Method and kit for measuring two-component analyte, and uses thereof

Abstract

To provide a method and the like for measuring a parameter of an analyte by using a dual-component detection method.SOLUTION: The method includes the steps of: a. performing a two-component detection method in a solution, including providing two binding components specific to non-immobilized analyte at known concentrations and contacting the two binding components specific to the analyte with the analyte to generate a signal dependent on the concentration of two-component / analyte complexes formed in the solution; b. providing a dissociation constant relation for the two-component detection method, which is a mathematical function on a relation of a dissociation constant of the two binding components specific to the analyte and the analyte; c. preparing a specimen and dilutions of the binding components specific to the analyte; d. applying the two-component detection method to the specimen and the dilutions; and e. determining the dissociation constant of the two binding components specific to the analyte by using a signal detected in the specimen and the one or more dilutions as constraint inputs for a mathematical compatibility about the dissociation constant relation.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to methods and kits for measuring parameters of an analyte using a two-component detection method and their uses. The claimed method includes measuring dilutions of an analyte with an extended measurable concentration range of the measurement. The measurement is based on a predetermined non-bijective calibration curve of the measurement, thereby providing a method for reading the non-bijective calibration curve of the two-component detection method for each measurement. This allows a mathematical relationship for calculating the concentration of the analyte to be accurately determined over a wider measurement range. In another aspect, the present invention relates to the application of a predetermined non-bijective calibration curve of the measurement method in a compartmentalized two-component detection method. In yet another aspect, the present invention relates to the application of a predetermined non-bijective calibration curve of the measurement method using unique molecular components identified in a compartmentalized two-component detection method. In yet another aspect, the present invention relates to the determination of the dissociation constant of the binding component specific for the analyte of the two-component method. The claimed method further relates to an extended measurable concentration range of the measurement, the determination of the dissociation constant of the component, and a better quantification of the two-component detection method in a parallel manner.

[0002] The present invention relates to a method and a kit for measuring an analyte parameter using a two-component detection method and their uses. Applications of the method and the kit are also disclosed. [Background technology]

[0003] Related Technology Considerations In particular, the methods and kits are suitable for determining the concentration of an analyte using two-component detection methods and relate to a larger measurement range using two-component detection methods and uses thereof.

[0004] The saturation effect or "hook effect" is a common phenomenon in detection systems that generally involves saturation of the reagent components used to capture the specific binding partner or analyte. However, the mathematical background is different for one-component or two-component detection methods.

[0005] Two-component detection systems are typically utilized in homogeneous assays, where two components are applied to generate detection signals. Many of these assays apply the proximity concept, and require the analyte and the component to be brought into close proximity to generate signals. Proximity assay techniques such as FRET (fluorescence resonance energy transfer), BRET (bioluminescence resonance energy transfer) (Pfleger, Seeber, & Eidne, 2006), Cyan Fluorescent Protein (CFP)-Yellow Fluorescent Protein (YFP) pair, PCA (protein complementation assay) (Morell, Ventura, & Aviles, 2009), Alphascreen (Taouji, Dahan, Bosse, & Chevet, 2009), and DNA labeling proximity methods (PLA-proximity ligation assay, PEA-proximity extension assay) (Soderberg et al., 2006), as well as non-proximity-based assays called emulsion coupling, are representative examples of bimolecular (two-component) detection systems and detection methods.

[0006] In one-component detection systems, only one assay component, called the tracer, is required to generate a signal. Filtration binding assays containing radioactive or fluorescent tracers are good examples of one-component detection systems. Calibration curves obtained using one-component detection systems show a typical S-shape that ends with a plateau (also commonly called the "hook") obtained at the saturating tracer concentration. These curves are also called saturation curves.

[0007] Calibration curves generated with bimolecular detection systems are atypical. These curves are non-bijective and characterized by an initial concentration-dependent signal increase followed by a plateau followed by a signal decrease (the "hook effect"). The range of target molecule concentrations where the signal starts to decrease is called the hook point. Below the hook point, the assay components are gradually saturated by the target molecules and an increase in signal is measured. At the hook point, both components are saturated with the target molecules and a maximum signal is detected. Above the hook point, excess target molecules oversaturate the components, which inhibits association and causes a gradual signal decrease.

[0008] In the prior art, the hook point of a two-segment non-bijective calibration curve must be determined before measurements of analyte concentrations are performed to ensure that all measurements are made in the range of the progressively saturated segments of the two-segment non-bijective calibration curve. Oversaturation is generally considered a measurement failure.

[0009] Failure to comply with this requirement can lead to erroneous measurements since the determined values ​​do not uniquely correspond to the analyte concentrations in a non-bijective calibration curve.

[0010] To avoid such errors, the dynamic range of the measurements is typically restricted to that of the progressively saturated segment of the non-bijective calibration curve.

[0011] Several approaches are known in the prior art to address the hook effect, especially in sandwich immunoassays.

[0012] Wu et al., 2018 discuss the occurrence of the hook effect in sandwich immunoassays due to excess capture and detection antibodies and suggest dilution of samples to avoid misinterpretation of results.

[0013] JP06109740A relates to a turbidimetric immunoassay (TIA) for quantitative analysis of target components in a sample utilizing antibody-antigen reactions. JP06109740A discusses the occurrence of prozones for excess antigen in known methods, in which case the method repeats the test with sample dilution. As an improvement, JP06109740A suggests obtaining two or more calibration curves for samples with different concentrations and determining which calibration curve is applicable based on deviations from a virtual curve. However, the disclosure of JP06109740A is conceptually limited to preventing the prozone effect to avoid false negative measurements, without enabling a wider assay dynamic range from a defined mathematical relationship.

[0014] EP 0 576 879 relates to a method for determining component concentrations in medical samples, where the hook effect in immunological detection schemes at antigen excess and the resulting non-monotonic non-bijective calibration curves, called "Heidelberger-Kurve", are considered. EP 0 576 879 discloses that it is known in the prior art to overcome the ambiguity of non-monotonic calibration curves using two different sample dilutions. As a further improvement, EP 0 576 879 suggests the use of learning algorithms and multivariate statistics to correlate the measured input values ​​with the correct regime of the calibration curve. However, EP 0 576 879 concerns the analysis of signal transduction in precipitation assays and therefore cannot be easily extended to two-component assays, especially since precipitation is a variable / multi-component reaction with ambiguous stoichiometry. Furthermore, using learning algorithms, results may vary depending on the experimental conditions, and different calibration curves may be required for each experimental condition.

[0015] CN106226516 also discusses a strategy to deal with the hook effect for a detection method involving a hyperbolic calibration curve.CN106226516 suggests carrying out separate reactions of standards of analytes with different concentrations using equal amounts of reactants to construct a calibration curve at the inflection point from the pre-calibration and post-calibration curves.

[0016] Binder et al 2008 disclose a calibration algorithm for GeneChip microarrays that takes into account the Hook effect and may involve co-processing log differences and sums of perfect match and mismatch probe intensities.

[0017] Therefore, in view of the teachings of the prior art, there is room for improvement to provide methods and kits suitable for determining the concentration of an analyte using a two-component detection assay, which can reliably, effectively, and with little additional effort provide a defined mathematical relationship that extends the dynamic range of such two-component detection assays and eliminates the need to use approximation methods.

[0018] In another embodiment, the methods and kits are suitable for determining the dissociation constant of an analyte binding component using a two-component detection method and relate to measuring dissociation constants in parallel using a two-component detection method and their uses.

[0019] In chemistry, biochemistry, medical technology and pharmacology, the dissociation constant (Kd) is a specific type of equilibrium constant that measures the tendency of a larger complex to reversibly dissociate into smaller components (Friguet, Chaffotte, Djavadi-Ohaniance, & Goldberg, 1985). The dissociation constant is the reciprocal of the association constant. In the particular case of two-component assays, the dissociation constant is the dissociation constant between the two components and the analyte. Since the components can be antibodies, there is a wide interest in determining the dissociation constant.

[0020] Several approaches are known in the prior art for determining dissociation constants.

[0021] Rossant ET AL 2015 and Xiong Y. et al. (2017) disclose approaches for determining the equilibrium parameters (Kd) of affinity complexes using FRET-based assays, but for binary complexes.

[0022] Rey et al. 2017 explores the role of cooperativity in the design of potent proteolytic targeting chimeras (PROTACs) for degrading Bruton's tyrosine kinase (BTK). Rey et al. propose that maximizing PROTAC activity against BTK is related to maximizing the ternary complex of BTK:PROTAC:CRBN, and disclose TR-FRET analysis of the ternary complex with members of a PROTAC library serving as analytes and BTK and CRBN as ligands, each labeled with a FRET (donor / acceptor) fluorescent molecule. Rey et al. further present non-bijective curves of ternary complex formation (and resulting FRET signal) versus analyte concentration, as well as the dissociation constants (K D They disclose a numerical fit to derive the σ(σ) value.

[0023] EP 2837937 discloses a mathematical model of binding equilibrium in ternary affinity complexes. EP 2837937 discusses features of ternary complex formation, including a "bell-shaped" binding curve or "hook effect". Equations are also disclosed that relate the equilibrium dissociation constant to the analyte concentration, binding moiety and / or ternary complex concentration.

[0024] Rey et al. 2017 and EP 2 837 937 A1 address the estimation of equilibrium parameters in ternary complex formation and mention the occurrence of Hook effects, but do not provide a solution that properly takes these effects into account.

[0025] EP1460414 relates to a homogeneous time-resolved FRET assay for identifying potential lead antibodies in the drug discovery process. EP1460414 discloses an example of using HFRET assay to analyze protein-protein interactions. As a drawback of direct binding assay, EP1460414 discloses that screening undiluted samples can increase the number of false negatives as a result of the "hook" effect. To limit the effect, screening with multiple sample dilutions is proposed, but this can affect time and cost.

[0026] Zorba et al. have disclosed the principle of an amplified luminescent proximity homogeneous assay (AlphaLISA) modified from a binding competition assay to detect the interaction of the transcription activation domain p53 with its ligand. They consider the occurrence of the hook effect at excessive concentrations and propose a suitable titration to avoid it.

[0027] Douglass et al. 2013 discusses various assays for monitoring protein-protein interactions and identifying small molecules as inhibitors, including AlphaScreen or AlphaLISA. Douglass et al. also discuss the hook effect and suggest titration to select a protein concentration that avoids the hook point of the assay.

[0028] However, EP 1 460 414 A1, Zorba and Douglass et al., 2013 do not address the estimation of dissociation constants in these assays.

[0029] In light of the teachings of the prior art, it would be desirable to provide improved methods and kits suitable for measuring dissociation constants, particularly in a parallel or high-throughput format, using two-component detection methods.

[0030] Furthermore, the methods for determining the dissociation constants of two-component detection methods in the prior art are complex and expensive, but are severely limited, especially in parallel formats. The most frequently used methods, such as displacement ELISA, fluorescence polarization, and surface plasmon resonance, require parallelization of single measurements. Therefore, there is a need for simplified reaction-level parallel yet robust methods. Summary of the Invention

[0031] In light of the prior art, the technical problem underlying the present invention is to provide alternative and / or improved means for determining the concentration of an analyte using two-component detection, as well as for determining the dissociation constant of a binding component specific for the analyte in a two-component detection method.

[0032] In particular, it is an object of the present invention to provide a method or kit for measuring an extended range of analyte concentrations using two-component detection methods.

[0033] It is also an object of the present invention to provide methods and kits for determining the concentration of an analyte and the dissociation constant of an analyte binary complex using a compartmentalized binary detection assay, and uses thereof.

[0034] In particular, it is an object of the present invention to provide methods and parallel detection and uses thereof for determining analyte concentrations with absolute quantification and extended dynamic range in compartmentalized two-component detection methods.

[0035] This object is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims.

