emulsion was collected into 50 μL aliquots of the total volume and incubated overnight at 4°C.

[0372] Droplet dispensing was performed at the droplet concentration, adjusted to 1×10 6 drops / ml, and a single first compartment was delivered to the wells of a 96-well microplate. QIAGEN's QIAcuity probe master mix (42 μL) was added to the 96-well microplate, 1 drop of the droplet was dispensed into the well, and after mixing, it was loaded onto a QIAcuity nanoplate 26k 24-well plate. A negative control containing only the labeled trastuzumab antibody and pertuzumab antibody was also set using an antibody at a concentration of 4.00E-11 M (antibody binding control). Digital PCR was performed using a QIAGEN QIAcuity digital PCR system, with cycling parameters according to the PCR conditions of performing 40 cycles of denaturation at 95°C for 15 seconds and annealing at 58°C for 30 seconds with a 2-minute hot start at 95°C. Imaging conditions: P8 label - FAM green channel, integration time 500 milliseconds, gain 6, and BL label - HEX yellow channel, 400 milliseconds integration time, gain 6.

[0373] The number of complexes and the number of antibodies were obtained as described elsewhere (Example 6). The number of antibodies was predicted to be 4320 average copies per reaction of a QIAcuity nanoplate 26k 24-well plate (correcting for antibody labeling ratio and labeling error), and the number of detected complexes was on average 40 (correcting for antibody labeling ratio and labeling error). When the antibody concentration was 4.e-11 M, the 4320 detected copies of antibody corresponded to droplets of 0.18 nL size, so the concentration (average) of the complexes was 3.7e-13 M. Based on the concentration of the antibody and certain binding characteristics including the Kd of the antibody (exemplary Kds are PTZ = 7.4E-11 and TTZ = 1.2E-10, see Example 6 for apparent kd), a calibration curve can be created. However, the complexes can be in the range of 10 - 150, and correspondingly can be repeatedly used in the range of 9.2e-14 M - 1.3e-12 M. The calculated concentration of the HER2 protein target was 4.4e-12 M (average), 476 copies in 0.18 nL, 476 copies of HER2 per cell (average), and the HER2 per cell was in the range of 114 copies - 1992 copies.

[0374] Example 9 This method forms a physically separated first compartment and is advantageous for determining the absolute amount of a target analyte using the method of the invention that uses protein labels instead of nucleic acid labels.

[0375] Protein label In an exemplary embodiment of practicing the present invention using protein labeling, the antibody capture agent attaches to the surface of paramagnetic beads 2.7 μm in diameter, each containing 250,000 attachment sites. Typically, 500,000 beads are added to 100 μL of sample. At low target concentrations, when the number of beads is greater than the number of target molecules in the sample, each bead captures 0 or 1 target molecule. The fraction of beads capturing target molecules in a given sample follows a Poisson distribution. A single bead is confined in a femtoliter-sized well on the Simoa Disc, allowing each individual bead to be read "digitally" to determine whether it is bound to the target analyte.

[0376] The beads were washed to remove non-specifically bound proteins and incubated with two detection antibodies labeled with β-galactosidase and alkaline phosphatase, respectively. In this way, each bead that captured a single protein molecule was detected with two enzymes. Beads that did not capture molecules remained label-free. The beads were loaded into an array of 216,000 femtoliter-sized wells, each well holding one or fewer beads. The beads were added in the presence of substrate, followed by sealing the wells with oil and imaging. When the target analyte was captured (i.e., when an immune complex was formed), the substrate was converted to a fluorescent product by the captured enzyme label. The reaction was evaluated according to the concept of two-component evaluation (Example 6), counting all compartments and reading single-positive and double-positive compartments.

[0377] Example 10 For multiple target analytes, a two-component readout / analysis can be performed by nucleic acid sequencing such as NGS, in which case the label must be a nucleic acid sequence containing a unique sequence for each type of binder. In embodiments, molecular-specific barcodes (UMIs) are added to the label of each binder during the first binder labeling reaction or at a later time to each partition. This example describes the detection of a target using the inventive method that uses NGS as a readout method. This method is advantageous for determining the absolute amount of a target analyte using the inventive method of determining the absolute conditions in a first compartment using next-generation sequencing methods.

