Methods for increasing the dynamic range of a bioassay

Abstract

This invention relates to methods for improving the dynamic range of bioassays. Compositions and methods are provided to extend the range of bioassays such as immunoassays. In one embodiment, multiple discrete test sites are used. Data indicating the proportion of test sites present with the analyte of interest and data providing statistical values ​​for the signals generated by the population of discrete test sites are collected. The results of these numerical and statistical methods are aggregated to provide an extended dynamic range. In another embodiment, reagent additions are provided two or more times sequentially to a single test site or container. Such additions present different reagent groups at different dilutions of the same sample and allow for the simultaneous characterization of both high-abundance and low-abundance analytes in a single multiplex assay, while avoiding the high-dose hook effect of high-abundance analytes observed at low sample dilutions. These methods can be combined to provide further improvements in dynamic range.

Description

[0001] This application is a divisional application. The original application was filed on September 6, 2018, with application number "201811035838.9" and invention title "Method for Improving the Dynamic Range of Bioassays".

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 554,889, filed September 6, 2017. These and all other foreign references are incorporated herein by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition provided herein, the definition provided herein shall prevail. Technical Field

[0003] The field of this invention is bioassays, particularly immunoassays. Background Technology

[0004] The background description includes information that can be used to understand the invention. This is not an admission that any information provided herein is prior art or related to the currently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0005] Bioassays, such as immunoassays (e.g., EIA, FIA, etc.), nucleic acid amplification assays (e.g., rtPCR, linear amplification assays, etc.), and cell-based assays, are the backbone of modern biological research and clinical practice. Historically, these bioassays have been performed by treating samples with analyte-specific reagents and obtaining readings of signal intensity (e.g., absorbance, fluorescence intensity, luminescence intensity, etc.) from a bulk mixture. This signal intensity is compared to a standard or dose / response curve of the signal intensity obtained from a bulk mixture prepared from a similar sample treated with the same reagents to derive the concentration of the analyte of interest. In the case of immunoassays, such analyte-specific reagents typically comprise antibodies or antibody fragments that form specific, detectable complexes with the analyte of interest. The dynamic range of such immunoassays is generally limited to about two orders of magnitude. However, in some applications, the concentration range of the analyte may extend beyond this range. Similarly, in multiplex assays, the concentration range of each analyte can vary widely.

[0006] Attempts have been made to develop bioassays with extended measurement or dynamic range. For example, Bell's U.S. Patent No. 6,551,788 describes a fluorescence-based immunoassay in which particles of different sizes are used as the initial capture phase. The patent states that larger particles provide more efficient capture of analyte molecules at low concentrations than smaller particles; unfortunately, it does not provide a theoretical framework or data to support this assertion.

[0007] U.S. Patent Application Publication No. 2014 / 0065722 by Goix et al. describes a method of some degree of similarity, in which different populations of particles are conjugated with either low- or high-affinity antibodies against the same analyte and used as a capture phase. The different populations of particles are read separately to generate differentiated dose / response profiles that can be used to provide an extended measurement range. U.S. Patent Application Publication No. 2015 / 0046114 by Dowell et al. describes a similar method in which a co-capture antibody is used in combination with different labeled antibodies with different affinities for the analyte in a sandwich immunoassay. However, these methods require the availability of antibodies with the same specificity and significantly different affinities, which may not be suitable for the analyte of interest.

[0008] In another approach, test devices have been proposed that incorporate physically distinct regions or sensors with different binding affinities to the analyte of interest. For example, U.S. Patent No. 9,671,398 (Campbell et al.) describes the use of two different immunosensors, one constructed to demonstrate attenuation of binding of the analyte relative to the other, to derive overlapping dose / response profiles that provide an effective extended measurement range. However, the construction of such devices is complex and becomes increasingly complicated when applied to multiplex assays.

[0009] In another method described in Drukier's U.S. Patent No. 7,604,956, an extended measurement range is achieved by systematically reducing background noise sources in other routine assays. This reduction in background noise effectively increases the sensitivity of the assay without affecting performance at high analyte concentrations, resulting in an increased dynamic range. However, it is unclear whether the method for minimizing background noise sources is suitable for a wide range of bioassays. Furthermore, since the method for minimizing background noise sources is specific to each analyte, it seems unlikely that this method could be used for multiplex assays.

[0010] In yet another method described in U.S. Patent No. 7,723,127 (Talebpour and Leanord), an immunoassay is performed using multiple additions of a sample containing the analyte of interest to a capture reagent. Each subsequent addition is performed using a large volume of diluted capture reagent. The resulting composite dose / response curve falls between those generated using a small initial sample volume and those generated as a single bolus application of the total sample volume. However, while this is positioned to provide a dose / response curve with an extended dynamic range, the comparative data provided show a significant response to a wider range of analyte concentrations compared to composite dose / response curves provided by a single addition. The reported extended measurement range appears to be a result of unconventional calculations of the minimum detectable dose, which is chosen based on the signal strength of 10% of the maximum observed signal, rather than the ability to distinguish the observed signal from the signal achieved with a blank sample.

[0011] Recently, various techniques have been developed to utilize the ability to encode individually occurring results in such assays, especially in the case of multiplex assays. For example, multiplex immunoassays can be performed using two-dimensional surface microarrays (which provide positional encoding of individual results) or suspended arrays of microbeads (which can be encoded using dyes, fluorophores, etc., different from those used to provide analyte-specific results). Such methods are commonly used for multiplex assays because the encoding of results provides a means of distinguishing results from different assays performed simultaneously on the same sample. However, even without multiplexing, deriving quantification from such encoded results is challenging. For appropriate scaling, individual coded results are typically provided on relatively small and discrete regions—such as the surface of individual microbeads or individual printed “dots” on microarrays. Historically, analysis of single discrete coded results has not provided sufficiently accurate quantification. Statistical methods have been applied to aggregated data from multiple discrete coded results to provide quantification.