[0036] Thus, the present invention provides a. providing two binding moieties specific for an analyte at known concentrations for carrying out a two-component detection method in solution, comprising contacting the two binding moieties specific for the analyte with the analyte to generate a signal that is dependent on the concentration of a two-component / analyte complex formed in the solution; b. providing a standard curve of analyte concentration for the two-component detection method that is a mathematical function reflecting the dependence of the signal on the concentration of analyte, the standard curve of analyte concentration being not a bijective function, but preferably exhibiting monotonic segments of increase and decrease; c. preparing one or more dilutions of the sample using a defined dilution factor; d. applying the two-component detection method to the sample and to the one or more dilutions; e. determining the concentration of the analyte in the sample using the signal detected in the sample and one or more dilutions as a constraining input for a mathematical fit to a standard curve of the non-bijective analyte concentrations at different analyte concentrations; The present invention relates to a method for determining the concentration of an analyte, preferably in a sample containing an unknown concentration of the analyte, using a two-component detection method comprising:

[0037] In some embodiments, the method includes, after applying the two-component detection method, comparing the signal detected in the sample and in one or more dilutions to a standard curve of analyte concentrations to determine which point of the standard curve of analyte concentrations is applicable to the analyte concentration in the sample, where a defined dilution factor is taken into account. In some embodiments, determining the analyte concentration in the sample includes comparing a signal reflecting the formation of a two-component / analyte complex in the sample to an applicable point of the standard curve of analyte concentrations.

[0038] Preferably, the two binding components specific for the analyte and the analyte are non-immobilized and in solution, so that the two component / analyte complex is also formed in solution. In some embodiments, the method includes providing an unknown concentration of the analyte to a sample. In some embodiments, the method is an in vitro method, and preferably, providing the sample does not include surgical or invasive treatment of a human or animal living body.

[0039] The methods of the present invention allow for the determination of unknown concentrations of known analytes using non-compartmental or compartmentalized two-component detection methods with extended dynamic range.

[0040] As mentioned above, the reference curve of a saturable two-component detection method is a non-bijective function with two segments separated by a so-called hook point. A bijective function means that a one-to-one correspondence is possible. In mathematical terms, a bijective function is a function between the elements of two sets, where each element of one set pairs with exactly one element of the other set and vice versa. For the reference curve or calibration curve of a saturable two-component detection method, this is not the case. The same signal reflecting the concentration formation of a binary component / analyte complex can result in at least two different analyte concentrations. Thus, the same detectable signal generated by the formation of a binary component / analyte complex can be attributed to two different actual analyte concentrations.

[0041] Therefore, to avoid ambiguity, prior art techniques estimate the hook point of a two-segment non-bijective basis curve before performing measurements of analyte concentration, which are then typically performed only within one of the segments, severely limiting the dynamic range.

[0042] The present method eliminates such limitations.Instead, the present method prepares one or more sample dilutions with known dilution ratios, and applies two-component detection method to the sample and one or more dilutions.Applying two-component detection method preferably means that two binding components specific to a known concentration of analyte are contacted with the sample and one or more dilutions, so as to generate a signal that depends on the concentration of the two-component / analyte complex formed in the solution.

[0043] As detailed herein, the signal can take various forms and depends on the two-component detection system and method used. For example, in the case of a proximity assay such as a FRET assay, the signal can refer to a fluorescent signal from a FRET donor-acceptor pair that indicates the formation of a two-component / analyte complex. In the case of a compartmentalized two-component method such as emulsion coupling, the signal can also refer to a signal resulting from ddPCR or NGS sequencing using fluorescently tagged PCR products that indicates the presence of a two-component / analyte complex based on deviation from the expected Poisson distribution in the compartmentalized droplets.

[0044] Since a signal reflecting the formation of a binary component / analyte complex is obtained in the sample as well as in one or more dilutions, the signal and the dilution factor can be used to determine which point on a standard curve of analyte concentrations is applicable to the analyte concentration in the sample. To this end, a comparison is made between the signals resulting from the sample and one or more dilutions and the standard curve.

[0045] As shown in Figure 1, the reference curves of two-component detection methods typically exhibit a bell-shaped curve with a monotonically increasing segment for lower analyte concentrations and a monotonically decreasing segment for higher analyte concentrations (saturation region). Thus, the increase or decrease relates to the change in signal depending on the analyte concentration.

[0046] Various scenarios can be envisaged for the signals detected in the sample and diluent. For example, the signal due to the sample can be higher than the signal due to the diluent (with lower analyte concentration). From this information, it can be deduced that either both signals are due to a monotonically increasing segment, or that the signal from the sample is due to a monotonically decreasing segment and the signal from the diluent is due to a monotonically increasing segment. The difference between the two scenarios can be determined by matching the dilution factors of the sample and the diluent (see Example 3).

[0047] Note that based on the dilution factor, the expected ratio of the analyte concentration in the sample to that of one or more dilutions is known. In the curve shown in Figure 1, this corresponds to the distance on the horizontal axis.

[0048] Advantageously, the mathematical fit of the two signals with a known difference / ratio of analyte concentrations allows for accurate optimal positioning of the signals on the non-bijective standard curve, thereby allowing for the determination of the applicable concentration. In other words, diluting the sample to detect multiple signals with known dilution factors provides additional constraints on the mathematical fit of the obtained data, thereby allowing for a robust estimation of the applicable corresponding concentration value of the signal due to the sample.

[0049] It should be noted that additional signal resulting from one or more dilutions can be determined, and that information can be used to increase the accuracy of the analyte concentration measurement.

[0050] It is therefore possible to determine the non-bijective mathematical signal-concentration relationship of the two-component method, i.e. the reference curve or calibration curve, and to use defined analyte dilutions to limit the measurements in order to avoid failure to determine the hook point of the two-segment non-bijective calibration curve. Thus, in typical cases, the dynamic range of the signal of the two-component method can be used in both segments of the non-bijective reference curve. In prior art methods, this results in an effect of much more than doubling the dynamic range, since it is important to leave a significant distance from the hook point.

[0051] While the prior art suggests a dilution concept to some extent to avoid the ambiguity associated with the hook effect, the method described herein preferably allows for determining the concentration by considering the calculation requirements of a non-bijective curve, and therefore preferably requires two independent inputs for mathematical fitting. In other words, beyond the general broad dilution concept, the present invention preferably provides a mathematical understanding, which brings about further important advantages. In particular, preferably, a reference curve of the entire non-bijective analyte concentration can be used as a calibration curve to determine the concentration of the analyte. Although the dilution concept is generally constrained to empirically find a dilution that allows a measurement that avoids ambiguity, the dilution does not consider the entire non-bijective curve as a calibration curve. Instead, the method described herein preferably provides the necessary parameters for calculating a non-bijective curve to determine the analyte concentration using a two-component detection reaction, and the entire non-bijective curve can be considered as a calibration curve.

[0052] In one embodiment, a standard curve is obtained experimentally by providing a reference sample of known analyte concentration, making a series of known dilutions of the reference sample, and performing the two-component detection method on the reference sample and each of its known dilutions.

[0053] Advantageously, the reference curve of a given two-component detection method must be experimentally determined only once. For this purpose, a two-component detection system is provided that includes a component with a given affinity for a given concentration of analyte, as well as a reference sample with a known analyte concentration. For the reference sample, a series of dilutions are made, and the two-component detection method is applied to each dilution, so that a series of signals reflecting the formation of two-component / analyte complexes for a given analyte concentration are generated. The experimentally obtained dependence of the signals reflecting the formation of two-component / analyte complexes on analyte concentration can be interpolated to form a reference curve or calibration curve for use as described herein.

[0054] However, it is also possible to provide the reference curve by analytical methods or numerical simulations.

[0055] In one embodiment, a standard curve of analyte concentration is calculated analytically by solving chemical equilibrium and mass conservation equations or is provided by a numerical solution based on defining the dissociation constants of the analyte with each of two binding moieties specific for the analyte.

[0056] A demonstration of the definition of a standard curve based on analytical methods or numerical simulations is provided in Example 1.

[0057] Generally, analytical and numerical methods for providing a reference curve require some additional parameters that define the two-component detection method with respect to the analyte. In particular, in order to provide a reference curve as a numerical value, the dissociation constant between the analyte and each of the two binding components specific to the analyte must be estimated. As detailed in the following example, this information can essentially be used to provide a reference curve of the signal that reflects the formation of the two-component / analyte complex in an efficient and robust manner.

[0058] In one embodiment, the method can be used to determine the concentration of an analyte using a compartmentalized two-component detection method, and relates to a larger measurement range using the compartmentalized two-component detection method and its use. Compartmentalized two-component detection methods are highly preferred because they can determine the absolute concentration of an analyte.

[0059] A typical compartmentalization method is digital PCR (ddPCR) (Quan, Sauzade, & Brouzes, 2018) that utilizes a water-oil emulsion droplet system. Droplets are formed in a water-oil emulsion to form partitions (compartments) that separate template DNA molecules. The droplets essentially perform the same function as individual test tubes or wells in a plate where the PCR reaction occurs. Large-scale sample partitioning is a key aspect of ddPCR technology.

[0060] The ddPCR technology is digital in that the droplets support the PCR amplification of the template molecules they contain, generating a signal based on each individual DNA template molecule. Following PCR, each droplet is analyzed or read to determine the fraction of PCR-positive droplets in the original sample. These data are then analyzed using Poisson statistics to determine the absolute concentration of DNA template in the original sample.

[0061] In another embodiment, the method can be used to apply the principle of absolute quantification (Quan et al., 2018) to binary measurements: the analyte signal is based on the detection of individual molecules of a compartmentalized analyte-binary complex and Poisson statistics, and the analyte-binary complex is determined by a chemical equilibrium, i.e., dissociation constant, based on the concentrations of the molecules and their analytical properties.

[0062] In this embodiment, the generation of tens, thousands or millions (or even more) of droplets and their application to bi-competent detection methods means that a single sample measurement is obtained that is absolutely quantitative, highly linear, and with the droplet number as the primary dynamic range of the bi-competent method, rather than using a single, inherently non-linear signal as in the case of non-compartmentalized measurements. This brings the absolute quantitation and statistical characteristics inherent to compartmentalized methods to the detection of analyte concentrations using bi-competent methods. These compartmentalization techniques can be combined with the methods described herein to provide an extended range of non-bijective two-segment reference curves for bi-component measurements, effectively doubling the available dynamic range of the measurement.

[0063] In some embodiments, these methods are preferred for multi-analyte measurements, since they usually have vast concentration differences. In such embodiments, a suitable compartmentalized two-component method, such as emulsion coupling, can be used to measure DNA copy number (low amount) and RNA / protein copy number (high amount) in the same sample (see examples below and EP 3224360).

[0064] When applying the digital linear signal generation method, there is no upper limit to the number of compartments in terms of the principle of signal generation. The measurement range is expanded by using the method described herein, and a signal-based dynamic range is doubled for measurement while maintaining the sensitivity and accuracy characteristic of the compartmentalized two-component method.

[0065] Thus, the compartmentalized two-component method combined with the method described herein provides unparalleled precision, since large-scale sample partitioning allows reliable measurement of small fold differences in target analytes. This increases the signal-to-noise ratio, since the dominant template in the sample does not prevent the detection of rare targets. The compartmentalized two-component method also provides a low signal dropout error rate, since the inherent high dilution of the method removes substances that may interfere with effective signal generation.

[0066] According to the invention, the above method is also suitable for determining the dissociation constant of a binary component / analyte complex.