[0378] NGS readout MCF7 (ATCC™ HTB-22T) cells were cultured in DMEM, GlutaMAX Supplement (31966021, Gibco) supplemented with 10% FBS (10270106, Gibco) and 1% Pen / Strep (15140122, Gibco). Cells were cultured in a cell culture incubator (Heracell™ 150i CO2 incubator, 50116048, Thermo Scientific™) until they reached approximately 90% confluence in a 5% CO2 atmosphere at 37 °C. Cells were harvested by scraping. Cells were washed twice with DPBS (14040133, Gibco) and counted (Countess™ II Automated Cell Counter, Invitrogen™) including live / dead staining with trypan blue (T10282, Invitrogen™).

[0379] The antibody was conjugated with a DNA-amplifiable label using Actome GmbH's proprietary procedure in Germany. The achieved labeling efficiency was 80% - 100%, which was confirmed by SDS-PAGE electrophoresis. The antibody preparation has 3% free label and 25% labeling error (see Example 5). Trastuzumab (TTZ) and pertuzumab (PTZ) are recombinant humanized monoclonal antibodies, both targeting the extracellular region of the HER2 tyrosine kinase receptor with non-overlapping epitopes. The conjugated antibody has two different labels, the trastuzumab BL label and the pertuzumab P8 label, with a 10-bp UMI incorporated. The dissociation constants of the antibodies were determined according to Example 6 and were TTZ = 0.49E-10 M and PTZ = 0.93E-10, respectively.

[0380] Using the instant drop-on-demand technology (I.DOT One; Dispendix, Stuttgart, Germany) equipped with an I.DOT PURE plate with 90 μm orifice (Dispendix, Stuttgart, Germany) [50, 51], 0.5 μl of LBTW was dispensed into a 384-well V-bottom plate (0030623304, Eppendorf). Before dispensing, the I.DOT was calibrated according to the liquid to be applied to ensure reliable dispensing. Diluted crude cell lysate ("cl") or DPBS was dispensed using the liquid class "H2O".

[0381] In the case of non-dissolved cells, the cell dispensing procedure was performed as described above (T. Gross, 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 cell concentrations of both MCF7 and BT-474 were adjusted to 1×10e6 cells / ml using an improved version of the single cell printer (SCP), the F.SIGHT single cell dispenser (CYTENA GmbH, Freiburg, Germany)

[43] , and loaded into a dispensing cartridge (CYTENA GmbH, Freiburg, Germany). The settings for MCF7 cells were a cell size of 10 μm to 25 μm (BT-474: 10 μm to 30 μm) and a circularity of 0.5 to 1 (the same as BT-474), respectively (Fig. 1a, S3a, and S3b). F.SIGHT can reliably dispense single cells with a minimal amount of liquid. The single cell dispensing efficiency (excluding single cell isolation success events from the target events) is usually about 90% and is further controlled by cell images clearly assigned to each dispensing event. Therefore, events other than single cell dispensing events such as doublets or empty droplets can be excluded.

[0382] The master mix was prepared as follows: Loaded into the Sapphire Chip reaction of 1×PerfeCTa™ qPCR ToughMix™ (Quantabio) and naica™ Crystal Digital PCR system using primers with cell-specific barcodes (optimized set-up of linkage PCR primers obtained by state-of-the-art optimization methods), and read with a Prism3 reader (Stilla Technologies, Virgijf, France). Linkage PCR was performed with cycling parameters of (PCR conditions of 2 minutes of hot start at 95°C, 15 seconds of denaturation at 95°C, and 240 cycles of 30 seconds of annealing at 58°C). Imaging conditions were P8-labeled - FAM green channel, 500 milliseconds integration time, gain 6, and BL-labeled - HEX yellow channel, 400 milliseconds integration time, gain 6.

[0383] Evaluate the two-component signal as described (Example 6). Record the number of amplification compartments or calculate using the quantified tracer linkage PCR amplicons.