[0012] In one approach, signal intensities from a large number of individual discrete test regions are measured, and these signal intensities are used to generate a frequency distribution of the intensity values. Typically, the mean or median of the distribution is used as a value associated with the analyte concentration in the sample. For example, U.S. Patent Application No. 2012 / 0308997 (Ruan et al.) describes a method for immunoassay using a population of particles. The distribution of signal intensities from the test particles is used to calculate an average signal intensity, which is then used to derive the concentration of the analyte in the test sample. All publications herein are incorporated by reference to the same extent as each individual publication or patent application is specifically and individually indicated as incorporated by reference. Where the definition or usage of a term in the incorporated references is inconsistent with or contrary to the definition of the term provided herein, the definition of the term provided herein shall apply, and not the definition of the term in the references. However, in such methods, the assay sensitivity is essentially both the average signal intensity and the variation (i.e., the signal-to-noise ratio) found within this distribution. Because the signal-to-noise ratio is low at low analyte concentrations, the assay sensitivity using this method is necessarily limited.

[0013] In another approach, individual discrete test regions are binary classified as "positive" (i.e., indicating the presence of analyte) or "negative" (i.e., indicating the absence of analyte). In such "numerical" methods, the analyte concentration of the sample is derived using the relative ratio of positive to negative test regions, typically by comparison with a dose / response curve generated using a sample containing a known analyte concentration. For example, UK Patent No. 2510653 (Shim et al.) describes a method in which individual droplets in an oil:water emulsion prepared using a test sample are classified as containing or not containing β-galactosidase labeling, based on enzyme activity. The amount of β-galactosidase labeling present in the test sample is determined by the ratio of positive to negative droplets determined from an image of a monolayer of such droplets. Similarly, U.S. Patent Application Publication No. 2015 / 0293102 (Shim) describes various immunoassays using fluorescently or enzyme-labeled antibodies in a femtodrop suspension, wherein the concentration of the analyte in the test sample is derived using the ratio of positive to negative droplets. U.S. Patent Application Publication No. 2004 / 0126899 (Lee and Yanavich) describes the use of a fixed amount of magnetic particles that form a relatively stable complex with a porous solid phase if analyte is present. Negative particles that do not form complexes with the solid phase are removed by applying a weak magnetic field, and quantification is provided by enumerating the remaining bound particles. U.S. Patent Application Publication No. 2010 / 075862 (Duffy et al.) describes the use of a complex array of optical fibers terminating in a test region. Immunoassays are performed on the coated ends of the fibers, and the result for each fiber is determined as positive or negative based on signal intensity. The proportion of positive fibers is used to determine the analyte concentration in the test sample. International Patent Application Publication No. WO2017 / 034925 (by Zur Megede and Karlin-Neumann) describes a “digital readout assay” in which multiple “zones” are identified and classified as positive (containing a marker indicating the presence of an analyte) or negative (lacking the marker). The ratio of positive to negative zones is used to derive the concentration of the analyte present in the test sample. Unfortunately, the dynamic range of assays using such methods is essentially a function of the occupancy rate of the discrete test regions used, with an upper limit of full or near-full occupancy. Therefore, the dynamic range of such assays is necessarily limited.

[0014] Therefore, methods for improving the dynamic range of bioassays are still needed. Summary of the Invention

[0015] The present invention provides compositions and methods for extending the dynamic range of bioassays.

[0016] One embodiment of the present invention is a method for quantifying results derived from bioassays (such as immunoassays, nucleic acid amplification assays, enzyme assays, and / or bioactivity assays) by performing a first bioassay against a first analyte on a sample using a plurality of first discrete test sites, wherein the first bioassay provides a change in a first signal (e.g., fluorescence, enzyme activity, absorbance, phosphorescence, and / or luminescence) indicating the presence of the first analyte. A plurality of quantities representing this first signal are obtained, each of which is associated with a value of the first signal observed from an individual test site among the first discrete test sites. A first proportion comprising an enumeration of first discrete test sites having a signal indicating the presence of the analyte in the sample is determined. Additionally, a total statistical value of the first signal is derived using a statistical distribution of the plurality of quantities derived from the first discrete test sites. The first proportion is used to determine the mass of the first analyte in the sample when it falls within a first cutoff range, and the statistical value of the first signal is used to determine the mass of the first analyte in the sample when it falls outside the first cutoff range. In some embodiments, such a cutoff range is selected to divide a population in which less than 90% of the first discrete test sites include the first analyte.

[0017] In some embodiments, a second bioassay is performed in conjunction with the first bioassay to quantify the second analyte in a similar manner using a second set of discrete test sites. In such embodiments, the first and second discrete test sites can be encoded separately. Suitable test sites include planar microarrays, microparticles, cells, microdroplets, nanodroplets, femtodroplets, micelles, molecules, and molecular complexes.

[0018] Another embodiment of the present invention is a method for improving the dynamic range of a biobinding assay by contacting a first test surface comprising a first trapping molecule with a first volume of sample, wherein the sample contains a first target molecule that binds to the first trapping molecule and also includes a second target molecule. This first volume provides a first sample dilution. The first test surface and the sample are incubated for a first period of time sufficient to form a first complex comprising the first trapping molecule and the first target molecule. A second volume comprising a second test surface is then added to the first volume to generate a second sample dilution. This second test surface comprises a second trapping molecule, and in some embodiments, the second volume exceeds the first volume. The second test surface and the sample (diluted at the second sample dilution) are incubated for a second period of time sufficient to form a second complex comprising the second trapping molecule and the second target molecule. A first signal and a second signal are then obtained from the first complex, and a second signal is obtained from the second complex. In some embodiments, the first test surface comprises a first microparticle population, and the second test surface comprises a second microparticle population. In some embodiments, the second target molecule is present in the sample at a concentration exceeding that of the first target molecule. In some embodiments, the first target molecule and the second target molecule are indistinguishable, and the first trapping molecule and the second trapping molecule have the same specificity. In such embodiments, the dynamic range of the assay for a single analyte is extended. In some embodiments, the methods described above can be used to quantify the first signal and / or the second signal in order to further extend the dynamic range of the bioassay (one or more).