[0067] In another aspect, the invention provides a method for determining the dissociation constant of a binding component specific for an analyte in a two-component detection method, comprising the steps of: a. providing two binding moieties specific for an analyte at known concentrations for performing a two-component detection method comprising contacting two binding moieties specific for the analyte with a solution containing the analyte to generate a signal dependent on the concentration of a two-component / analyte complex formed in the solution; b. providing a dissociation constant relationship for the two-component detection method that is a mathematical function of the relationship of the dissociation constant of the analyte-specific binding component in dependence on the signal reflecting the concentration of the two-component / analyte complex and the concentration of the analyte and / or the analyte-specific binding component; c. preparing one or more dilutions of the sample or a binding component specific for the analyte; d. applying the two-component detection method to the sample and to the one or more dilutions; e. determining the dissociation constants of two binding moieties specific for the analyte using the detected signals of the sample and one or more dilutions as constraint inputs for a mathematical fit of the dissociation constant relationship at different analyte concentrations and / or analyte-specific binding moiety concentrations; The present invention relates to a method comprising the steps of:

[0068] The method of the present invention allows the determination of the dissociation constant of a two-component detection system by preparing one or more sample dilutions with a known dilution factor, and applying the two-component detection method to the sample as well as to one or more dilutions. Applying the two-component detection method to determine the dissociation constant of an analyte-specific binding component preferably means contacting two binding components specific to a known concentration of an analyte with a sample of known concentration to generate a signal that depends on the concentration of the two-component / analyte complex formed in the solution. The concentration of either the analyte-specific binding components or the sample is changed by dilution, providing a dilution with a known dilution factor. The signals detected in the sample and in the one or more dilutions, as well as the concentrations of the analyte and / or the analyte-specific binding components, can be used as constraint inputs for the mathematical fit.

[0069] Preferably, the two binding components specific for the analyte and the analyte are non-immobilized and present in solution, so that the binary / analyte complex is formed in solution as well. In some embodiments, the method includes providing a sample with a known concentration of the analyte. In some embodiments, the method is an in vitro method, and preferably, the step of providing the sample does not include surgical or invasive treatment of a human or animal living body. As detailed above, the signal can take a variety of forms and depends on the binary detection system used. For example, in the case of a proximity assay such as a FRET assay, the signal may refer to a fluorescent signal from a FRET donor-acceptor pair indicating a formed binary / analyte complex. In the case of a compartmentalized binary method such as emulsion coupling, the signal may also refer to a signal resulting from ddPCR and / or NGS sequencing that indicates the presence of a binary / analyte complex based on deviations from an expected Poisson distribution in the compartmentalized droplets.

[0070] As shown in the examples (e.g., see Examples 2, 5 and Figure 2), using the signals detected in the sample and one or more dilutions as constraining inputs for a mathematical fit allows for robust determination of the dissociation constant between the analyte and two analyte-specific binding components.

[0071] The dissociation constant relationship can be provided by analytically solving the chemical equilibrium and mass conservation equations (see Example 2, Equation 3). In general, it can be written as f(Kd1, c12, a0) = Kd2, where "Kd1" and "Kd2" are dissociation constants, "c12" is the measured concentration of the ternary complex determined by the signal reflecting the formation of the binary / analyte complex, and "a0" is the known concentration of the analyte. As additional inputs, f(Kd1, c12, a0) = Kd2 can further include the concentrations of the analyte, and both binary components, which are also known. These terms are valid for binding moieties specific for a fixed, known concentration of the analyte.

[0072] When the concentration of the analyte-specific binding component varies, another advantageous aspect of the determination of the dissociation constant relationship can be provided, which can be written as f(Kd1, c12, b10, b20) = Kd2, where "Kd1" and "Kd2" are dissociation constants, "c12" is the measured concentration of the ternary complex determined by the signal reflecting the formation of the binary / analyte complex, and "b10" and "b20" are the known concentrations of the analyte-specific binding components. As an additional input, f(Kd1, c12, b10, b20) = Kd2 can further include the concentration of the analyte, which is also known. These terms are valid for the case of a known concentration of analyte that does not vary.

[0073] To determine the dissociation constants of the binding components ("Kd1" and "Kd2"), Equation 3 can be applied. When there are two or more solutions containing known amounts of analyte, two components, and a determined amount of the ternary antibody / binary complex, the two Kd's can be calculated according to Example 2.

[0074] Different dilutions of the sample containing the analyte correspond to different concentrations of "c12", and therefore the "f" function has different forms (see Figure 2). However, because Kd is a material constant, all forms of the "f" function must be satisfied for a particular pair of Kd values.

[0075] In fact, when the dissociation constant relationship with Kd1 and Kd2 is graphed, the curves of different known concentrations intersect near one point that is the applicable pair of Kds to be determined (see Example 2). In other words, the mathematical solution is preferably to find the intersection point between the curves, which reflects the relationship of dissociation constants (Kd1-Kd2) that change with different input concentrations provided by applying the two-component detection method to the sample and one or more dilutions.

[0076] In one embodiment, determining two or more relationships of dissociation constants using a variety of experimental conditions, for example by providing different dilutions of the analyte, can help determine values ​​of the dissociation constants that satisfy these relationships.

[0077] In a preferred embodiment, since measurement errors are confounded in different measurements, statistical models and / or other mathematical tools can be used to determine the value of the dissociation constant.

[0078] In another embodiment, the method is suitable for determining the dissociation constant of an analyte binding component using a two-component detection method in parallel and for use in the determination of the dissociation constant of an analyte binding component using a two-component detection method.

[0079] In one embodiment, the method for determining the dissociation constant of a component that binds to an analyte is based on an absolutely quantifiable compartmentalized two-component detection method.

[0080] In another embodiment of the method described herein, it is preferred to use parallel reading technology.In some embodiments, next-generation DNA sequencing is preferred as two-component reading technology.In particular, these two-component methods are suitable for reading based on DNA sequencing, which generates unique DNA signal as part of the detection principle.These methods include, but are not limited to, proximity ligation, extension assay and emulsion coupling.

[0081] In another embodiment, when the two-component method is a compartmentalized two-component method, the two-component measurement method based on DNA sequencing reading is preferred.Under compartmentalized conditions, compared with non-compartmentalized measurement, the generation of unique DNA signals is unbiased.

[0082] In another embodiment, compartmentalized generation of DNA signals refers to unbiased DNA signals that enable signal readout through the use of unique molecular identifiers (UMIs) (Parekh, Ziegenhain, Vieth, Enard, & Hellmann, 2017).

[0083] Various methods have been developed to improve the accuracy of quantification of different polynucleotides in a sample using next-generation sequencing technologies, including methods such as competitive polymerase chain reaction (PCR) as described in U.S. Pat. No. 5,213,961 and deep barcode sequencing using unique molecular identifiers (UMIs) as described in (Smith et al., 2009).

[0084] Unique molecular identifiers or molecular barcodes are advantageous for quantifying the unique DNA signals of two-component assays in samples. However, when UMIs are involved in second and subsequent detections, the same UMI may be introduced into different targets, leading to counting errors. Also, the original UMI method is based on an ideal but unrealistic situation, i.e., both PCR and sequencing techniques are perfect and do not introduce errors. The UMI strategy works on the assumption that both PCR and sequencing steps report the underlying target and UMI fragments without errors. These conditions are difficult to provide under non-compartmentalized conditions, but are smoothly provided under compartmentalized conditions. The UMI-labeled components are single or a few molecules per compartment in compartmentalized two-component assays, which means that the UMI barcodes are effectively incorporated into the unique DNA signals.

[0085] In another embodiment, unique molecular identifiers (UMIs) that represent unique DNA signals can be incorporated into unique DNA signals and further amplified by compartmentalized two-component method.However, without using unique molecular identifiers, the quantification of unique DNA signals will be biased.Amplification can be specific to certain unique DNA signals to balance their ratio with other unique DNA signals that are not further amplified.

[0086] In another embodiment, the unique DNA signal incorporated into the unique molecular identifier can be used to detect analytes with two or more identical binding sites or analytes with three or more non-identical binding sites. These configurations are particularly preferred for detecting interacting analytes such as proteins or other molecules.

[0087] In another embodiment, the method and the kit of the two-component assay using parallel reading technology can measure both the concentration of the analyte and the dissociation constant of the component in parallel.Today, next-generation sequencing technology can provide billions of independent readings, which are directly proportional to the achievable parallelism of readings.

[0088] In a further embodiment, the analyte is selected from the group consisting of proteins, peptides, nucleic acid segments, carbohydrates, lipids, antibodies (monoclonal or polyclonal), antigens, oligonucleotides, specific receptor proteins, ligands, molecules, cells, microorganisms, and fragment products or combinations thereof.

[0089] In one embodiment, the two binding components specific for the analyte are selected from the group consisting of nucleic acids, preferably RNA and / or DNA oligonucleotides, antibodies, peptides, proteins, aptamers, molecularly imprinted polymers, cells or combinations thereof.

[0090] In one embodiment, the sample contains two or more different types of analytes.

[0091] In one embodiment, two or more pairs of binding moieties specific for different types of analytes are used.

[0092] A particular advantage of the methods described herein is that it is even possible to include more than one type of analyte (e.g., DNA; RNA; proteins, interacting proteins and their chemical modifications) and likewise more than one type of binding moiety in the same assay.

[0093] The parallel approach described herein can be used to determine in parallel the analyte concentrations of different types of analytes, as well as the dissociation constants of binding moieties specific for the different types of analytes.

[0094] In one embodiment, the two-component method involves using a proximity-based assay to generate a concentration-dependent signal of the two-component / analyte complex, where the proximity-based assay uses two binding components specific for the analyte that generate a detectable signal depending on their proximity.

[0095] In one embodiment, the two-component method comprises using a resonance energy transfer assay, preferably a Förster resonance energy transfer (FRET) assay or a bioluminescence resonance energy transfer (BRET) assay, a protein complementation assay (PCA), Alphascreen or a DNA labeling proximity assay, preferably a proximity ligation assay (PLA) or a proximity extension assay (PEA).

[0096] In one embodiment, a two-component detection method involves using a compartmentalized assay to generate a concentration-dependent signal of a two-component / analyte complex, the signal reflecting the presence of two binding components specific for the analyte within a single compartment.

[0097] In one embodiment, the compartmentalization assay uses an emulsion droplet method, where each droplet in the emulsion represents a separate compartment.

[0098] In one embodiment, the compartmentalization assay is emulsion coupling.As described in detail below, emulsion coupling refers to the digital assay concept based on the detection of the individual ternary molecular complex of dual-labeled (two components) in emulsion, which can be identified by, for example, digital droplet PCR (ddPCR) or next-generation sequencing (NGS) using fluorescently tagged PCR product.

[0099] Advantageously, such an approach allows for absolute quantification of analyte concentrations and parallel determination of multiple analyte concentrations in a robust and efficient manner.

[0100] In one embodiment, the two-component detection method comprises using an analysis method based on absolute molecular number. Such an analysis method based on absolute molecular number preferably refers to the application of digital detection methods such as digital PCR or next-generation sequencing.

[0101] In one embodiment, the two-component detection method involves using a droplet digital PCR assay.

[0102] In one embodiment, the two-component detection method involves the use of analyte-specific binding components associated with unique amplifiable nucleic acid labels, and employs a compartmentalized assay, where nucleic acid amplification is performed for each compartment using fluorescently tagged amplification products.

[0103] In a preferred embodiment, the nucleic acid amplification is PCR and the fluorescently tagged amplification products are fluorescently tagged PCR products.

[0104] The analyte-specific binding components (such as antibody pairs) are preferably labeled with a unique DNA label that is PCR amplifiable, i.e., two analyte-specific binding components, e.g., two antibodies, can preferably be labeled with a single-stranded DNA that uniquely identifies the binding components (e.g., antibodies). The labeled binding components (e.g., antibodies) are added to the sample or one or more dilutions to allow for the formation of a two-component / antibody complex.

[0105] The reaction is then preferably highly diluted, for example by a dilution factor of more than 20000, preferably more than 50000, 100000, to achieve single-complex separation upon compartmentalization, for example by emulsification into droplets.

[0106] For nucleic acid amplification, a fluorescently tagged amplification product is used that recognizes a unique nucleic acid tag that is specific and amplifiable for the binding component (e.g., an antibody). For example, a fluorescently tagged PCR product, such as a FAM or VIC-labeled real-time PCR probe, that is complementary to a single-stranded DNA that uniquely identifies the binding component may be used.

