High sensitivity immunoassay

Abstract

The present disclosure provides a high sensitivity assay to detect the presence of a target analyte in a sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. patent application No. 63 / 335,379 filed on April 27, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] High-sensitivity immunoassays typically employ a matching pair of specific high-affinity antibodies oriented in a sandwich format. One antibody serves as an analyte capture molecule, and a second antibody serves as a detection molecule, which can be measured directly (e.g., fluorescently labeled), or indirectly measured via binding to a labeled reporter molecule directed to the detection molecule or a label connected to the detection molecule (e.g., streptavidin-fluorescein or streptavidin-enzyme bound to a detection antibody molecule with a biotin tag). Examples of high-sensitivity immunoassays include those performed on Luminex platforms and Quanterix platforms.

[0004] An additional example of a highly sensitive immunoassay is SiMREPS, which is based on single-molecule detection using TIRF microscopy (see, e.g., US2019 / 0339266; Johnson-Buck et al., Nat Biotechnol. 33(7): 730-732, 2015). SiMREPS provides the ability to resolve nonspecific target binding interactions and specific target binding interactions, which improves sensitivity by reducing / eliminating background. The SiMREPS technology uses detection molecules, such as antibody Fabs with fast association rate and fast dissociation rate kinetics, so that repeated time measurements of detection probe binding can be used to distinguish specific binding from nonspecific binding. Summary of the invention

[0005] The present disclosure provides an assay that uses both a high affinity binder targeting an analyte of interest and a binder with a fast dissociation rate and a relatively low affinity for binding to the target analyte to detect and quantify the analyte in a sample. Therefore, in one aspect, the present disclosure provides a method for quantifying a target analyte, the method comprising incubating: (i) a solution containing a complex, the complex containing a target analyte bound to a detector, wherein the detector binds to the target analyte with high affinity and is labeled with a detectable marker; and (ii) a capture agent population fixed to a solid support in a channel or compartment; wherein these capture agents specifically bind to the analyte and have a high dissociation rate for binding to the target analyte, and then the analyte: detector complex migrates through the channel or compartment; based on the migration rate of the complex relative to the unbound detector through the solid support, the complex is spatially resolved from other molecules in the solution to provide an eluate containing a complex substantially free of unbound detectors; and the amount of the detector complexed with the analyte is quantified, thereby determining the amount of the target analyte present in the solution. On the other hand, the capture agent population is fixed in discrete areas on a solid support (e.g., a membrane). In some embodiments, the capture agent is a Fab or an aptamer. In some embodiments, the high-affinity detector is an aptamer, an antibody or a ligand. In some embodiments, the detectable marker is specific for the analyte. In some embodiments, the detectable marker is an oligonucleotide. In some embodiments, the oligonucleotide comprises a primer binding site, or comprises a sequence of a primer binding site of a detection oligonucleotide targeted for detection in an amplification reaction. In some embodiments, the oligonucleotide comprises an analyte identification region and / or a sample identification region. In some embodiments, the quantitative step comprises amplifying the target region of the oligonucleotide to obtain an amplicon. In some embodiments, the amplification step comprises PCR, such as quantitative PCR (qPCR) or digital PCR (dPCR). In some embodiments, quantitatively comprising sequencing the region of the oligonucleotide specific for the analyte to determine the amount of the oligonucleotide present in the detector-analyte complex. In some embodiments, the complex comprises a fluorescent marker. In some embodiments, quantitatively comprising detecting the level of the fluorescent signal generated by the fluorescent marker. In some embodiments, the channel or compartment is a capillary. In some embodiments, the solid support is a plurality of beads present in the channel or compartment. In some embodiments, the channel or compartment accommodates a solid support composed of a polymer. In some embodiments, when the solution flows through the channel or compartment, pressure or an electric field is applied to the solution. In some embodiments, the channel or compartment is a channel of a microfluidic device. In some embodiments, the channel is a capillary present in a micropore. In some embodiments, the solid support is a membrane or a wicking matrix, and a solution containing a complex flows through the membrane or the wicking matrix by a lateral flow, wherein the complex comprises a target analyte bound to a detection agent.

[0006] In another aspect, a kit is described herein, comprising a capture agent as described herein, and a high affinity antibody bound to a solid support. In some embodiments, the solid support is a microbead or microparticle. In some embodiments, the solid support is a membrane or a wicking material. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 An overview of one illustrative embodiment of an immunoassay using a binding agent with fast off-rate and on-rate kinetics as a capture agent attached to a solid support is provided.