[0019] Various objects, features, aspects and advantages of the subject matter of the invention will become more apparent from the following detailed description of preferred embodiments, together with the accompanying drawings, in which similar reference numerals denote similar parts. Attached Figure Description

[0020] Figures 1A to 1G: Figures 1A to 1G show the dose-response curves of median fluorescence intensity (MFI) for different particle-based cytokine assays performed in conjunction with a C-reactive protein (CRP) assay using two different sample dilutions applied sequentially in the same test well. The cytokine assay reagents were applied during initial incubation at a low sample dilution (1:3). Figure 1A shows the dose / response curves generated for IL-1β. Figure 1B The dose / response curves generated for IL-10 are shown. Figure 1C The dose / response curves generated for IFN-γ are shown. Figure 1D The dose / response curves generated for IL-8 are shown. Figure 1E The dose / response curves generated for hMCP-1 are shown. Figure 1F The dose / response curves generated for IL-4 are shown. Figure 1GThe dose / response curves generated for IL-6 are shown.

[0021] Figure 2 : Figure 2 The dose / response curves of median fluorescence intensity (MFI) for C-reactive protein (CRP) assays performed in conjunction with multiplex cytokine assays were plotted using two different sample dilutions applied sequentially in the same test well. CRP assay reagents were provided in volumes that provided the second sample dilution (1:60) after the addition of the cytokine assay reagents. Detailed Implementation

[0022] The following description includes information that can be used to understand the invention. This is not an admission that any information provided herein is prior art or related to the currently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0023] This invention provides apparatus, systems, and methods for determination using multiple discrete test regions. Suitable test regions include microparticles, spatially defined portions of planar arrays, droplets (including microdroplets and / or flydrops) in emulsions and / or suspensions, individual pixels within a digitized image of the test region, cells, individual molecules in a solution, and / or results otherwise encoded to represent different regions or volumes of space. Discrete test regions can be characterized individually for signal intensity derived from the identification of an analyte of interest within the discrete test regions. All or some of the discrete test regions used in a test of a sample are analyzed (e.g., in a flow cytometer) to determine analyte-specific signal intensities. The analyte-specific signal intensities of the discrete test regions are used for statistical analysis of the signal intensities of a test population of discrete test regions, and for determining whether each characterized discrete test region includes the analyte in a binary manner (i.e., positive or negative "digital" data). The digital data can be used to derive the proportion of discrete test regions that include the analyte relative to discrete test regions that do not include the analyte. For example, a gated signal intensity value indicates a positive result for the presence of the analyte in a single test region (e.g., a microparticle).

[0024] The percentage of a population of particles that meets this criterion can be correlated with the analyte concentration. For example, at medium to high analyte concentrations, virtually all test areas will indicate the presence of the analyte, corresponding to a 100% occupancy rate. As the analyte concentration decreases, the percentage of individual test sites that increase will provide a signal indicating that the analyte was not detected at that test site, and the occupancy rate decreases. For example, if 20% of the characterized individual test sites indicate that they did not identify the presence of the analyte, the occupancy rate would be considered 80%. For numerical quantification purposes, once the occupancy rate is less than 100% but above the background level, a correlation can be established between the level of occupancy and the analyte concentration. For example, below a predetermined cutoff value (e.g., approximately 90% occupancy), such numerical data can be used to calculate the concentration of the analyte in the test sample. Above the predetermined cutoff value, statistical data collected from the population of characterized discrete test areas can be used to determine the concentration of the analyte in the test sample. In a preferred embodiment, discrete test areas can be planned (e.g., by selecting antibody and / or antibody content) such that these ranges overlap and provide a continuous measurement range.

[0025] It should be understood that the disclosed techniques provide many advantageous technical effects, including providing measurements with an extended dynamic range relative to conventional measurements using signal strength derived from bulk solutions, statistical calculations of the average signal strength from individual test result groups, and / or binary classification of individual members of test result groups.

[0026] The following discussion provides numerous exemplary embodiments of the subject matter of this invention. While each embodiment represents a single combination of inventive elements, the subject matter of this invention is considered to encompass all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, the subject matter of this invention is also considered to encompass other remaining combinations of A, B, C, or D, even if not explicitly disclosed.

[0027] As used herein, and unless the context otherwise requires, the term “coupled to” is intended to include both direct coupling (where two coupled elements are in contact with each other) and indirect coupling (where at least one additional element is located between the two elements). Therefore, the terms “coupled to” and “coupled with” are used synonymously. In some embodiments, the numbers representing quantities, properties such as concentrations, reaction conditions, etc., of the expressed components used to describe and claim certain embodiments of the invention should be understood to be modified by the term “about” in certain circumstances. Therefore, in some embodiments, the numerical parameters set forth in the written description and appended claims are approximate values, which may vary depending on the desired properties sought to be obtained in a particular embodiment. In some embodiments, numerical parameters should be interpreted according to the number of significant figures reported and by applying common rounding techniques. Although the wide range of numerical ranges and parameters describing some embodiments of the invention are approximate values, the values ​​set forth in particular instances are reported as precisely as possible. The numerical values ​​presented in some embodiments of the invention may contain some errors necessarily caused by the standard deviation found in their respective test measurements.

[0028] As used herein and throughout the appended claims, unless the context clearly specifies otherwise, the terms "a / an," "a / an," and "the" include the plural reference objects. Additionally, as used herein, unless the context clearly specifies otherwise, "in" includes both "in" and "on".