[0107] Nucleic acid amplification can be performed in each compartment, e.g., emulsion droplet, and signal readout can be performed by detecting the fluorescence signal of the compartment, e.g., the "color" of the droplet.

[0108] Preferably, ddPCR is used, and according to the standard evaluation of ddPCR, droplet group can be determined according to the fluorescent signal of droplet.Here, the number of labeled binding components (e.g., antibodies) is determined in each reaction (counting all the labeled positive droplets for a given label, and using the same defined droplet group for all reactions).In addition, the number of double staining (having two different binding component labels) is also determined.

[0109] If no ternary binary / analyte complexes are formed, the partitioning of the labeled antibodies follows a Poisson distribution, resulting in a calculable number of doubly colored droplets (containing two binding components in one compartment based on mere chance). If a ternary complex is present in the reaction, the number of doubly colored droplets (containing an additional ternary complex) detected will be higher than would be expected by Poisson distribution alone. Based on such an analysis, the number of ternary complexes can therefore be calculated. Advantageously, this embodiment allows absolute quantification of the ternary analyte complexes formed.

[0110] In one embodiment of the methods described herein, multiple analytes are determined in parallel.

[0111] In one embodiment, a two-component detection method includes using binding components specific for multiple analytes that contain nucleic acid barcodes, and using a compartmentalized assay, nucleic acid amplification is performed for each compartment generating a concatenated nucleic acid barcode, the compartments are recombined in a common pool, and parallel nucleic acid sequencing techniques are used to generate a concentration-dependent signal of the two-component / analyte complex.

[0112] A preferred parallel nucleic acid sequencing technology for use herein is next generation sequencing technology.

[0113] In a preferred embodiment, the binding moiety containing the nucleic acid barcode refers to an antibody labeled with a unique PCR-amplifiable DNA label that also contains a unique label for the named antibody type and a label for the individual molecule (unique molecular identifier - UMI) (see also Parekh et al., 2017). The antibody thus labeled is added to the sample to allow the formation of a binary / antibody complex.

[0114] Carrying out nucleic acid amplification for each compartment to generate linked nucleic acid barcodes can be achieved by highly diluting the sample before nucleic acid amplification, for example, with a dilution factor of more than 20000, preferably more than 50000, more preferably more than 100000. In a preferred embodiment, the nucleic acid amplification is PCR. PCR reagents can be added to achieve single-complex separation and nucleic acid amplification per water-in-oil emulsion droplet. For this purpose, standard protocols for droplet digital PCR can be particularly suitable.

[0115] The compartments, e.g., emulsion droplets, can then be recombined in a common pool, and antibody-specific dimerized UMI labels can be evaluated using parallel nucleic acid sequencing techniques. Herein, the number of labeled antibodies can be determined by counting all unique UMI labels for a given antibody in each reaction (the counting is, for example, limited to a given antibody in a given labeling context). Possible multiple labeling of the same antibody can be eliminated using preferentially dimerized sequences, since multiple labels per antibody are colocalized in the same droplet, thus indicating a double UMI-labeled dimer in a labeling context specific to a given antibody.

[0116] Ternary binary / antibody complexes can be counted based on their dimerized double UMI-labeled PCR products (two different antibody-specific labels, called heterodimers) and compensated according to the antibody multiplex labeling.

[0117] If no ternary complexes are formed, the partitioning of the labeled antibodies follows a Poisson distribution, resulting in a calculable number of ternary complexes (based on the detection of heterodimers) in the droplet. If ternary complexes are present in the reaction, the number of detected heterodimers will be higher than would be expected by a pure Poisson distribution. Based on this measurement, the number of complexes can be calculated.

[0118] In a compartmentalized two-component approach, it would be advantageous to incorporate UMIs into distinct targets as an encoding step for colocalization of unique DNA signals.

[0119] In a preferred embodiment, the present invention provides a kit for carrying out a method for determining the concentration of an analyte, preferably using the two-component detection method described herein, comprising: a. two binding moieties specific for a known concentration of an analyte for carrying out a two-component detection method comprising contacting two binding moieties specific for the analyte with the analyte to generate a signal that is dependent on the concentration of a two-component / analyte complex formed in the solution; b. a standard curve of analyte concentration for a two-component detection method, the standard curve being a mathematical function reflecting the dependence of the signal on the concentration of the analyte, the standard curve of analyte concentration being a non-bijective function and exhibiting monotonic segments of increase and decrease; c. optionally, instructions for preparing one or more dilutions of the sample and applying a two-component detection method to the sample and the one or more dilutions; d. a computer program when executed on a computer configured to perform a computational step of comparing the signal detected in the sample and in one or more dilutions to a non-bijective standard curve of analyte concentrations to determine the concentration of the analyte in the sample; and The present invention relates to a kit comprising:

[0120] In a further preferred embodiment, the present invention relates to a kit for carrying out a method for determining a dissociation constant, preferably in a two-component detection method as described herein, comprising: a. two binding moieties specific for a known concentration of an analyte for carrying out a two-component detection method comprising contacting two binding moieties specific for the analyte with the analyte to generate a signal that is dependent on the concentration of a two-component / analyte complex formed in the solution; b. a dissociation constant relationship of the two-component detection method that is a mathematical function of the relationship of the dissociation constants (Kd1 and Kd2, respectively) of the analyte and the analyte-specific binding component in dependence on the signal reflecting the concentration of the two-component / analyte complex and the concentration of the analyte and / or the analyte-specific binding component; c. optionally, instructions for preparing one or more dilutions of the sample and applying a two-component detection method to the sample and the one or more dilutions using a defined dilution factor; d. a computer program, when executed on a computer, configured to perform a calculation step of determining the dissociation constants kd1 and kd2 of two binding moieties specific for the analyte using the signals detected in the sample and one or more dilutions as constraint inputs for a mathematical fit of the dissociation constant relationship at a given analyte and / or analyte-specific binding moiety concentration; The present invention relates to a kit comprising:

[0121] The technical features disclosed for the methods described herein also apply to kits for use in such methods, and thus, those skilled in the art will recognize that preferred features of the methods described herein may be used to advantage in the context of a kit as well.

[0122] The methods of the present invention may in some embodiments also relate to a computer program product, such as a software product.

[0123] The software may be configured to run on a general computing device and is configured to perform one or more of the steps of the methods described herein.

[0124] In one embodiment, the computer program may be configured to perform step e) of claim 1, or a further preferred embodiment of the calculation step disclosed herein, of comparing the signal detected in the sample and in one or more dilutions with a standard curve of analyte concentrations to determine the concentration of the analyte in the sample.

[0125] In one embodiment, the computer program may be configured to perform step e) of claim 2, or a further preferred embodiment of the calculation step disclosed herein, which performs a calculation step to determine the dissociation constants kd1 and kd2 of two binding components specific for the analyte using the signals detected in the sample and in one or more dilutions as constraint inputs for a mathematical fit for the dissociation constant relationship at a given binding component and analyte concentration.

[0126] In one embodiment, a concentration standard curve for a two-component detection method or a dissociation constant relationship for a two-component detection method may be provided in the form of reference data.

[0127] In this specification, reference data for a concentration standard curve of a two-component detection method preferably relates to any data that makes it possible to provide a mathematical function reflecting the dependence of a signal reflecting the formation of a two-component / analyte complex formed in the solution on the analyte concentration.

[0128] In this specification, reference data for the dissociation constant relationship of a two-component detection method preferably relates to any data that makes it possible to provide a mathematical function for the relationship between the dissociation constants (kd1 and kd2, respectively) of an analyte-specific binding component and the analyte depending on the concentration of the two-component / analyte complex and the signal reflecting the concentration of the analyte and / or the analyte-specific binding component.

[0129] Typically, the reference data may be stored in the computer usable medium or computer readable medium of the control device. Any format used in the industry may be suitable. The reference data may be stored in a separate file and / or integrated into computer code or software (e.g., source code) to perform the calculation step for determining the analyte concentration or dissociation constants kd1 and kd2 in the sample as described above.

[0130] Thus, a computer program product or kit comprising a computer program of the invention also encompasses or directly relates to the features described for the methods provided herein. Further details regarding preferred computer-based techniques are provided in the examples and associated references described herein.

[0131] Detailed Description of the Invention Before the present invention is described with reference to examples, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

[0132] The term "sample" or "sample solution" as used herein preferably refers to a solution containing an unknown concentration of an analyte. Examples of samples include biological fluids such as serum, plasma, urine, tears, cells, cell mixtures, cell culture supernatants, or cell lysates containing one or more biological target molecules. In addition, the sample can also include any conditioning reagents (e.g., permeabilization reagents) required to make the analyte soluble and accessible for detection and quantification. Such conditioning reagents can be added to the sample at any time before or after carrying out the methods described herein.

[0133] The term "analyte" refers to a substance that is detected, quantified or assayed by the method of the present invention. Typical analytes can include, but are not limited to, proteins, peptides, nucleic acid segments, carbohydrates, lipids, antibodies (monoclonal or polyclonal), antigens, oligonucleotides, specific receptor proteins, ligands, molecules, cells, microorganisms, fragments, products and combinations thereof, or any substance that can express an attachment site, binding member or receptor (such as an antibody). Analyte can also refer to a complex derived from the entity. For example, analyte can refer to an aggregate or complex formed from multiple entities or molecules, such as a protein-protein complex, where the interaction of the entities / molecules is of interest.

[0134] By "protein" is meant an amino acid sequence of sufficient chain length to produce higher levels of tertiary and / or quaternary structure. By "peptide" is preferably meant a smaller molecular weight protein.

[0135] The term "nucleic acid" refers to any nucleic acid molecule, including, but not limited to, DNA, RNA and hybrids or modified variants and polymers thereof ("polynucleotides") in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid molecule / polynucleotide also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly stated. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Nucleotides are designated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G).

[0136] As used herein, the term "nucleic acid amplification" refers to a process in which a limited amount of nucleic acid undergoes a biochemical reaction in which a larger amount of nucleic acid is produced.Thus, nucleic acid amplification relates to the production of additional copies of nucleic acid sequences, and is generally carried out using polymerase chain reaction (PCR) or ligase chain reaction (LCR) or other techniques well known in the art (see, for example, Dieffenbach, CW and GS Veksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NY).

[0137] As used herein, the term "two-component detection method" or "two-molecular detection method" refers to a method that uses two binding components specific to an analyte and determines a signal reflecting the formation of a two-component / analyte complex when the two binding components specific to the analyte are contacted with a solution containing the analyte. The term "two-component detection system" or "two-molecular detection system" preferably refers to the components or reagents required to carry out a two-component detection method. This includes two binding components, preferably specific to an analyte, provided in a single or separate solution, preferably of known concentration, and any sample solution containing the analyte to be analyzed.

[0138] Preferably, the two binding components specific for the analyte and the analyte are non-immobilized, i.e., provided in solution, so that the two-component / analyte complex is formed in solution as well. Thus, the term two-component detection method as used herein preferably refers to the liquid phase formation of the ternary complex, and is distinct from the common sandwich immunoassays, which include a (primary) capture binding agent and a (secondary) detection binding agent immobilized as solid phases.

[0139] Thus, the term "non-immobilized" preferably refers to a moiety, such as an analyte-specific binding moiety, that is freely diffusing in (liquid) solution, such that the binding kinetics to an analyte that is equally freely diffusing in said liquid solution are governed by the law of conservation of mass in solution and the equations for the law of mass action described herein.

[0140] The term "analyte-specific binding moiety" or "analyte-specific binding partner" preferably refers to one member of a binding pair, the second member being the analyte, and the term "binding pair" also includes any class of immune-type binding pairs, such as antigen / antibody or hapten / anti-hapten systems, as well as any class of non-immune-type binding pairs, such as biotin / avidin, biotin / streptavidin, folic acid / folate binding protein, complementary nucleic acid segments, such as complementary DNA or RNA strands, protein A or G / immunoglobulin, and binding pairs that form covalent bonds, such as sulfhydryl-reactive groups, including maleimides and haloacetyl derivatives, and amine-reactive groups, such as isotriisocyanates, succinimidyl esters, and sulfonyl halides.