[0384] After cycling, remove the Sapphire chip from the cycler, remove the blue cap, recover the emulsion, and use 20% perfluorooctanol (PFO) in chloroform or hydrofluoroether (HFE) to break the emulsion and purify the cDNA library. Briefly, 125 μL of recovery agent (20% PFO in HFE), 55 μL of GITC buffer (5 M GITC, 25 mM EDTA, 50 mM Tris-HCl pH 7.4), and 5 μL of 1 M DTT were added to separate aliquots of 50 μL of 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 pelleted with an Nd magnet and washed twice with 80% EtOH. Elution was performed with 30 μL of T8.5-DTT-Tween (10 mM DTT, 0.1% Tween-20 in T8.5 (10 mM Tris-HCl, pH 8.5)).

[0385] Linear PCR amplification (40 μL library, 50 μL 2×KAPA HiFi (Roche), 10 μL of 10 μM TSO-PCR handle primer) was performed. PCR cycling was carried out according to (3 minutes at 95°C, [20 seconds at 98°C, 20 seconds at 63°C, 3 minutes at 72°C] for 13 cycles, 5 minutes at 72°C), followed by holding at 4°C finally. 2 μL of 1 M DTT was added and 0.6×Ampure XP purification was performed according to the manufacturer's recommendation.

[0386] To the library, adapter ligation was performed at 20 °C for 15 minutes using NEBNext Ultra II Adapter Ligation Mix (35 μL fragmented library, 30 μL NEBNext Ultra II Ligation Master Mix, 1 μL NEBNext Ligation Enhancer, 2.5 μL NEBNext Adapter for Illumina) and kept at 4 °C. 28.5 μL of T8.5 was added and 0.8× Ampure purification was performed according to the manufacturer's recommendation and eluted in 30 μL. The eluted library was amplified with PCR Master Mix (50 μL 2× KAPA HiFi, 10 μL of 10 μM Hy-i7 primer, 10 μL of 10 μM Hy-i5 primer, 30 μL eluted library) using the following thermocycling program: 3 minutes at 95 °C, (20 seconds at 98 °C, 30 seconds at 64 °C, 30 seconds at 72 °C) for 13 cycles, 5 minutes at 72 °C, and finally held at 4 °C. The sequencing-compatible library was purified using 0.8× Ampure purification and eluted in 30 μL of T8.5.

[0387] The library was sequenced on an Illumina NextSeq2000 system using 50 cycles for Read 1, 10 cycles for Index 1 (sample index, custom i7 read primer), 10 cycles for Index 2 (sample index), and 58 cycles for Read 2.

[0388] The library was processed using a custom pipeline. Briefly, cells and UMIs were deconvolved, and droplets were deconvolved according to the description of such library deconvolution as described in WO 2020 / 260277. Briefly, droplet deconvolution was achieved based on the unique pairing between UMIs for each droplet during the linkage PCR step, and such pairing created randomly droplet-local UMI pairs, and the network of such pairs was very specific and could clearly define the droplets, enabling the reconstruction of the droplet content representing the types of antibodies present. Since the distribution of antibodies can be derived based on the content of the droplets, droplets containing one type of antibody and droplets containing both antibodies can be counted. The total number of droplets can be counted directly or derived using the concept of a tracer. The obtained droplet information can be evaluated using the method described for dPCR readout (Example 6).

Explanation of Signs

[0389] Drawing Translation Figure 1 Number of couplexes Number of complexes rHER2 direct rHER2 direct Figure 2 Couplex [M] Complex [M] Figure 3A Couplex [M] Complex [M] Figure 3B Couplex [M] Complex [M] Figure 4A Couplex [M] Complex [M] Figure 4B Couplex [M] Complex [M] Figure 5 Anti-GAPDH mouse labeled Anti-GAPDH mouse labeled Figure 6 Couplex HER2 / TTZ / PTZ [M] Complex HER2 / TTZ / PTZ Mean HER2=1.01E-11 Mean HER2 = 1.01E-11

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 binder and a second type of binder, each containing a label specific to the type of binder, wherein the first type of binder and the second type of binder specifically bind to a first target analyte; b. determining the binding characteristics of the first type of binder and the second type of binder to the first target analyte; c. preparing a sample containing cells of known concentration; d. contacting the first type of binder and the second type of binder with the sample, wherein the first type of binder and the second type of binder bind to the first target analyte to form an analyte-binder complex; e. compartmentalizing single cells of the sample into a plurality of first partitions; f. complementing each first partition containing a single cell with a lysis buffer, thereby lysing the single cell within each of the first partitions; g. compartmentalizing a single analyte-binder complex into a plurality of second partitions; h. performing a two-component detection method to determine the number and / or absolute concentration of complexes and binders in the second partitions; i. determining the absolute concentration of the first target analyte per cell in the first partitions, 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 complexes and binders in the second partitions in step h; A method comprising the above steps.