[0029] The description of ranges of values ​​in this document is intended only as a shorthand method of individually referring to each individual value falling within that range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if it were described separately herein. Unless otherwise indicated herein or the context clearly contradicts this, all methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such as”) provided with reference to certain embodiments herein is intended only to better illustrate the invention and not to limit the scope of the invention as otherwise protected. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0030] The grouping of optional elements or embodiments of the invention disclosed herein is not to be construed as limiting. Each member of a group may be mentioned and claimed individually or in combination with other members of that group or other elements found herein. For convenience and / or patentability reasons, one or more members of a group may be included in or removed from the group. In the event of any such inclusion or removal, the specification herein is deemed to contain the modified group, thus fulfilling the written description of all Markush groups used in the appended claims.

[0031] The inventors have considered that the systems and methods described herein are applicable to a wide variety of bioassays, which can be based on the formation of a variety of bio-derived molecules that act as binding partners based on specific pairings. Suitable binding partners include immunoglobulins, lectins, cell surface receptors, aptamers, and nucleic acids. In some embodiments, the bioassay can be an immunoassay, for example, utilizing a labeled antibody or antibody analog (e.g., antibody fragment, single-chain antibody, recombinant antibody, etc.) that binds (directly or indirectly) to the analyte of interest and / or a labeled analyte of interest that competes with the analyte in the test sample from discrete test sites for direct (e.g., “sandwich”) or competitive assay. Suitable labels include fluorescent molecules, luminescent molecules, phosphorescent molecules, chromophores, microparticles, metal particles or colloids, enzymes, and nucleic acids. In some embodiments, such labels are directly incorporated by covalent attachment and / or genetic manipulation. In other embodiments, such labels are incorporated by affinity means such as by using biotin (or biotin analogs) and avidin / streptoavidin, by using protein A and / or protein G, and / or by using labeled antibodies against components of the assay system.

[0032] In other embodiments, the systems and methods conceived in this invention relate to nucleic acid binding and / or amplification assays. Such assays may include one or more polynucleotides that are at least partially complementary to a nucleic acid sequence representing or associated with the nucleotide sequence of interest. In such embodiments, a detectable signal indicating the presence of the polynucleotide of interest can be generated by any suitable means—including binding or substitution of a labeled probe sequence, formation of a complex between a dye and a nucleic acid structure (e.g., a double-helix region), etc. Suitable labels include fluorescent molecules, luminescent molecules, phosphorescent molecules, chromophores, microparticles, metal particles or colloids, enzymes, nucleic acids, and mass labels. In some embodiments, such labels are directly incorporated through direct synthesis, covalent attachment, and / or genetic manipulation. In other embodiments, such labels are incorporated through affinity means such as by using biotin (or biotin analogs) and avidin / streptavitin, by using protein A and / or protein G, and / or by using labeled antibodies against components of the assay system.

[0033] Members of a binding couple can be localized to a test site by any suitable means, such as a flow cytometry-compatible coded particle or a distinguishable site on a test surface. Typically, this can be achieved through non-covalent binding (e.g., adsorption), covalent coupling (e.g., via an available amine, thiol, or aldehyde group on the binding couple molecule), or indirect coupling (e.g., by using protein A / protein G, streptavidin / avidin:biotin pair formation, etc.). Numerous coupling chemistry processes and schemes are known in the art. Examples of suitable coupling methods include coupling an amine group via a hydroxysuccinimide or N-hydroxysuccinimide ester, coupling a thiol group via an N-ethylmaleimide group, coupling a hydroxyl, amine, or thiol group via an epoxy group, and coupling an aldehyde group via an acylhydrazine group (e.g., generated by oxidation of the carbohydrate or carbohydrate side chain of a glycoprotein).

[0034] In other embodiments, the systems and methods conceived in this invention relate to cell-based assays. Such assays may, for example, include cells that produce measurable changes in signal intensity in the presence of an analyte of interest. In such embodiments, individual cells may serve as discrete test sites, which can then be identified by recognizing specific cell surface markers. Such cells may be grown in suspension or as a layer on a surface. In some embodiments, cells may be genetically modified to incorporate a reporter construct that generates a detectable signal. Suitable signals include fluorescence, phosphorescence, luminescence, and / or enzyme activity. In some embodiments, cells in suspension may be characterized using flow cytometry, where a microscopy system (which may then provide digital images) may be used to characterize cells presented as a layer on a surface.

[0035] As described above, a wide variety of discrete test sites are suitable for the systems and methods of the present invention. In some embodiments, discrete test sites can be a collection of individual reactive sites on a planar microarray. Such test sites can be individually identified (i.e., encoded) by their location on the microarray. Alternatively, such arrays can be assembled by allowing particles or other discrete bodies to precipitate or assemble on a flat or substantially flat surface. In such embodiments, the particles or other discrete bodies may include identifying features. In such embodiments, the results can be characterized using a digital imaging system accompanied by suitable image analysis software.

[0036] In other embodiments, discrete test sites can be an aggregate of particles held in a suspension. Such particles can be identified by the incorporation of one or more detectable dyes (selected such that they do not interfere with analyte-specific signals), size, density, diffraction pattern, response to a magnetic field, and / or configuration. Such aggregates of particles can be characterized, for example, using flow cytometry.

[0037] In other embodiments, discrete test sites can be collections of micelles or droplets (e.g., microdroplets, nanodroplets, and / or filaments) held as suspensions or emulsions. Such discrete test sites can be identified by incorporating one or more detectable markers within the micelles or droplets. Such collections of micelles or droplets can be characterized, for example, using flow cytometry or microchannel devices.

[0038] In other embodiments, discrete test sites can be collections of individual molecules or molecular complexes held in solution. Such molecules or molecular complexes can be identified by incorporating one or more detectable markers associated with them.