[0008] Figure 2 An illustrative assay is depicted.

[0009] Figure 3 A top view of an illustrative lateral flow assay is depicted.

[0010] Figure 4 A side view of an illustrative lateral flow assay is depicted.

[0011] Figure 5 An example of a "double Y" lateral flow assay is depicted. DETAILED DESCRIPTION

[0012] the term

[0013] The terms "a" or "an" are intended to mean "one or more" or "one or more". The term "comprises" and variations such as "comprising" and "having", when preceding the recitation of a step or element, are intended to indicate that the addition of additional steps or elements is optional and is not excluded. In the practice of the present invention, any methods, apparatus, and materials similar or equivalent to those described herein may be used. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not meant to limit the scope of the present disclosure.

[0014] The term "about" refers to the recited number and any value that varies from the recited number by less than 10%.

[0015] As used herein, the term "binding agent" refers to a binding molecule that specifically binds to a target molecule (e.g., an analyte to be detected in a sample). Examples of binding agents include, but are not limited to, antibodies or antibody fragments, aptamers, haptens, ligands, scaffold-based polypeptides, or other molecules that specifically bind to an analyte of interest.

[0016] As used herein, "affinity" is generally used to describe the strength of the interaction of a binding agent with an analyte or with a molecule that interacts with the binding agent. A binding agent may have a single or multiple binding sites for an analyte or with an alternative molecule that interacts with the binding agent. In the present disclosure, the affinity of a binding agent for its bound analyte is represented by the equilibrium dissociation constant (K D ) represents the dissociation rate of the binding agent from the analyte (k d or k off ) and the binding rate (k a or k on ), that is, K D =k off / k on . K D is inversely related to binding affinity (e.g., the binding affinity of an antibody for an antigen), where K D The smaller the value, the greater the affinity of the binder for its target analyte.

[0017] The term "antibody" is used in the broadest sense herein and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies and antibody fragments, as long as they exhibit the desired antigen binding activity. Antibody fragments refer to a portion of an intact antibody that binds to a target antigen. Examples of antibody fragments include but are not limited to Fab, Fab', Fab'-SH, F(ab')2 and Fv; double-chain antibodies; linear antibodies; single-chain antibodies, such as single-chain variable fragments (scFv), fusions of light and / or heavy chain antigen binding domains with a linker or without a linker (optionally in series); and monospecific or multispecific antigen binding molecules formed by antibody fragments, for example, antibodies constructed from multiple variable domains lacking an Fc region.

[0018] "Protein" or "polypeptide" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, "protein" or "polypeptide" includes amino acid polymers in which one or more amino acid residues is a non-naturally occurring analog of a corresponding naturally occurring amino acid.

[0019] "Polynucleotide" or "nucleic acid" includes any form of RNA or DNA, including, for example, genomic DNA, complementary DNA (cDNA), amplified DNA molecules, or synthetically produced DNA or RNA molecules. The term includes chimeric molecules, and molecules containing non-standard bases, modifications, or nucleotide analogs. For example, an oligonucleotide can contain naturally occurring nucleotides and / or their analogs. A polynucleotide can be single-stranded or double-stranded, or have both single-stranded and double-stranded regions (e.g., hairpins).

[0020] As used herein, the term "analyte" refers to any molecule that can be detected in a sample by a binding agent that specifically binds to the molecule. Analytes include, but are not limited to, polypeptides, nucleic acids, carbohydrates, lipids, hormones, or other molecules of interest present in a sample, including macromolecules or complexes.

[0021] The terms "label" and "detectable marker" interchangeably refer to compositions that can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful labels include fluorescent dyes (fluorophores), fluorescence quenchers, luminescent agents, electron-dense reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin, digoxigenin, 32 P and other isotopes, haptens, proteins, nucleic acids, or other substances that can be made detectable, for example, by incorporating the marker into an oligonucleotide, peptide, or antibody that specifically reacts with a target molecule. The term includes combinations of single labeling agents, for example, combinations of fluorophores that provide a unique detectable signature at, for example, a specific wavelength or combination of wavelengths. As used herein, "detectable marker" includes reference to an oligonucleotide that can be detected by PCR, sequencing, or other biochemical reactions.