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

3. The first type of binder and the second type of binder each contain a nucleic acid label specific to the type of binder, and the two-component detection method in step h is amplifying the nucleic acid sequence label inside each of the second partitions, and / or combining the plurality of second partitions to perform nucleic acid sequence analysis in order to determine the number and identity of the nucleic acid sequence labels, thereby determining the number and / or concentration of the complexes The method according to claim 1 or 2, comprising

4. The method according to any one of claims 1 to 3, wherein the two-component detection method in step h includes performing a ligation step between the labels of the first type of binder and the second type of binder in each second partition. (Multimulti)

5. The method according to any one of claims 1 to 4, wherein the ligation step in step h includes ligating the nucleic acid sequence label of the first type of binder to the nucleic acid sequence label of the second type of binder inside each second partition when both form a complex with the first target analyte. (Multimulti)

6. Before contacting the sample with the first type of binder and the second type of binder, after contacting the sample in step d, permeabilizing the cells in the sample to allow one or more of the first type of binder and the second type of binder to enter one or more cells, The method according to any one of claims 1 to 5. (Multimulti)

7. The first type of binder and the second type of binder each contain a nucleic acid label specific to the type of binder, The plurality of first partitions in step f are further supplemented with PCR reagents, optionally together with reverse transcription reagents, and nucleic acid amplification primers containing a barcode specific to each first partition and the single cell therein, An additional nucleic acid amplification step is performed after cell lysis within each first partition, Optionally, the plurality of first partitions in step f are further supplemented with a protein crosslinking agent and / or a protease inhibitor and / or a nuclease inhibitor. The method according to any one of claims 1 to 6. (Multimulti)

8. The method according to any one of claims 1 to 7, wherein the first type of binder and the second type of binder each contain a nucleic acid label specific to the type of binder, and each nucleic acid sequence label contains an identifier sequence (barcode) specific to each label molecule and a unique molecular identifier (UMI). (MultiMulti)

9. The method according to any one of claims 1 to 8, wherein the sample contains RNA, the plurality of first partitions in step f are further supplemented with reverse transcription reagents, RNA is simultaneously converted to cDNA during any one or all of steps d to g, and when nucleic acid sequence analysis is performed in step h, the analysis includes next-generation sequencing (NGS) or qPCR analysis of the cDNA generated in steps d to g.

10. The method according to any one of claims 3 to 9, wherein the first type of binder and the second type of binder each contain a nucleic acid label specific to the type of binder, and the amplification in step h includes performing PCR and / or droplet PCR and / or digital PCR and / or real-time PCR and / or RT-PCR. (MultiMulti)

11. The method according to any one of claims 1 to 10, wherein the at least one target analyte includes at least a first target analyte and a second target analyte, and in step a, at least a first type of specific binder and a second type of specific binder are prepared for each of the at least first target analyte and the second target analyte. (MultiMulti)

12. When the number of different target analytes is less than 5 and nucleic acid sequence analysis is performed in step h, the analysis includes digital PCR and / or real-time PCR, or When the number of different target analytes is 5 or more and nucleic acid sequence analysis is performed in step h, the analysis includes next-generation sequencing (NGS). The method according to any one of claims 1 to 11. (MultiMulti)

13. The method according to any one of claims 1 to 12, wherein the binder is selected from the group consisting of antibodies, artificial protein scaffolds, and aptamers. (MultiMulti)

14. The method according to any one of claims 1 to 13, wherein the target analyte is selected from the group consisting of peptides, proteins, chemically modified peptides or proteins, nucleic acids, chemically modified nucleic acids, and any complexes thereof. (MultiMulti)

15. The method according to any one of claims 1 to 14, wherein the sample is 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 optically blocked controlled single cell printing, and then prepared in step c. (Multi-multi)

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