[0039] In other embodiments, discrete test sites can be a collection of pixels obtained from a digital image of a large number of test sites. For example, if an immunoassay is performed on a test surface and the immunoassay produces a visible signal, a digital image of the test surface can be obtained, which includes signal intensity data related to the concentration of the analyte of interest. The digital image includes pixels, each of which represents a discrete region within the overall test surface. In some embodiments of the inventive concept, such individual pixels (or a defined collection of pixels) can be considered as discrete test sites and analyzed as discrete test sites to obtain the benefits of the systems and methods of the inventive concept.

[0040] In a preferred embodiment, the bioassay is an immunoassay performed on the surface of an aggregate of particles held in a suspension. The immunoassay provides a fluorescent signal using labeled antibodies, which, if the analyte of interest is present, is retained on the particles (in a direct assay). Such particles can be encoded by integrating one or more fluorescent dyes that provide signals distinguishable from analyte-specific signals for particle identification. Such particles can also be encoded by size. In some embodiments, different populations of particles are applied to a single sample to provide results for more than one analyte species from a single assay (i.e., multiplex assay). In such preferred embodiments, results are preferably obtained using a flow cytometer configured to identify and acquire analyte-specific signal data from individual particles as they pass through the instrument.

[0041] The analyte-specific signal data is subjected to at least two different analytical operations. In the statistical operation, the collected data is aggregated, and estimates of “typical” discrete test site data are generated from the aggregated data. For example, a frequency distribution of the analyte-specific signal intensity can be generated from the set of discrete test sites, and the mean or median is determined and reported from this aggregated data. In the differentiated numerical operation, based on measurements of the analyte-specific signal associated with individual discrete test sites, it is determined whether an individual discrete test site includes the analyte of interest (i.e., a “positive” discrete test site) or lacks the analyte of interest (i.e., a “negative” discrete test site). The relative quantities of positive and negative discrete test sites are then determined (e.g., using a ratio) to generate a numerical result.

[0042] In the systems and methods conceived in this invention, statistical and numerical results are combined to provide aggregated results. At low concentrations of the analyte of interest, for example where the relative amount of positive discrete test sites is about 90% or less of the total number of discrete test sites characterized, the aggregated results are highly dependent on the numerical results. If the relative amount of positive discrete test sites exceeds this value (or another applicable cutoff value), the aggregated results are highly dependent on the statistical results. This effectively increases the dynamic range of the bioassay. In some embodiments, the dynamic range is about 2, 5, 10, 25, 50, 100, or greater than 100 times the dynamic range of the same bioassay performed using only statistical data or only numerical data.

[0043] Table 1 shows typical results for particle-based immunoassays targeting IL-10.

[0044]

[0045] Table 1

[0046] As shown, statistical data (median fluorescence intensity or MFI) derived from immunoassay particle populations cannot provide differentiation between standards at concentrations below 2.06 pg / mL, and only a small difference between standards at 2.06 pg / mL and 6.17 pg / mL. However, numerical data (in the form of particle gating %) show a large difference between standards at 2.06 pg / mL and 0.69 pg / mL, and a significant difference between standards at 0.69 pg / mL and 0.00 pg / mL. Aggregation results provide a continuous measurement range from at least 0.69 pg / mL to 500.00 pg / mL.

[0047] Table 2 shows the results of similar studies that determined TNFα concentrations. As shown, statistical data failed to provide differentiation between standards containing less than 2.47 pg / mL, while numerical data provided clear differentiation between standards with concentrations as low as 0.27 pg / mL. Aggregated data provided a continuous dynamic range from at least 0.27 pg / mL to 200.00 pg / mL, demonstrating an improvement of approximately 10 times compared to using statistical or numerical data alone.

[0048]

[0049] Table 2

[0050] Table 3 shows the results of another study utilizing the determination of IL-6. As shown, statistical data failed to provide differentiation between standards containing less than 12.35 pg / mL, while numerical data provided clear differentiation between standards with concentrations as low as 1.37. Aggregate data provided a continuous dynamic range from at least 1.37 pg / mL to 1,000 pg / mL, demonstrating an improvement of approximately 10 to 100 times compared to using statistical or numerical data alone.

[0051]

[0052] Table 3

[0053] Table 4 shows the results of GM-CSF determinations using the system and method of this invention; Table 5 shows the results from numerous particle populations exposed to a negative (0 pg / mL) control sample. As shown, statistical data do indeed differentiate between standards with concentrations less than 12.35 pg / mL GM-CSF, while numerical data provide differentiation between standards with concentrations as low as 1.37 pg / mL GM-CSF. Repeat determinations using a negative control indicating approximately 90% or higher of “negative” particle content can be considered background noise.

[0054]

[0055] Table 4

[0056]

[0057]

[0058] Table 5

[0059] In another embodiment of the analyte mixture of the present invention, one or more analytes are present in large (e.g., greater than 5, 10, 20, 50, 100, 200, 500, 1,000, or more than 1,000) excess amounts, or one or more second analytes, and both analytes are characterized simultaneously. Such multiplex assays are performed using the same test container and a common set of reagents, etc., for all tested analytes. Thus, in typical multiplex assays, various analytes are characterized within similar measurement ranges. However, in some cases, it is desirable to measure different analytes within the same sample, wherein those analytes are present at significantly different concentrations. For example, cytokines are typically present in human serum in small amounts (e.g., about 1 pg / mL to 100 pg / mL), while C-reactive protein is typically present in larger amounts (e.g., ng / mL to μg / mL). Since C-reactive protein (CRP) and certain cytokines are also inflammatory markers, it is desirable to quantify them from the same sample, and preferably from the same assay.