[0022] A molecule "linked to a marker (e.g., a labeled antibody)" is a molecule that is covalently bound to the marker via a linker or chemical bond, or non-covalently bound to the marker via ionic bonds, van der Waals bonds, electrostatic bonds, or hydrogen bonds, such that the presence of the molecule can be detected by detecting the presence of the bound marker.

[0023] Introduction

[0024] The present disclosure uses both high-affinity binding agents targeting analytes of interest and binding agents with fast dissociation rates and relatively low affinity for binding to target analytes to detect or quantify analytes of interest in samples. In the present disclosure, fast dissociation rate binding molecules (e.g., Fab fragments or aptamers specific to target analytes) are used for analyte capture, and high-affinity binding agents (e.g., high-affinity antibodies or aptamers) are used to detect the agent, such as by detecting signals from markers attached to or otherwise incorporated into high-affinity binding agents and / or by using oligonucleotides attached to high-affinity binding agents for amplification and / or sequencing. For the purposes of the present disclosure, binding agents with fast dissociation rates that bind to target analytes of interest are referred to as "capture agents", and high-affinity binding agents that bind to target analytes of interest are referred to as "detection agents". Therefore, as used herein, "capture" agents are repeatedly bound to dissociated substances from analyte-detection agent complexes.

[0025] In the present disclosure, the sample in which the analyte to be evaluated exists is hatched with an excess of a detection agent, so that when there is a target analyte in the sample, an analyte-detector complex is generated. Subsequently, under the condition of the compartment / channel that holds the immobilized capture agent, the analyte-detector complex is migrated by the analyte-detector complex, and the capture agent is hatched with the capture agent connected to a solid support (for example, connected to the beads or microparticles held in a channel or compartment, or connected to the compartment / channel or micropore of a microfluidic device). The capture agent can also be connected to or incorporated into a polymer matrix, wherein the polymer matrix is ​​such as polyacrylamide, agarose, or other cross-linked, branched or linear polymers that are known to allow screening of polypeptides and / or nucleic acids. The polymer matrix can be contained in a channel or a compartment. Samples can be applied to a separate channel or compartment, or in some embodiments, a solid support can be a larger form with a plurality of holes or a reservoir, for keeping different samples in a spatially resolved manner.

[0026] In some embodiments, the capture agent can be directly attached to the surface of a compartment or channel (e.g., a microfluidic chamber) having a small size (e.g., 2 to 5 microns). The capture agent can be attached to the surface using a cross-linked or linear polymer extending from the surface into the lumen of the channel. In such embodiments, a small amount of sample will be used, and the separation length will be determined to achieve sufficient resolution of bound and free detection agents.

[0027] The target analyte in the analyte-detector complex is bound to the capture agent, but because the capture agent has a fast dissociation rate, the target analyte dissociates rapidly. Therefore, the analyte-detector complex repeatedly binds to the capture agent and then dissociates from the capture agent, thereby slowing down the migration rate of the analyte-detector through the compartment / channel or matrix relative to the unbound detection part. Pressure or an electric field is usually applied to a solution containing the analyte-detector to cause the analyte-detector to flow through the compartment or matrix containing the immobilized capture agent, but other mechanisms can also be used to generate forces, such as centrifugal force. The reagents and methods disclosed herein are further described in detail below.

[0028] Binder

[0029] The binding agent can be any molecule that exhibits specific binding to the analyte of interest. In some embodiments, the binding agent comprises an antibody or binding fragment thereof that specifically binds to the target analyte. In some embodiments, the binding agent comprises an aptamer that specifically binds to the target analyte. In some embodiments, the aptamer is a peptide aptamer. In other embodiments, the aptamer is a polynucleotide aptamer. In some embodiments, the binding moiety comprises a ligand that binds to a site on the target analyte.

[0030] As used herein, the term "specific binding" when used with respect to an affinity agent refers to an affinity agent (e.g., an antibody) that binds to an antigen with an affinity that is at least 5 times greater than that of a non-antigen molecule, e.g., 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 25 times, 50 times, 10 ... 3 times, 10 4 times, 10 5 times, 10 6 times, 10 7 times, 10 8 times, 10 9 times, 10 10 times, 10 11 times, 10 12 times, 10 13 times, 10 14 times or 10 15 For example, an affinity agent that specifically binds a particular antigen will typically bind that antigen with an affinity at least 2 times greater than it binds to a non-antigenic molecule.