[0141] The binding component of the two-component detection method can preferably refer to any component that has the ability to bind to the analyte.In a preferred embodiment, the binding component specific to the analyte can be nucleic acid, preferably RNA and / or DNA oligonucleotide, antibody, peptide, protein, aptamer, molecular imprint polymer, cell or combination thereof.

[0142] The term "antibody" as used herein encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

[0143] An "antibody fragment" comprises a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, bispecific antibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0144] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Moreover, in contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example.

[0145] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which portions of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567, and Morrison et al., Proc. NatL. Acad Sci. USA 81:6851-6855 (1984)).

[0146] Complementarity determining regions (CDRs) are parts of the variable chains of immunoglobulins (antibodies) produced by B cells, and these molecules bind to their specific antigens. As the most variable parts of the molecule, CDRs are important for the diversity of antigen specificities generated by immunoglobulins. There are three CDRs (CDR1, CDR2 and CDR3) arranged non-contiguously on the amino acid sequence of the variable domain of an immunoglobulin. Since immunoglobulins are typically composed of two variable domains (on two different polypeptide chains, heavy and light chains), there are six CDRs for each antigen receptor that can collectively contact the antigen.

[0147] Furthermore, fragments of a whole antibody can perform the function of binding to an antigen. Examples of binding fragments include (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains, (ii) an Fd fragment consisting of the VH and CH1 domains, (iii) an Fv fragment consisting of the VL and VH domains of a single antibody, (iv) a dAb fragment consisting of the VH domain (Ward, ES et al, Nature 341:544-546 (1989)), (v) an isolated CDR region, (vi) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments, (vii) a single chain Fv molecule (scFv) in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate and form an antigen-binding site (Bird et al, Science 242:423-426 (1988); Huston et al, PNAS USA 85:5879-5883 (1988)), (viii) bispecific single chain Fv dimers (PCT / US92 / 09965), and (ix) "bispecific antibodies," multivalent or multispecific fragments constructed by gene fusion (WO 94 / 13804; P. Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)).

[0148] An "antigen-binding domain" is a part of an antibody that contains an area that specifically binds to and is complementary to a part or all of an antigen. If the antigen is large, the antibody can only bind to a specific part of the antigen, which part is called an epitope. An antigen-binding domain can be provided by one or more antibody variable domains. An antigen-binding domain can include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0149] In some embodiments, the binding moiety may be based on engineered protein scaffolds. Protein scaffolds are derived from stable and soluble natural protein structures that have been modified to provide binding sites for the target molecule of interest, i.e., the analyte. Examples of engineered protein scaffolds include, but are not limited to, affibodies based on the Z domain of Staphylococcus aureus protein A, which provides binding interfaces for two of its alpha helices (Nygren, PA (2008). FEBS J275(11):2668-76), anticalins derived from lipocalins that incorporate binding sites for small ligands at the open end of a beta barrel fold (Skerra, A. (2008) FEBS J275(11):2677-83), nanobodies, and DARPins. Engineered protein scaffolds are typically targeted to bind to the same antigenic proteins as antibodies. Short peptides can also be used to bind target proteins. Philomers are natural structured peptides derived from bacterial genomes. Such peptides represent a diverse array of protein structural folds and can be used to inhibit / disrupt protein-protein interactions in vivo (Watt, PM (2006). Nat Biotechnol 24(2):177-83)].

[0150] In the two-component detection method, preferably, two binding components specific to the analyte are used, both of which exhibit the ability to bind to the analyte. Preferably, the two binding components specific to the analyte are selected and designed so that they do not compete with each other for binding to the analyte, but allow simultaneous binding of the components to form a two-component / analyte complex. As mentioned above, the analyte may refer to an aggregate or complex formed from multiple entities, such as a protein-protein complex. One of the two binding components specific to the analyte may bind to one entity of the analyte complex, such as a first protein, and the other may bind to a second entity of the analyte, such as a second protein. Thereby, a two-component / analyte complex is formed only in the case of the interaction of both proteins, and then labeled with the binding components.

[0151] In some embodiments, the binding component can be an aptamer. Aptamers are synthetic oligonucleotides (DNA or RNA) that recognize target molecules 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 obtained in vitro and can be developed to recognize a wide variety of different molecular classes ranging from simple ions (e.g., Pb2+, Liu & Lu, 2003, J Am Chem Soc, 125, 6642-6643) to nucleotides, small molecules, proteins, viruses, and cells to whole organisms (Menger et al., 2006. Handbook of Experimental Pharmacology, 359-373). High-binding affinity aptamers have been used to detect low molecular weight molecules such as theophylline (Jenison et al., 1994. Science, 263, 1425-1429), L-arginine (Geiger et al., 1996. Nucl. Acids Res., 24, 1029-1036), moenomycin (Schuerer et al., 2001. Bioorg. Med. Chem., 92, 2557-2563), 17b-estradiol (Kim et al., 2007. Biosens. Bioelectron., 22, 2525-2531), but also thrombin (thrombin-binding aptamer, 5'-GGTTGGTGTGGTTGG-3') (Baldrich et al., Anal. Chem. 2004, 76, 23, 7053-63), and larger molecules such as cholera toxin or the HIV-1 tat protein have also been selected by the well-known SELEX method (Ellington & Szostak, 1990. Nature, 346, 818-822) (for review see Tombelli et al., 2007, Biomolec Eng., 24, 191-200).

[0152] Some of the above-mentioned aptamers have been used in ELISA-like assays on microplates or on biosensor transducer (QCM, SPR) surfaces. An aptamer-modified AuNP colorimetric system has also been developed for the determination of the protein PDGF in a sandwich-based assay (Huang et al., 2005, 77, 5735-5741).

[0153] As used herein, the term "analyte / binary complex" or "binary / analyte complex" refers to a ternary complex that includes an analyte and both binding components specific for the analyte. Such binary / analyte complexes are formed when applying a binary detection method to a solution containing the analyte. More specifically, the formation of a binary / analyte complex in binary detection directly reflects the presence (and concentration) of the analyte in a given solution. To use the formation of a binary / analyte complex to determine the actual concentration of the analyte, a signal reflecting the formation of a ternary complex should be obtained.

[0154] As used herein, the phrase "signal dependent on the concentration of a binary component / analyte complex" or "signal reflecting the formation of a binary component / analyte complex" refers to any quantifiable information that allows for the estimation of the amount of ternary binary component / analyte complex formed.

[0155] Of course, the signal depends on the two-component detection method applied. For example, when Förster resonance energy transfer (FRET) assay is used, the signal can refer to the fluorescence signal emitted by the FRET acceptor when the FRET donor absorbs the excitation beam in close proximity. In two-component system, the FRET acceptor or the FRET donor is preferably bound to two binding components specific for the analyte. Therefore, the fluorescence signal from the FRET donor-acceptor is only when the respective binding components are in close proximity, so the signal directly reflects the formation of the ternary two-component / analyte complex.

[0156] Similarly, Alphascreen (Amplified Luminescent Proximity Homogeneous Assay), developed by PerkinElmer and widely marketed, is a further prototypic two-component detection method, where signal generation depends on the proximity of "donor" and "acceptor" beads coated with a hydrogel layer that provides functional groups for bioconjugation reactions. When biological interactions between molecules bring the beads into close proximity, a cascade of chemical reactions is initiated, generating a greatly amplified signal. Upon laser excitation, a photosensitizer in the "donor" bead converts ambient oxygen to a more excited singlet state. The singlet state oxygen molecules diffuse and react with chemiluminescent species in the "acceptor" bead, further activating a fluorophore contained within the same bead. The fluorophore then emits light at 520 nm to 620 nm. The functional groups of the beads may represent two binding components specific for the analyte, for example in the form of specific antibodies. Only when a ternary complex containing the analyte and two components specific for the analyte is formed will the donor bead and acceptor bead be in close proximity, resulting in a detectable fluorescent signal.

[0157] Because FRET assay or Alphascreen assay depends on the proximity of donor and acceptor, and thus on the binding moiety specific for the analyte, such assay is called proximity-based detection assay.Many different proximity-based assays in the context of two-component detection methods are known in the art and can be used in the methods described herein.

[0158] As used herein, the term "proximity-based binary detection method" refers to any assay in which a signal reflecting the formation of a binary / analyte complex is based on the proximity of two analyte-specific binding components. Statistically, two analyte-specific binding components are more likely to be in close proximity in solution to produce a detectable signal if they form part of the same ternary complex.

[0159] Non-limiting examples of proximity-based two-component methods include methods using a resonance energy transfer assay, preferably a Förster resonance energy transfer (FRET) assay or a bioluminescence resonance energy transfer (BRET) assay, a protein complementation assay (PCA), Alphascreen or a DNA-labeled proximity assay, preferably a proximity ligation assay (PLA) or a proximity extension assay (PEA).

[0160] These proximity assay techniques are well known in the art. For further references on FRET or BRET, see, for example, (Pfleger, Seeber, & Eidne, 2006), for protein complementation assays, see Morell, Ventura, & Aviles, 2009, for Alphascreen, see Taouji, Dahan, Bosse, & Chevet, 2009, and for DNA labeling proximity methods such as PLA (proximity ligation assay) or PEA (proximity extension assay), see Soderberg et al., 2006.

[0161] Proximity-based assays are particularly preferred for "non-compartmentalized two-component detection method" as used herein, which refers to a two-component detection method, in which no compartmentalization of the sample solution is performed when applying the two-component detection method. Some non-compartmentalized assays may also be called homogeneous assays. In other words, non-compartmentalized two-component methods preferably include a step of contacting the analyte and two components in a homogeneous reaction solution, and the detection step for obtaining a signal reflecting the formation of the ternary two-component / analyte complex can be performed in the homogeneous sample solution without separating the unbound components or the analyte.

[0162] In certain preferred embodiments, the methods described herein use compartmentalized two-component detection methods. As used herein, the term "compartmentalized two-component detection methods" refers to two-component detection methods in which a sample is compartmentalized after contacting an analyte with a binding component specific to the analyte to quantify the formation of a ternary analyte / binding component.

[0163] In general, compartmentalized two-component detection methods are based on compartmentalizing the solution into small compartments such that without the formation of a ternary analyte / binding component approach, the probability of both binding components being present in a compartment is low and follows a Poisson distribution.

[0164] For compartmentalization, different assays can be envisioned. For example, emulsion droplet method can be used to form droplets in emulsion (e.g., water in oil), each droplet representing a separate compartment. Compartmentalization can include location or physical compartment, or diffusion-limited environment.

[0165] The term "droplet" as used herein preferably refers to an isolated portion of a first fluid surrounded by a second fluid. The first fluid preferably comprises a hydrophilic fluid such as water, an aqueous medium, or a buffer, and preferably comprises a sample solution or one or more dilutions thereof to which a two-component detection system or other reagent is added. The second fluid is preferably a hydrophobic fluid such as a hydrocarbon, silicone oil, mineral oil, organic solvent, etc. Emulsion techniques for compartmentalizing sample solutions are well known in the art.

[0166] In a particularly preferred embodiment, the compartmentalized two-component detection method is emulsion coupling, which refers to a digital assay concept based on the detection of dual-labeled (two-component) individual ternary molecular complexes in an emulsion, which can be identified, for example, by droplet digital PCR (ddPCR) or next-generation sequencing (NGS).

[0167] Droplet digital PCR (ddPCR) refers to a method for performing digital PCR, preferably based on water-oil emulsion droplet technology. The oil droplets can be created using a droplet generator that applies a vacuum to each well (see, for example, Pinheiro et al. Analytical Chemistry 84(2):1003-11). Typically, a sample can be fractionated into 20,000 or more droplets, and PCR amplification of template molecules occurs in each droplet. Advantageously, ddPCR technology uses reagents and workflows similar to those used in most standard TaqMan probe-based assays.