[0060] In such embodiments of the inventive concept, such as those applied to biobinding assays (e.g., immunoassays, hybridization assays, etc.), a first binding coupler having a specific affinity for a first analyte present at low concentrations is provided in the reagent mixture. This first binding coupler is coupled to or otherwise associated with a first analyte identifier, such as a fluorescently labeled microparticle that can be provided in a first liquid suspension. The first binding coupler is exposed to a sample containing an analyte mixture including the first analyte at a relatively low sample volume to first liquid suspension ratio (e.g., from 2:1 to 1:5), and incubated for a period sufficient to allow molecules of the first analyte in the sample to recombine with the microparticles carrying the first binding coupler. The time required for this is a function of the affinity of the first binding coupler, the concentration of the first analyte, temperature, etc., and can range from about 15 minutes to 24 hours at temperatures in the range of 1°C to 50°C or higher (e.g., in nucleic acid binding assays).

[0061] Following this initial binding step, a suspension of the second binding pair (e.g., a particulate suspension of the particle-coupled second binding pair) is added to the mixture at a volume sufficient to provide a relatively high sample-to-total-reagent volume dilution (e.g., from 1:10 to 1:1,000). This step effectively reduces the concentration of the second abundant analyte in the sample to a range permissible by characterization with the second reagent. Simultaneously, the presence of the second binding pair only at this higher dilution effectively prevents saturation of the second binding pair. This lack of saturation provides a more linear dose / response profile than that provided by exposing the second binding pair to extremely high concentrations of the second analyte, as it provides partial occupancy of the total population of the second binding pair over a wider range of second analyte concentrations.

[0062] After a second incubation period suitable for the formation of a complex between the second abundant analyte and the second binding coupler, the extent to which the first analyte:first binding coupler and second analyte:second binding coupler complex has been formed can be characterized, for example by adding a second labeled antibody or probe nucleic acid sequence followed by optical characterization (e.g., in a fluorescence-excited cell sorter or similar apparatus). In some embodiments, this second incubation period is shorter than the first incubation period. In other embodiments, the second incubation period is similar to the first incubation period. In still other embodiments, the second incubation period is longer than the first incubation period. Similarly, the first and second incubation periods can be performed at similar or different temperatures. In some embodiments, the addition of the second binding coupler is accompanied by a component that alters the pH, ionic strength, and / or ionic composition of the reaction mixture. For example, in nucleic acid binding assays, the temperature and ionic strength of the reaction mixture during the second incubation can be changed from those of the first incubation to adjust hybridization efficiency and / or fidelity.

[0063] Surprisingly, the inventors have discovered that at least some of the first analyte: first binding coupler complex established during the first incubation is not lost upon dilution, despite the expectation of reequilibration at a significantly lower free first analyte concentration resulting from the addition of a second volume containing the second binding coupler. This allows for the generation of time-sequentially separated dose / response curves representing two different sample dilutions in a single multiplex assay.

[0064] In such methods, a first volume of an aqueous suspension of one or more highly sensitive bead-based capture reagents (e.g., beads conjugated with high-affinity antibodies specific for analytes present at low concentrations) is added to a test well or container. In some embodiments, the liquid portion of the dispensed suspension may be removed (e.g., by using a filter microplate) to minimize the dilution of the sample for subsequent application. A volume of sample is then added to the highly sensitive bead-based capture reagent along with a volume of assay buffer, and the minimally diluted sample is incubated with the highly sensitive bead-based reagent for an appropriate period of time (e.g., from 15 minutes to 24 hours) and at a temperature (e.g., from 4°C to 50°C or higher). During this initial capture incubation, the typical dilution of the sample, expressed in terms of the volume of highly sensitive bead-based reagent and assay buffer, can range from about 10:1 to 1:10, and in some applications may be outside this range.

[0065] Following the initial capture incubation, a volume of suspension containing one or more low-sensitivity bead-based capture reagents (e.g., beads conjugated to antibodies specific to analytes present in high concentrations in the sample) is added directly to the test well or container, resulting in a second capture incubation of both the sample and the diluted high-sensitivity bead-based reagent for a suitable time period (e.g., 15 minutes to 24 hours) and at a suitable temperature (e.g., 4°C to 50°C or higher). During this second capture incubation, the dilution of the sample relative to the total volume represented by the assay buffer, the high-sensitivity bead-based reagent, and the low-sensitivity bead-based reagent can be in the range of about 1:10 to 1:100 or greater. In some embodiments, the second capture incubation is performed for a shorter period than the first capture incubation. After the second capture incubation, the amounts of bound low-abundance analytes and bound high-abundance analytes can be characterized, for example, by incubation with a specific antibody conjugated with a detectable label (e.g., biotin, fluorophore, etc.). Surprisingly, while conventional steady-state equilibrium models for non-covalent interactions suggest that such dilution should result in the loss of analytes captured from highly sensitive bead-based reagents, the inventors have found that such analytes are retained after dilution.

[0066] In an example of this implementation, 45 μL of a bead suspension conjugated with antibodies specific to different cytokines is added to the wells of the microplate, and the suspension buffer is removed. After removing the buffer, 10 μL of assay buffer and 5 μL of serum sample or serum-based cytokine calibrator are added to the wells to provide a 1:3 sample dilution in the assay buffer. After a first capture incubation of 1 hour at room temperature, 285 μL of a bead suspension conjugated with antibodies specific to CRP (a high-abundance analyte) is added to the wells to provide a 1:60 sample dilution in buffer + reagent. After a second incubation of 30 minutes at room temperature, the liquid contents of the wells are removed, and the beads are washed three times with washing buffer. It should be understood that no additional step is required between the first and second capture incubations. 25 μL of a biotinylated detection antibody solution specific to cytokines and CRP is added to each well and incubated at room temperature for 30 minutes. The liquid contents of the wells were then removed, and the beads were washed three times with washing buffer. 25 μL of streptavidin-PE solution was then added to each well. After incubation at room temperature for 10 minutes, the liquid contents of the wells were removed, and the beads were washed twice with washing buffer before analysis on a flow cytometer.