[0031] In some embodiments, the binding agent is an antibody. As mentioned above, the term "antibody" covers full-length antibody forms, such as IgG, and antibody functional fragments that bind to the target antigen, including multimeric forms and monomeric forms. Therefore, antibodies that can be used include double-chain antibodies, three-chain antibodies, tetrameric forms, single-domain antibodies, and the like. The functional fragment can be a part of an antibody, such as F(ab')2, Fab', Fab, Fv, or can be an engineered binding fragment, such as scFV. In some embodiments, the binding portion can be a mimetic antibody. Examples include polypeptides based on fibronectin scaffolds (such as Adnectin), ankyrin repeat scaffolds (such as DARPin), polypeptides based on lipocalin scaffolds (such as Anticalin), and affinity bodies (see, for example, Engineered Protein Scaffolds as Next-Generation Therapeutics, Annual Review of Pharmacology and Toxicology 2020, Vol. 60: 391-415).

[0032] In some embodiments, the binding agent is a ligand that binds to a specific site of a target cell molecule (e.g., a target protein analyte), including a ligand of a receptor, enzyme, or other protein. The ligand can be a polypeptide molecule, a small molecule, or any molecule that binds to a cognate cell binding partner.

[0033] In some embodiments, the binding moiety can be a nucleic acid or peptide aptamer. Aptamers interact with their targets by recognizing specific three-dimensional structures. Peptide aptamers consist of short variable peptide loops attached to a protein scaffold (such as the bacterial protein thioredoxin-A) at both ends. Peptide aptamers specific for a target of interest can be selected using any method known to the skilled person (such as a yeast two-hybrid system, ribosome display, or phage display). Peptide aptamers can be produced by chemical synthesis, or recombinant production.

[0034] In some embodiments, the aptamer is a nucleic acid aptamer. Nucleic acid aptamers are a class of small nucleic acid ligands, composed of RNA or single-stranded DNA oligonucleotides folded into a three-dimensional structure, and have high specificity and high affinity to their targets. For example, the systematic evolution of ligands by exponential enrichment (SELEX) technology can be used to obtain aptamers that are specific to specific molecular targets. Nucleic acid aptamers can be produced by chemical synthesis or in vitro transcription of RNA aptamers. Nucleic acid aptamers include DNA aptamers, RNA aptamers, XNA aptamers (nucleic acid aptamers comprising xeno nucleotides) and L-RNA aptamers.

[0035] High affinity binders

[0036] As described herein, a binding agent that exhibits high affinity binding to an analyte is contacted with a sample comprising a target analyte. In some embodiments, the binding agent is an antibody, e.g., a bivalent or multivalent antibody. In some embodiments, the binding agent is an aptamer or ligand that binds to a target analyte (e.g., a receptor).

[0037] In typical embodiments, the high affinity binder has a K of about 1 nM or less. D binds to the target analyte, or in some embodiments, binds to the target analyte with a K of 100 pM or less D Binding to the target analyte. Any technique can be used to measure K D , including, for example, ELISA-based methods, as well as other biophysical methods such as microscale thermophoresis (MST), surface plasmon resonance (SPR), and biolayer interferometry (BLI). By way of illustration, SPR can be used. SPR has several steps, for example, analyte is immobilized on the device surface and then interrogated with a binding agent, thereby forming a time-lapse pattern that can be used to calculate the binding rate (k on The next step is usually to replace the binding agent with a buffer solution, and when the signal returns to baseline, the rate at which the binding agent dissociates (dissociation rate, k) is measured. off ). D By k off / k on The ratio of is calculated.

[0038] High affinity binders as used in the methods described herein typically have a low dissociation rate, so that the high affinity binder remains bound to the analyte during the assay process. For example, the dissociation rate of the high affinity detection agent from the analyte can be 10 times, 100 times, 1000 times, or lower times the dissociation rate of the capture agent (see, e.g., Chang et al., J. Immunol. Methods 378: 102-115, 2012).

[0039] The following table shows the advantageous K D value and a capture agent with fast association and dissociation kinetics.

[0040] Table 1. Illustrative capture agent kinetics

[0041]

[0042] In some embodiments, the detector is provided in molar excess relative to the amount of analyte to maximize the formation of a detector-analyte complex. In some embodiments, the detector is provided at a concentration relative to the concentration of the analyte in the sample that exceeds about 1 pM to about 1 mM, most commonly exceeding about 0.1 nM to about 1 μM, or about 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, or 1 μM.