[0168] "Next generation sequencing (NGS)" as used herein is intended to encompass recently developed technologies for nucleic acid sequencing that typically allow for much higher throughput than traditional Sanger techniques (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 may allow for sequencing of clonally extended or unamplified single molecule nucleic acid fragments. Particular platforms include, for example, sequencing by ligation of dye-modified probes (including circular ligation and cleavage), pyrosequencing and single molecule sequencing. Nucleotide sequence species, amplified nucleic acid species and detectable products generated therefrom can be analyzed by such sequence analysis platforms. In the methods of the invention, next generation sequencing can be used to quantify PCR amplifiable unique DNA labels, for example, to assess the formation of two component / analyte complexes as described below.

[0169] Details of emulsion coupling as a preferred compartmentalized two-component detection method are described in EP 3224360, which is incorporated herein by reference.

[0170] For ddPCR detection in an emulsion coupling assay, analyte-specific binding components (such as antibody pairs) are labeled with a unique PCR-amplifiable DNA label, and the thus labeled binding components are added to the sample (or a dilution thereof, see e.g., Example 3).

[0171] Prior to emulsification of the sample, the reaction is highly diluted (approximately 100,000-fold) and PCR reagents are added to achieve single-complex isolation and PCR amplification per water-in-oil emulsion droplet. ddPCR can be performed using standard ddPCR protocols.

[0172] The evaluation of the reaction can be based on the division of the label in a ddPCR reaction using a fluorescently tagged PCR product (e.g., using a FAM or VIC-labeled real-time PCR probe). With ddPCR, the standard evaluation of the droplet population can be determined according to the fluorescent signal of the droplet. Here, the number of labeled binding components (e.g., antibodies) is determined in each reaction (counting all labeled positive droplets for a given label and using the same defined droplet population for all reactions).

[0173] In addition, the number of double-colored droplets (with two different binding moiety labels) is also determined. In the absence of ternary complexes, the partitioning of the labeled antibodies follows a Poisson distribution, resulting in a calculable number of double-colored droplets (containing two binding moieties in one compartment based on mere chance). If ternary complexes are present in the reaction, the number of double-colored droplets (containing additional ternary complexes) detected is higher than expected by a Poisson distribution.

[0174] Based on this measurement, the number of complexes can be calculated (for further reference, see, for example, EP 3224360 or Karakus et al., 2019), which allows absolute (number of molecules) quantification of the ternary analyte complex.

[0175] For next generation sequencing detection in emulsion coupling assays, the binding components may be preferably labeled with a label specific for the binding component (e.g., an antibody) and preferably also with an individual label for the molecule, i.e., a PCR amplifiable unique DNA label that includes a unique molecular barcode or unique molecular identifier-UMI (see Parekh et al., 2017). The labeled binding components may be added to the sample or a dilution thereof (see Example 3).

[0176] After binding of the binding component, e.g., an antibody, and prior to emulsification of the sample, the reaction can be highly diluted (e.g., about 100,000-fold) and PCR reagents can be added to achieve separation of single-complexes. PCR amplification can be performed for each water-in-oil emulsion droplet. ddPCR can be performed using the ddPCR protocol.

[0177] The evaluation of the reaction may be based on NGS reading of dimerized UMI labels specific for binding components, e.g., antibodies, generated according to standard protocols for emulsion coupling. The number of labeled binding components, e.g., antibodies, may be determined by counting all unique UMI labels for a given binding component, e.g., antibody, in each reaction (the count is limited to a given binding component). Possible multiple labeling of the same binding component, e.g., antibody, can be eliminated using preferentially dimerized sequences (multiple labels per binding component are colocalized in the same droplet, so that a double UMI-labeled dimer is always obtained in a labeling situation specific for a given antibody).

[0178] Ternary antibody / binary complexes are counted based on their dimerized double UMI-labeled PCR products (in the context of labels specific to two different binding components (e.g., antibodies), called heterodimers). Following the above concept, a correction for multiple labeling of binding components (e.g., antibodies) can be used, which takes into account double UMI-labeled dimers with labels specific to a given component (e.g., antibody).

[0179] Further evaluation of the sample can be performed as in the case of fluorescently tagged PCR (droplet digital PCR), briefly, without ternary complexes, the partitioning of the labeled antibodies follows a Poisson distribution, resulting in a calculable number of ternary complexes (based on the detection of heterodimers) in the droplets (which by chance contain just two antibodies). If ternary complexes are present in the reaction, the number of detected heterodimers is higher than would be expected by a pure Poisson distribution. Based on this measurement, the number of complexes can be calculated (EP 3224360, (Karakus et al., 2019)). This allows for absolute (number of molecules) quantification of the ternary complexes.

[0180] Dilutions of samples can be measured in the same sequencing reaction using DNA barcodes specific for the sample (e.g., barcoded primers), and because the antibodies have labels specific to distinguishable components (e.g., antibodies), many measurements (using different antibody pairs against different antigens) can be performed in parallel.

[0181] In emulsion coupling embodiments, the binding moieties may be provided as a library of binding moieties suitable for detection of multiple analytes, in these embodiments, each member of the binding moiety library may be associated with a unique nucleotide sequence that can be used to identify the binding moiety.

[0182] "Associated" in the context of emulsion coupling can mean that the presence of the binding moiety in the complex can be detected by the presence of a nucleic acid sequence in the linking sequence generated by the method. The nucleotide sequence can be attached to the binding moiety as a label, can be part of the binding moiety itself, e.g., an aptamer, or can be present within the nucleic acid in the binding moiety, e.g., a phage. For example, each member of the library can be labeled with a unique nucleotide sequence that is the nucleotide sequence attached to the binding moiety.

[0183] Methods for attaching nucleotides to binding moieties such as antibodies or compounds are known in the art. Alternatively, if the binding moiety library is a phage display library, the unique nucleotide sequence can be a sequence that codes for one or more CDR regions or the displayed binding domain. For example, the display library can be created by inserting the sequence that codes for the amino acid sequence to be displayed into a phage at a known position. The binding sequence can then be identified using a universal primer that amplifies the inserted sequence. Alternatively, if the binding moiety can be an aptamer, the aptamer itself can be a unique nucleotide sequence.

[0184] The nucleotide sequence may be an oligonucleotide and may include single-stranded or double-stranded RNA or DNA. The nucleotides used to label the binding agent or target may preferably be 5-150 bases long, for example 10-40 or 40-80 bases long. The nucleotides forming the nucleic acid may be chemically modified to increase the stability of the molecule, improve its bioavailability, or to confer additional activity to the molecule. For example, pyrimidine bases may be modified at the 6- or 8-position, and purine bases may be modified at the 5-position with CH3 or halogens such as I, Br or CI. Modifications or pyrimidine bases also include 2NH3, 0-CH3, N-CH3 and N2-CH3. Modifications at the 2' position are sugar modifications and typically include NH2, F or OCH3 groups. Modifications may also include 3' and 5' modifications, such as capping.

[0185] Alternatively, modified nucleotides such as morpholino nucleotides, locked nucleic acids (LNA) and peptide nucleic acids (PNAs) can be used. Morpholino oligonucleotides are constructed from different morpholino subunits, each containing one of the four genetic bases (adenine, cytosine, guanine and thymine) linked to a six-membered morpholine ring. The subunits are linked by non-ionic phosphorodiamidate intersubunit bonds to obtain morpholino oligonucleotides. LNA monomers are characterized in that the conformation of the furanose ring is restricted by a methylene linker that connects the 2'-0 position to the 4'-C position. PNA is an analogue of DNA in which the backbone is a pseudopeptide rather than a sugar.

[0186] Each member of the library is associated with a unique nucleotide sequence, i.e., each member may have a unique detectable nucleic acid identification label. Preferably, the unique nucleic acid identification labels may be linked. Linked may mean that the linking process has the potential to form random multimeric nucleic acid products based on the co-localization of these nucleic acid identification labels under suitable assay conditions. Preferably, the multimeric products are dimers. Suitable assay conditions may include amplification of the labels, where specific consensus amplification of unique sequences produces linkable amplicons. Linking of linkable amplicons, e.g., component-specific nucleic acids, forms linked identification labels, which code for the co-localization information of the identification labels. The linking reaction may be based on amplification or may include other techniques. Amplification-based linking may utilize two or more amplification primer pairs with the same binding capacity but with complementary 5' tags or dimeric linker sequences that form a nucleic acid duplex capable of polymerase extension. The tag or dimeric linker sequence means that the sequence amplified by one primer pair hybridizes to the sequence amplified by the second primer pair, thereby linking the identification label.

[0187] Since the identification labels, i.e., the relevant unique nucleotide sequences used in libraries such as protein display libraries and antibody libraries, may differ in their biological background, the amplification and linking process is based on two different primer pairs, for example, one primer pair amplifies the target sequence, such as the identification label based on cDNA, and the second primer pair amplifies the nucleotide sequence specific to the binding agent used as the identification label. Linking of different labels allows the binding agent specific information to be linked to the target information, for example, the protein encoded by the display cDNA.

[0188] The methods, kits, or other computer-implemented aspects of the invention may, in some embodiments, comprise and / or use one or more conventional computing devices having a processor, input devices such as a keyboard or mouse, memory such as a hard drive and volatile or non-volatile memory, and computer code (software) for performing the functions of the invention.

[0189] The components of the computing device may be conventional, although the device may be custom configured for a particular implementation. The computer-implemented method steps or systems may run on any particular architecture, such as personal / microcomputers, minicomputers, or mainframe systems. Exemplary operating systems include Apple Mac OS X and iOS, Microsoft Windows, and UNIX / Linux, SPARC, POWER, and Itanium-based systems, and z / Architecture. Computer code for carrying out the method steps described herein may be written in any programming language or model-based development environment, such as, but not limited to, Python, C / C++, C#, Objective-C, Java, Basic / VisualBasic, MATLAB, Simulink, StateFlow, Lab View, or Assembler. The computer code may include subroutines written in a proprietary computer language specific to the manufacturer of the circuit board, controller, or other computer hardware component used in conjunction with the invention.

[0190] The information processed and / or produced by the method, i.e., the digital representation of the signal reflecting the formation of the ternary binary / analyte complex, can use any type of file format used in the industry. Any suitable computer readable medium can be utilized. The computer usable or computer readable medium can be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, device, or propagation medium. More specific examples of computer readable media (not all of which are illustrative) include an electrical connection with one or more wires, a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disk read-only memory (CD-ROM), an optical storage device, a transmission medium such as one that supports the Internet or an intranet, a cloud storage, or a magnetic storage device.

[0191] All cited patent and non-patent literature is incorporated herein by reference in its entirety.

[0192] As used herein, the terms "comprising" or "comprises" are used in reference to expression cassettes, reporter vectors, and their respective components, which accommodate the inclusion of unspecified elements.

[0193] The term "consisting of" refers to an expression cassette, a reporter vector and each of its components as described herein, excluding any optional element not recited in the description of the embodiment.