[0067] Typical results from this type of study are shown in Figures 1A to 1B. Figure 1G (Low-abundance analytes - cytokines) and Figure 2 (High abundance analyte - CRP). Figure 1A shows the dose / response curve for IL-1β; the background fluorescence intensity is 14.07 MFI, and the LDD is approximately 2 pg / mL. Figure 1B The dose / response curves generated for IP-10 are shown; the background fluorescence intensity is 1.86 MFI, and the LDD is approximately 2 pg / mL to 4 pg / mL. Figure 1C The dose / response curves for IFN-γ are shown; the background fluorescence intensity is 5.09 MFI, and the LDD is approximately 5 pg / mL to 10 pg / mL. Figure 1D The dose / response curves for IL-8 are shown; the background fluorescence intensity is 1.36 MFI, and the LDD is approximately 1 pg / mL to 1.5 pg / mL. Figure 1E The dose / response curves for human MCP-1 (hMCP-1) are shown; the background fluorescence intensity is 1.00 MFI, and the LDD is approximately 2 pg / mL to 5 pg / mL. Figure 1F The dose / response curves for IL-4 are shown; the background fluorescence intensity is 1.45 MFI, and the LDD is approximately 5 pg / mL to 10 pg / mL. Figure 1G The dose / response curves for IL-6 are shown; the background fluorescence intensity is 1.45 MFI, and the LDD is approximately 5 pg / mL to 10 pg / mL. Figure 2 The dose / response curves for CRP are shown; the background fluorescence intensity is 4.61 MFI, and the LDD is approximately 18 pg / mL. Figure 1A to 1B Figure 1G (Indicating low-abundance analytes) show dose-response curves with a dynamic range of approximately 1 pg / mL to 500 pg / mL or approximately 10 pg / mL to 1,000 pg / mL (depending on the cytokine being tested), while Figure 2 (Indicating high-abundance analytes) Dose-response curves show a dynamic range of approximately 18 pg / mL to 13,000 pg / mL. In general, in this example of an embodiment of the inventive concept, an immunoassay range exceeding five orders of magnitude is achieved in a single test well (considering that serum samples are diluted 60-fold for CRP assays) (e.g., a dynamic range of five to six orders of magnitude, such as 1–1.5 pg / mL to 780,000 pg / mL, when dilution is taken into account). The inventors believe that, with the use of high-resolution flow cytometry, and by employing a combination of serial dilution and digital / statistical analysis techniques, the assay dynamic range can be extended to seven to eight orders of magnitude or higher.

[0068] It should be understood that more than two sets of capture reagents can be used to generate three or more sample incubations at different dilutions in a single test well or container. In some embodiments, capture phases with the same specificity but differentiating labels can be used, allowing different sets of capture reagents to target the same analyte. Such embodiments can be used to quantify or characterize a single analyte appearing over a very wide concentration range. For example, a first set of specific capture reagents can be used for small sample dilution incubation to quantify small concentrations of the analyte, while large sample dilution incubation can be performed using a second set of capture reagents with the same specificity but different labels that allow differentiation from the first capture reagent. At low analyte concentrations, the capture reagent added at low dilutions will provide useful data, while the second set of capture reagents provided at high dilutions will show a very small to no signal and can be ignored. At high analyte concentrations, the first set of capture reagents provided at low dilutions will be saturated and unable to provide useful information (and therefore ignored), while the second set of capture reagents provided at high dilutions will be able to provide useful data. In some implementations, such specific capture reagent sets may target the same analyte but utilize different binding partners with different affinities and / or cohesions.

[0069] In another embodiment of the inventive concept, the combined use of statistical and numerical data analysis can be combined with successive sample dilutions as described above to further extend the assay range. Examples of this combined method are provided in Tables 6 to Y, with exemplary data used as described above with respect to Figures 1A to Y. Figure 1G and Figure 2 The described measurement scheme is generated.

[0070] Table 6 shows both statistical data in the form of median fluorescence intensity (MFI) and numerical data in the form of gated % per bead for IL-2 concentrations ranging from 0.00 to 100 pg / mL. In this successive dilution assay, the LDD for IL-2 provided by MFI was approximately 11 pg / mL, while the LLD provided by gated % in this mixed form was approximately 1 pg / mL.

[0071] [IL-2] pg / mL MFI Gated negative events % 100 46.14 0.00％ 33.33 23.71 8.15％ 11.11 14.07 32.12％ 3.70 6.98 71.83％ 1.23 6.49 85.71％ 0.41 6.21 94.00％ 0.14 6.85 93.51％ 0.05 6.32 98.61％ 0.00 6.32 98.36％

[0072] Table 6

[0073] Table 7 shows both statistical data in the form of mean fluorescence intensity (MFI) and numerical data in the form of gating % per bead for IL-1β concentrations ranging from 0.00 to 100 pg / mL. In this successive sample dilution assay, the LDD of IL-1β provided by MFI was approximately 1 pg / mL (consistent with previous examples), while the LLD provided by gating % in this mixed form was approximately 0.1 pg / mL. Based on the replication of the assay protocol and the LDD results using MFI, the inventors believe that the extended dose / response curve will replicate the dose / response curve shown in Figure 1A.

[0074]

[0075]

[0076] Table 7

[0077] Table 8 shows both statistical data in the form of median fluorescence intensity (MFI) and numerical data in the form of gating % per bead for IL-22 concentrations ranging from 0.00 to 100 pg / mL. In this successive sample dilution assay, the LDD of IL-22 provided by MFI was approximately 1 pg / mL, while the LLD provided by gating % in this mixed form was approximately 0.15 pg / mL.