[0043] In some embodiments, a high affinity detection reagent (e.g., an antibody) is conjugated to an oligonucleotide that is specific for the target being analyzed and / or comprises a sample identification region. In some embodiments, the oligonucleotide comprises one or more labels for detection purposes, such as a fluorescent label.

[0044] In some embodiments, the oligonucleotide comprises a primer binding site, or a region hybridized with a target primer binding site of a detection oligonucleotide to be amplified, which can be used, for example, to detect and quantify the analyte in a sample using PCR, qPCR and / or DNA sequencing. In some embodiments, the oligonucleotide also comprises an analyte identification region that is specific to the analyte, for example, to identify each analyte detected in a multiplex reaction by sequence analysis. In some embodiments, the oligonucleotide also comprises a sample identification region. In some embodiments, the oligonucleotide also comprises a region or joint that allows removal of the oligonucleotide from a high-affinity detection reagent for subsequent analysis or identification.

[0045] In some embodiments, qPCR or digital PCR (dPCR) is used to quantify the amount of target nucleic acid in a sample. Both techniques are capable of detecting as few as 1 target copy in a sample containing a large number of sequences of no interest. Quantification using qPCR requires the creation of a standard curve from samples containing a known number of copies of the target nucleic acid of interest. Once the curve is established, the concentration of the target nucleic acid sequence can be calculated based on the C value derived from each experimental sample. t (threshold cycle number) value is extrapolated. Digital PCR allows direct quantification of target nucleic acid sequences by counting the partitions determined to be positive. Typically, this will require correction for possible multiple occupancy of the target of interest within the partition. Although dPCR allows absolute quantification, it cannot provide the same dynamic range as qPCR. Direct quantification, dynamic range and cost are the main differences between qPCR and dPCR in terms of target nucleic acid quantification.

[0046] In some embodiments, the oligonucleotide connected to the detection agent comprises one or more primer binding sites, for example, for amplification reaction. The amplification reaction can be any amplification reaction. In some embodiments, the amplification reaction is a quantitative PCR reaction. Determine that the alternative amplification reaction of the positive pool includes T7 amplification, rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) or any other suitable amplification reaction. For example, LAMP or RCA amplification reactions can be used to generate quantitative fluorescent amplification products.

[0047] In some embodiments, the oligonucleotides are hybridized to complementary oligonucleotides attached to beads, for example to facilitate sequence analysis, or to alternative surfaces, for example for image analysis.

[0048] Fast off-rate binders

[0049] Fast off-rate binding agents that specifically bind to the analyte of interest are used herein as capture agents to capture the analyte-detector complex as it migrates through the channel, thereby slowing the mobility of the complex compared to the uncomplexed detector. It will be apparent to one skilled in the art that the equation K d =k off / k on Characterizes transient binding under fast off-rate conditions.

[0050] Fast off-rate binders (e.g., Fab) that bind to target analytes typically have an off-rate of about 0.1 s -1 The kinetic rate constant koff and / or greater than 1×10 5 M -1 s -1 The kinetic rate constant k on For example, in some embodiments, the kinetic rate constant k describing the association of the capture agent with the analyte is onGreater than 1×10 5 M -1 s -1 , or 1×10 6 M -1 s -1 , or higher. Most commonly, the upper limit of kon is related to the diffusion coefficient and may be close to 1×10 8 M -1 s -1 Up to 1×10 9 M -1 s -1 In some embodiments, the kinetic rate constant k describing the dissociation of the complex off More than 0.01s -1 , for example, greater than 0.1s -1 , greater than 1s -1 or more than 10s -1 In some embodiments, the kinetic rate constant koff describing the dissociation of the complex is greater than 0.01 s -1 , for example, greater than 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds. -1 In some embodiments, the binding rate of such a binding agent may be 10 5 or 10 6 M -1 s -1 In some embodiments, the K of the binding agent D From about 10 nM to about 10 μM.

[0051] Solid carrier

[0052] In one embodiment, the fast dissociation rate capture agent (e.g., Fab) is fixed on a solid support surface. Such surfaces include, but are not limited to, channels, nanoparticles, microspheres, beads, or polymer matrices. In typical embodiments, the capture agent is fixed to the surface of beads or microparticles filled into the channel, or to a polymer matrix (such as agarose or polyacrylamide, for example, a linear or cross-linked polymer that allows macromolecules to pass).