[0194] The invention will be explained in more detail by referring to the accompanying drawings. [Brief description of the drawings]

[0195] [Figure 1]Figure 1 shows the curves and the basic principle of the mathematical background of the extended dynamic range of the measurement based on the simulation of a compartmentalized saturable two-component process. The following parameters were used: number of compartments = 200000, input volume = 20 microL, dilution factor before emulsification = 192000, concentration of target nM - variation, concentration of component A, nM = 1, concentration of component B, nM = 0.3, dissociation constant of component A = 0.1 nM, dissociation constant of component B = 0.1 nM, (1) number of analyte two-component, ternary complexes (molecules), (2) - number of double positive compartments without analyte (Poisson background), (3) - number of double positive compartments with target (Poisson background), and (4) - sum of (1) and (3). [Diagram 2] Figure 2 shows the determination of the dissociation constants of the components. Graphical representation of the simulation results. Intersection of Equation 3 at different antigen (Ag) concentrations (indicated) and component concentrations of 1 nM and 5 nM, respectively. Droplet number: 10000, dilution factor 10000 and reaction volume 20 microL. Using these input parameters, the chemical equilibrium, Poisson process, was simulated and the number of ternary complexes formed was determined. The value of Kd calculated using Equation 3 is identical to the value used for the simulation as input (see Example 3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0196] example Example 1 According to the laws of mass action and conservation of mass in solution, a saturable two-component reaction between two binding components (e.g., antibodies) and an analyte / target molecule (e.g., antigen) can be expressed as the following simultaneous equations:

number

[0197] When we solve the simultaneous equation 1 for a0, we get two radical roots

number

[0198] Both of these are true positive roots with non-bijective basis curves, i.e. they do not allow a one-to-one correspondence between the detection signal (c12) and the analyte (a0) concentration. Taking into account that the entire concentrations of these analytes are not available for the reaction, but only a portion of them, the ratios of active / inactive (in terms of binding capacity) antigens and antibodies can also be added to these equations. These ratios can be determined using concepts similar to those described in Example 2.

[0199] Example 2 Simulation of Kd determination using emulsion coupling (EC) technique The reaction of an antigen (Ag) with two different Ag-reactive antibodies (Ab1 = 5 nM and Ab2 = 1 nM) was simulated in a volume of 20 microliters (according to the chemical equilibrium and mass conservation equation (Equation 1) with Kd1 = 0.2 nM and Kd2 = 0.1 nM set). The simulated reaction volume was diluted (1e+5 times) and the reactants were sorted into 100000 droplets (total volume: 20 microliters) using a simulation based on a Poisson distribution. All droplets are sorted and counted according to the reactants encapsulated. The kd1-kd2 function (Equation 3) was calculated at three different Ag concentrations [a0] with the following inputs: total concentrations of Ag[a0], Ab1[b10], and Ab2[b20]. The Ag-Ab1-Ab2[c12] complexes determined by simulation were substituted into the equation and the intersection point of the function was determined (see Figure 2).

number

[0200] Equation 3. Substitute Ag[a0], Ab1[b10], Ab2[b20] and Ag-Ab1-Ab2[c12] into the equation. For [c12], its value is determined by simulation, others are input values ​​of the simulation.

[0201] Figure 2 shows the intersection of the curves of Equation 3 (using the above equation) at different Ag concentrations. The calculated Kd values ​​are identical to those used as input in the simulations (see above).

[0202] Simulation results show that by determining the intersection point of Equation 3, both Kds can be calculated from the initial amount of antigen, as well as the antibody and measured concentrations of the analyte-binary (ternary) complex. In experiments, it is useful to calculate the pairwise intersections as approximations to the experimental Kds and treat the given values ​​with statistical methods, such as Gaussian mixtures.

[0203] It should be noted that not all combinations of input parameters result in a solvable system, and suitable conditions can always be found by varying not only the antigen concentration but also the antibody concentration. Also, only one of the equations (Eq. 3) gives two positive roots that need to be determined in each individual case.

[0204] Considering that the total concentrations of these analytes are not available for the reaction, but only a portion of them, the ratios of active / inactive (in terms of binding capacity) antigens and antibodies can also be added to these equations. These ratios can be determined using more dilutions, extending the concepts described in Example 2.

[0205] Example 3 Exemplary Measurements of Analyte Concentration A sample containing an unknown concentration of a known analyte is provided. In one example, the analyte is a protein, as well as in one example the EGFR (epidermal growth factor receptor) receptor variant HER2. To detect the analyte using a two-component method, Alphascreen is applied. Alphascreen is a prototypic two-component detection method in which signal generation depends on the proximity of "donor" and "acceptor" beads coated with a hydrogel layer that provides functional groups for bioconjugation reactions. When biological interactions between molecules bring the beads into close proximity, a cascade of chemical reactions is initiated, generating a greatly amplified signal. Upon laser excitation, a photosensitizer in the "donor" bead converts ambient oxygen to a more excited singlet state. The singlet state oxygen molecules diffuse and react with chemiluminescers in the "acceptor" bead, further activating a fluorophore contained within the same bead. The fluorophore then emits light at 520 nm to 620 nm. The functional groups involved in biological interaction are provided by two antibodies, as illustrated in the ErbB2 / Her2 antibody pair (Novus Biologicals DP0061), which are conjugated to the surface of donor beads and acceptor beads, respectively.When donor beads and acceptor beads are contacted with a sample, the antibody binds to HER2 protein to form a ternary complex, which holds donor beads and acceptor beads in close proximity, and according to the above principle, detectable fluorescent signals are obtained.

[0206] To measure an unknown concentration of a HER2 protein analyte, it can be compared to a set of predefined signals of different known concentrations of HER2 protein. Such a reference curve for a two-component assay is most often characterized by a specific signal value corresponding to two analyte concentration values, e.g., a non-bijective reference curve. Thus, the curve does not have a one-to-one correspondence between analyte concentration and detection signal. To determine the concentration of HER2, one or more predefined dilutions are made from an unknown amount of HER2 sample and the determined signal. Each signal of the dilutions has two corresponding concentrations when compared to the reference curve. The ratio of the estimated concentration and their predefined dilution factors must be equal, so that the concentration of the HER2 sample can be determined. Mathematically, f(x)=s1, where the reference curve function f(x) is a function of the concentration x, and s1 is the measured signal. f(x)=s1 can be satisfied with two concentrations x1 or x2. However, x can be determined if f(x / c)=s2 is determined, where c is the predefined dilution factor and s2 is the measured signal. Similarly, as above, f(x / c)=s2 can be satisfied by x3 or x4. x1 / x3 or x1 / x4 or x2 / x3 or x2 / x4 must be c, and only one or two of them are equal to c, and if two of them are equal, it can be proven that only the same x can be satisfied, and thus the ambiguity can be resolved. For example, if s1=1000 fluorescent units (FU), this corresponds to 100nM (x1) and 5nM (x2) of HER2 protein in the hypothetical standard curve, and the original HER2 sample is diluted 10 times, a signal of 2000 (FU) is obtained, which corresponds to 90nM (x3) and 10nM (x4) of HER2 protein in the hypothetical standard curve. Simply let x1 / x4=c, then x1 / x3=100 / 90 or x1 / x4=100 / 10 or x2 / x3=5 / 90 or x2 / x4=5 / 10, and the result is x=x1=100nM.

[0207] Example 4 Exemplary Determination of Analyte Concentration Using a Compartmentalized Two-Component Method A sample is provided containing an unknown concentration of a known analyte. In one example, the analyte is a protein, also in one example, the EGFR receptor mutant HER2, and an ErbB2 / Her2 antibody pair (Novus Biologicals DP0061) is used as a detection reagent, i.e. as a binding component specific for the analyte. A compartmentalized two-component method is emulsion coupling (EP 3224360, Karakus et al.). Emulsion coupling is a digital assay concept based on the detection of individual ternary molecular complexes of double-labeled (two components) in an emulsion, which are identified, for example, by ddPCR. For ddPCR detection, the ErbB2 / Her2 antibody pair (Novus Biologicals DP0061) is labeled with a unique DNA label that can be PCR amplified. As in example 3, the sample is diluted at a defined ratio and the labeled antibody is added to the sample. Prior to emulsification of the samples, the reactions are highly diluted (approximately 100,000-fold) and PCR reagents are added to achieve single-complex isolation and PCR amplification per water-in-oil emulsion droplet. ddPCR is performed using standard ddPCR protocols (Biorad QX200 ddPCR).

[0208] The evaluation of the reaction is based on the partitioning of the label in a ddPCR reaction using fluorescently tagged PCR products (using FAM or VIC-labeled real-time PCR probes). According to the ddPCR standard evaluation, the droplet population is determined according to the fluorescent signal of the droplets and the number of labeled antibodies is determined in each reaction (counting all labeled positive droplets for a given label and using the same predefined droplet population for all reactions). In addition, the number of double-colored (with two different antibody labels) is also determined. In the absence of ternary complexes (HER2 protein bound by two antibodies), the partitioning of the labeled antibodies follows a Poisson distribution, resulting in a calculable number of double-colored droplets (containing two antibodies based on mere chance). If ternary complexes are present in the reaction, the number of detected double-colored droplets (containing additional ternary complexes) is higher than would be expected by a Poisson distribution. Based on this measurement, the number of complexes can be calculated (for further references, see EP 3224360, Karakus et al., 2019). This allows the absolute (number of molecules) quantification of the ternary analyte complexes. According to Equation 1, the original absolute concentration of the analyte (HER2) can also be calculated using the known dissociation constant and the activity fraction of the applied antibody. However, Equation 1 has two power roots, only one of which is the concentration. Following a calculation similar to that given in Example 3, the correct concentration of an unknown HER2 sample can be determined (see Example 3 for a numerical example).

[0209] Example 5 Exemplary Determination of Dissociation Constants of Antibodies in Non-compartmentalized Two-component Assays A sample is provided containing a known concentration of a known analyte. By way of example, the analyte is a protein, also by way of example, the EGFR receptor mutant HER2, and the ErbB2 / Her2 antibody pair (Novus Biologicals DP0061) is used as an exemplary reagent. The two-component method is Alphascreen as in Example 3, using the dilutions and standard curves described in Example 3. To determine the relationship between the Alphascreen signal and the corresponding concentration of the ternary complex, the method described in Example 4 can be used in parallel with the Alphascreen standard curve measurement. This allows absolute (number of molecules) quantification of the ternary complex associated with the Alphascreen signal. To calculate the dissociation constant (Kd) of the antibody, Equation 3 is applied, and once two or more analyte dilutions have a known amount of analyte and a determined amount of ternary complex, the latter is determined using the determined corresponding relationship between the concentration of the ternary complex and the Alphascreen signal. The two Kds can be calculated according to Example 2. Mathematically, f(kd1, c12, a0)=kd2 applies, where kd1 and kd2 are dissociation constants, c12 is the measured concentration of the ternary complex of the analyte, and a is the concentration of the analyte. Different dilutions of the analyte correspond to different concentrations of c12, so the "f" function has different forms. However, Kd is a material constant, so as a result, all forms of the f function must be satisfied by a specific pair of Kd values, which can be determined, see Example 2.

[0210] Example 6 Exemplary Determination of Dissociation Constants of Antibodies in Compartmentalized Two-Component Assays A sample containing a known concentration of a known analyte is provided. In one example, the analyte is a protein, also in one example, the EGFR receptor mutant HER2, and the ErbB2 / Her2 antibody pair (Novus Biologicals DP0061) is used as an exemplary detection reagent, i.e., a binding component specific for the analyte. The compartmentalized two-component method is emulsion coupling with a Biorad QX200 ddPCR as a detection device (EP 3224360, (Karakus et al., 2019)) as in Example 4 using dilutions, and the number of ternary complexes can be calculated as in Example 4 (EP 3224360, Karakus et al., 2019). This allows the absolute (number of molecules) quantification of the ternary complex. To calculate the dissociation constant (Kd) of the antibody, Equation 3 is applied, and once two or more analyte dilutions have known amounts of analyte, antibody and a determined amount of ternary complex, the two Kds can be calculated according to Example 2. Mathematically, f(kd1, c12, a0)=kd2 applies, where kd1 and kd2 are dissociation constants, ac is the measured concentration of the ternary complex of the analyte, and a is the concentration of the analyte. Different dilutions correspond to different (concentration) values ​​of "ac", so the f function has different forms. However, Kd is a material constant, so as a result, all forms of the f function must be satisfied by a specific pair of Kd values, which can be determined, see Example 2.