[0078] [IL-22] pg / mL MFI Gated negative events % 100 491.37 0.00％ 33.33 209.08 0.00％ 11.11 88.17 9.33％ 3.70 41.42 52.08％ 1.23 25.95 78.16％ 0.41 19.99 88.41％ 0.14 15.68 93.08％ 0.05 16.55 95.45％ 0.00 12.41 96.23％

[0079] Table 8

[0080] Table 9 shows both statistical data in the form of median fluorescence intensity (MFI) and numerical data in the form of gating % per bead for IL-6 concentrations ranging from 0.00 to 100 pg / mL. In this successive sample dilution determination, the LDD of IL-6 provided by MFI was approximately 11 pg / mL (compared to...). Figure 1G (Consistent with the findings shown), while the use of gated % in this mixed formulation provides approximately 0.1 pg / mL of LLD. Based on the replication of the assay protocol and the LLD results using MFI, the inventors believe that the extended dose / response curve will replicate Figure 1G The dose / response curve is shown in the figure.

[0081]

[0082]

[0083] Table 9

[0084] Table 10 shows statistics in the form of median fluorescence intensity (MFI) for CRP concentrations ranging from 0.00 to 13,000 pg / mL. In this successive dilution assay, the LDD of CRP provided by MFI was approximately 6 pg / mL (compared to...). Figure 2 (Consistent with the findings shown in the text).

[0085] [CRP] pg / mL MFI 13,000 3,421.60 4,333.33 3,013.50 1,444.44 1,995.68 481.48 821.22 160.49 321.45 53.50 122.98 17.83 57.77 5.94 27.38 0.00 8.51

[0086] Table 10

[0087] It should be understood that various assay formats can utilize the serial dilution and / or numerical / statistical data analysis methods described above. For example, most binding / hybridization assays for biomolecules (e.g., proteins, nucleic acids, etc.) are adapted to microplate or other arrangements of test sites, which are suitable for multiple additions of reagent to a single discrete test site and subsequent removal of the used reagent (e.g., by filtration, centrifugation, aspiration, etc.). In some embodiments, microfluidic devices / microchannel devices can be used, where serial dilution can be achieved using flow channels of different sizes. In such embodiments, the test sites can be discrete and suspendable (i.e., capable of being suspended in the fluid test medium under assay conditions and during assay), such as microparticles, micelles, emulsions, etc. For example, initial sample dilution and interaction with a first set of test microparticles can be performed in a narrow, small-volume channel providing a relatively low sample dilution. After a period of flow sufficient for the initial complex set to form, this narrow, small-volume channel can be joined to a wide, large-volume channel carrying an additional set of test microparticles suspended in a volume of diluent. Subsequent mixing in the common wide, large-volume channel provides a relatively low sample dilution that can be used to quantify higher concentrations of the analyte. Coupled with the output of such microfluidic devices / microchannel devices to a flow cytometer, it allows for the combined digital / statistical quantification as described above. In such embodiments, the microfluidic device / microchannel device can be a single-use device / disposable device; in other embodiments, the microfluidic device / microchannel device can be a reusable device designed for use with two or more different samples.

[0088] It will be apparent to those skilled in the art that further modifications beyond those already described are possible without departing from the inventive concept of this document. Therefore, the subject matter of this invention is not limited except in the spirit of the appended claims. Furthermore, in interpreting both the specification and the claims, all terms should be interpreted in the broadest manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to an element, component, or step in a non-exclusive manner, indicating that the referenced element, component, or step may be present, utilized, or combined with other elements, components, or steps not explicitly referenced. When the specification and claims refer to at least one of A, B, C… and N, the text should be interpreted as requiring only one element from that group, rather than A plus N or B plus N, etc.

Claims

1. A method for performing multiplex biobinding assays, the method comprising: Within the test well, a first test surface containing a first capture molecule is brought into contact with a first volume of sample, wherein the sample contains a first target molecule at a first concentration and a second target molecule at a second concentration, thereby generating a first diluted sample at a first dilution, wherein the first target molecule binds to the first capture molecule. The first diluted sample is incubated for a period of time, which is sufficient to form a first complex comprising the first capture molecule and the first target molecule; A second volume containing a second test surface is added to the test well to generate a second diluted sample at a second dilution, wherein the second diluted sample is incorporated into the first diluted sample, wherein the second test surface contains a second capture molecule that binds to the second target molecule, and wherein the second dilution of the sample exceeds the first dilution of the sample; The second diluted sample is incubated for a second period of time, sufficient to form a second complex comprising the second capture molecule and the second target molecule; and A first signal is obtained from the first complex, and a second signal is obtained from the second complex, wherein the second concentration exceeds the first concentration by at least 5 times.

2. The method of claim 1, wherein the first test surface comprises a first microparticle population, and the second test surface comprises a second microparticle population.

3. The method according to claim 1, wherein the first target molecule is interleukin.

4. The method of claim 1, further comprising the additional step of washing the first test surface and the second test surface before obtaining the first signal and the second signal.

5. The method of claim 1, wherein the multiplex biobinding assay has an overall dynamic range of more than five orders of magnitude for the first target molecule and the second target molecule.

6. The method of claim 1, wherein the first test surface comprises a plurality of individual first discrete test sites, and wherein a change in the first signal indicates the presence of the first target molecule in the sample, and includes the following additional steps; A plurality of quantities of the first signal are derived, wherein each of the plurality of quantities is associated with the first signal observed from a single first discrete test site of a first group of the first discrete test sites; A first proportion is determined using at least a portion of the plurality of quantities, wherein the first proportion includes an enumeration of the first discrete test sites indicating the presence of the first target molecule; Calculate the statistical distribution of the plurality of quantities derived from the first discrete test site to derive the statistical value of the first signal; as well as When the first ratio falls within the first cutoff range of the first ratio, the first ratio is used to quantify the mass of the first target molecule in the sample, and when the first ratio falls outside the first cutoff range of the first ratio, the statistical value of the first signal is used to quantify the mass of the first target molecule in the sample.

7. The method of claim 1, wherein the first test surface and the second test surface are provided in a microfluidic device.

8. The method of claim 7, wherein the microfluidic device is a microchannel device.

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