[0211] Example 7 Exemplary Determination of Analyte Concentration Using a Compartmentalized Two-Component Method with Next-Generation Sequencing A sample is provided containing an unknown concentration of a known analyte. In one example, the analyte is a protein, also in one example, the EGFR receptor mutant HER2, and an ErbB2 / Her2 antibody pair (Novus Biologicals DP0061) is used as the detection reagent, i.e., the binding component specific for the analyte. A compartmentalized two-component method is emulsion coupling (EP 3224360, Karakus et al., 2019). Emulsion coupling is a digital assay concept based on the detection of doubly labeled (ternary) individual molecular complexes in an emulsion, which are identified, for example, by next-generation sequencing (NGS). For NGS detection, the ErbB2 / Her2 antibody pair (Novus Biologicals DP0061) is labeled with a PCR-amplifiable unique DNA label (label specific for the antibody), which also carries a molecular barcode (UMI) unique to the individual labeled molecule (Parekh et al., 2017). Defined analyte dilutions are made and labeled antibody is added to the sample as in Example 3. After antibody binding and prior to sample emulsification, the reaction is highly diluted (approximately 100,000-fold) to achieve single-complex dissociation, PCR reagents are added, and PCR amplification is performed per water-in-oil emulsion droplet as in ddPCR using the ddPCR protocol (Biorad QX200 ddPCR).

[0212] The evaluation of the reactions is based on the NGS readout of antibody-specific dimerized UMI labels produced according to the emulsion coupling concept (EP 3224360). The number of labeled antibodies is determined in each reaction by counting all unique UMI labels for a given antibody (counting exclusively in the labeling context specific for a given antibody), and possible multiple labeling of the same antibody was eliminated using preferentially dimerized sequences (multiple labels per antibody always have a double UMI-labeled dimer in the labeling context specific for the same antibody, since they colocalize in the same droplet). Ternary complexes are counted based on their dimerized double UMI-labeled PCR products (two different antibody-specific labels, called heterodimers), with correction according to antibody multiple labeling (following the concept described above to eliminate the effect of multiple labels per antibody). Further evaluation of the sample is performed according to Example 4, and briefly, in the absence of ternary complexes, the partitioning of the labeled antibodies follows a Poisson distribution, resulting in a calculable number of ternary complexes (based on the detection of heterodimers) in droplets (which by chance only have two antibodies). If ternary complexes are present in the reaction, the number of detected heterodimers is higher than would be expected by a pure Poisson distribution. Based on this measurement, the number of complexes can be calculated (EP 3224360). This results in an absolute (number of molecules) quantification of the ternary complexes. According to Equations 1-2, the original absolute concentration of the analyte (HER2) can also be calculated using the known dissociation constant and the activity fraction of the applied antibody (the latter is arbitrary). However, Equations 1-2 have two power roots, only one of which is always correct. Using dilutions, as described in Example 3, the exact concentration of an unknown HER2 sample can be determined (see Example 3 for a numerical example). Dilutions of the sample can be measured in identical sequencing reactions using DNA barcodes specific for the sample (e.g., barcoded primers).

[0213] Example 8 Exemplary Determination of Dissociation Constants of Antibodies in Compartmentalized Two-Component Assays Using Next-Generation Sequencing A sample is provided containing a known concentration of a known analyte. In one example, the analyte is a protein, as in one example, the ErbB2 / Her2 antibody pair (Novus Biologicals DP0061) labeled with a UMI label specific for the EGFR receptor mutant Her2, antibody. The compartmentalized two-component method was emulsion coupling (EP 3224360) using dilutions and formulas 1-2, using NGS as the detection method as in example 7. The number of ternary complexes can be calculated (EP 3224360). This results in an absolute (number of molecules) quantification of the ternary complex. To calculate the dissociation constant (Kd) of the antibody, formula 3 is applied, and once two or more analyte dilutions (which can be partially identical dilutions as above) have a known amount of analyte and a determined amount of ternary complex, the two Kds can be calculated according to example 2. Mathematically, f(kd1, c12, a0)=kd2 applies, where kd1 and kd2 are dissociation constants, c12 is the measured concentration of the ternary complex of the analyte, and a0 is the concentration of the analyte. Since different dilutions correspond to different (concentration) values ​​of c12, the f function has different forms, but since Kd is a material constant, all forms of the f function must be satisfied by a specific pair of Kd values, which can be determined, see Example 2. Dilutions of a sample can be measured in the same sequencing reaction using a DNA barcode (e.g., barcoded primer) specific to the sample.

[0214] Considering that the total concentrations of these analytes are not available for the reaction, but only a portion of them, the ratios of active / inactive (in terms of binding capacity) antigens and antibodies can also be added to these equations. These ratios can be determined using more dilutions, extending the concepts described in Example 8.

[0215] References JPEG2025072414000004.jpg217161 JPEG2025072414000005.jpg23161

Claims

1. 1. A method for determining the concentration of an analyte in a sample containing an unknown concentration of the analyte using a two-component detection method, comprising: a. performing a two-component detection method in solution, comprising providing two non-immobilized analyte-specific binding moieties at known concentrations and contacting the two analyte-specific binding moieties with the analyte to generate a signal that is dependent on the concentration of a two-component / analyte complex formed in solution; b. providing a non-bijective analyte concentration reference curve for the two-component detection method that is a mathematical function reflecting the dependence of the signal on the concentration of the analyte; c. preparing one or more dilutions of said sample using a defined dilution factor; d. applying the two-component detection method to the sample and to the one or more dilutions; e. determining the concentration of the analyte using the signal detected in the sample and the one or more dilutions as a constraining input for a mathematical fit to a standard curve of the non-bijective analyte concentrations at different analyte concentrations; The method includes:

2. 1. A method for determining the dissociation constant of a binding component specific for an analyte using a known concentration of the analyte in a sample in a two-component detection method, comprising: a. performing a two-component detection method in solution comprising providing two non-immobilized analyte-specific binding moieties at known concentrations and contacting the two analyte-specific binding moieties with the analyte to generate a signal that is dependent on the concentration of a two-component / analyte complex formed in solution; b. providing a dissociation constant relationship for said two-component detection method that is a mathematical function of the relationship between the dissociation constants (kd1 and kd2, respectively) of two analyte-specific binding components and the analyte in dependence on said signal reflecting the concentration of the two-component / analyte complex and the concentrations of the analyte and analyte-specific binding components; c. preparing one or more dilutions of said sample and / or said analyte-specific binding components; d. applying the two-component detection method to the sample and to the one or more dilutions; e. determining the dissociation constants kd1 and kd2 of two analyte-specific binding moieties using the signals detected in the sample and one or more dilutions as constraint inputs for a mathematical fit of the dissociation constant relationships at different analyte concentrations and / or analyte-specific binding moiety concentrations; The method includes:

3. 2. The method of claim 1, wherein the non-bijective analyte concentration standard curve is obtained experimentally by providing a reference sample of known analyte concentration, making a series of known dilutions of the reference sample, and performing the two-component detection method on the reference sample and each of its known dilutions, and / or the non-bijective analyte concentration standard curve is analytically calculated by solving chemical equilibrium and mass conservation equations or provided by a numerical solution based on a definition of a dissociation constant between the analyte and each of two binding components specific for the analyte.

4. The method of claim 2 , wherein the dissociation constant relationships are analytically calculated by solving chemical equilibrium and mass conservation equations.

5. The method of any one of claims 1 to 4, wherein the analytes are selected from the group consisting of proteins, peptides, nucleic acid segments, carbohydrates, lipids, antibodies (monoclonal or polyclonal), antigens, oligonucleotides, specific receptor proteins, ligands, molecules, cells, microorganisms, and fragment products or combinations thereof, and / or the sample comprises two or more different types of analytes.

6. The method according to any one of claims 1 to 5, wherein the two binding moieties specific for the analyte are selected from the group consisting of nucleic acids, preferably RNA and / or DNA oligonucleotides, antibodies, peptides, proteins, aptamers, molecularly imprinted polymers, cells or combinations thereof and / or two or more pairs of binding moieties specific for different types of analytes are used.

7. 7. The method of any one of claims 1 to 6, wherein the two-component method comprises using a proximity-based assay to generate a concentration-dependent signal of the two-component / analyte complex, the proximity-based assay using two binding components specific for an analyte that generate a detectable signal depending on their proximity, and / or the two-component method comprises using a resonance energy transfer assay, preferably a Förster resonance energy transfer (FRET) assay or a bioluminescence resonance energy transfer (BRET) assay, a protein complementation assay (PCA), Alphascreen or a DNA labelling proximity assay, preferably a proximity ligation assay (PLA) or a proximity extension assay (PEA).

8. 8. The method of any one of claims 1 to 7, wherein the two-component detection method comprises using a compartmentalisation assay to generate a concentration-dependent signal of a two-component / analyte complex, said signal reflecting the presence of two binding components specific for the analyte in a single compartment, said compartmentalisation assay preferably using an emulsion droplet technique, each droplet in the emulsion representing a separate compartment, more preferably said compartmentalisation assay is emulsion coupling.

9. The method of any one of claims 1 to 8, wherein multiple analytes are determined in parallel.

10. The method of any one of claims 1 to 9, wherein the two-component detection method comprises using an analysis method based on absolute molecular number and / or the two-component detection method comprises using a droplet digital PCR assay.

11. 11. The method of any one of claims 1 to 10, wherein the two-component detection method comprises using an analyte-specific binding component associated with a unique amplifiable nucleic acid label, using a compartmentalized assay, and nucleic acid amplification is performed for each compartment using fluorescently tagged amplification products.

12. 12. The method of any one of claims 1 to 11, wherein the two component detection method comprises using binding components specific for multiple analytes that contain a unique molecular identifier-barcode that identifies a nucleic acid molecule, using a compartmentalized assay, where nucleic acid amplification is performed for each compartment generating a unique molecular identifier-nucleic acid barcode that identifies a linked molecule, the compartments are recombined in a common pool, and parallel nucleic acid sequencing techniques are used to generate a concentration dependent signal of the two component / analyte complex.

13. 13. The method of claim 11 or 12, wherein the parallel nucleic acid sequencing technology is next generation sequencing technology (NGS).

14. 1. A kit for determining the concentration of an analyte using a two-component detection method, comprising: a. two or more non-immobilized, analyte-specific binding moieties of known concentrations for performing a two-component detection method comprising contacting the two analyte-specific binding moieties with a solution containing the analyte to generate a signal that is dependent on the concentration of a two-component / analyte complex formed in the solution; b. reference data for a non-bijective analyte concentration standard curve for said two-component detection method, said reference data being a mathematical function reflecting the dependence of said signal on analyte concentration, said non-bijective analyte concentration standard curve exhibiting monotonic segments of increase and decrease; c. optionally, instructions for preparing one or more dilutions of said sample and applying a two-component detection method to said sample and said one or more dilutions; d. a computer program when executed on a computer configured to perform a calculation step of comparing the signal detected in the sample and in one or more dilutions to a standard curve of non-bijective analyte concentrations to determine the concentration of the analyte in the sample; Kit including:

15. A kit for determining a dissociation constant in a two-component detection method, comprising: a. a reaction buffer for two or more binding moieties of known concentrations specific for an analyte for carrying out a two-component detection method comprising contacting two binding moieties specific for said analyte with a solution containing the analyte to generate a signal dependent on the concentration of a two-component / analyte complex formed in said solution; b. Reference data for the dissociation constant relationship of the two-component detection method, which is a mathematical function for the relationship of kd1 and kd2 depending on the signal reflecting the concentration of the two-component / analyte complex and the concentration of the analyte and / or the binding component specific for the analyte; c. Optionally, instructions for preparing one or more dilutions of said sample and applying a two-component detection method to said sample and said one or more dilutions using a defined dilution factor; d. a computer program when executed on a computer configured to perform a calculation step of determining the dissociation constants kd1 and kd2 of two analyte-specific binding moieties using the signals detected in the sample and one or more dilutions as constraint inputs for a mathematical fit of the dissociation constant relationships at different analytes and / or analyte-specific binding moiety concentrations; Kit including:

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