[0053] " solid carrier " refers to a material or a group of materials with one or more rigid or semi-rigid surfaces.In some embodiments, solid carrier is a matrix, such as a hydrophilic polymer, for example, a polymer that is insoluble in water but swells in water.Suitable polymer includes but is not limited to polyhydroxy polymers, for example, based on polysaccharides (such as agarose, dextran, cellulose, starch, amylopectin, etc.) polyhydroxy polymers, and fully synthetic polymers, such as polyacrylamide, polymethacrylamide, poly-(N, N-dimethylacrylamide), poly-(hydroxyalkyl vinyl ether), poly-(hydroxyalkyl acrylate) and polymethacrylate (for example polymethacrylate glycidyl), polyethylene glycol polymers, polyvinyl alcohol and based on styrene and divinylbenzene polymers, and including two or more copolymers corresponding to the monomers of the polymer mentioned above.Water-soluble polymers can be derived to become insoluble, for example, by cross-linking and by being coupled to insoluble bodies via adsorption or covalent bonding. Hydrophilic groups can be introduced onto hydrophobic polymers (e.g., copolymers of monovinylbenzene and divinylbenzene) by polymerization of monomers that exhibit groups that can be converted to OH, or by hydrophilization of the final polymer (e.g., by adsorption of suitable compounds such as hydrophilic polymers). In some embodiments, the carrier is a UNOsphere TM The carrier is a polymer produced by a water-soluble hydrophilic monomer (Bio-Rad, Hercules, CA). Alternatively, the matrix is ​​agarose (GE Sepharose, or Sterogene Superflow and Ultraflow). In some embodiments, the carrier is composed of glass fibers or synthetic fibers that have been activated with chemical groups that react with proteins or nucleic acids (such as Fab or aptamers) with a fast dissociation rate. In other embodiments, the carrier is a membrane material, such as nitrocellulose, which can non-covalently bind to a capture agent with a fast dissociation rate, as described in detail in the section of the lateral flow assay embodiment of the present invention.

[0054] In the present disclosure, the analyte-detector complex is allowed to contact the surface with the immobilized capture molecule and then interact with the fast dissociation rate binding agent instantaneously. The transient binding event slows down the migration rate across or through the surface or matrix. In some embodiments, the capture agent population of the heterogeneous mixture of the capture agent containing the analyte of interest is fixed on the solid support. In some embodiments, the homogeneous population of the capture agent is fixed to the solid support. In some embodiments, the capture agent population bound to the solid support comprises different types of binding agents, for example, a mixture of Fab, aptamer, ligand or other agents with fast dissociation rate binding kinetics to the target analyte. In some embodiments, the solid support comprises multiple regions with different capture agents, which are specific to different target analytes.

[0055] Many different methods can be used to identify the capture agent that is combined with the analyte at a fast dissociation rate. In some embodiments, such a binding agent is selected from a random library of peptides, small ligands, small molecules, aptamers, etc. In some cases, a capture agent, such as Fab, that is combined with an analyte of interest is generated using combinatorial techniques and library screening (e.g., phage or yeast display library screening) well known to those skilled in the art. For example, Fab can be generated using the HuCal technology from Bio-Rad Laboratories. In addition, a capture agent can be formed by a synthetic molecule. (Iterative In SituClick Chemistry Creates Antibody-like Protein-Capture Agents, HDAgnew et al., Angew. Chem. Int. Ed. 2009, 48, 4944-4948.) (Accurate MALDI-TOF / TOF Sequencing of One-Bead-One-Compound Peptide Libraries with Application to the Identification of Multiligand Protein Affinity Agents Using in Situ ClickChemistry Screening, Su Seongi Lee et al., Anal. Chem., 2010, 82(2), pp. 672-679).

[0056] Many different techniques can be used to fix the capture agent to a solid support. In some embodiments, the immobilized carboxylate groups on the amine reactive surface can be used to covalently connect the polypeptide capture agent to a substrate via an amine coupling reaction. Other illustrative reactive linking groups (for example, hydrazine, hydroxylamine, mercaptan, carboxylic acid, epoxide, trialkoxysilane, dialkoxysilane and chlorosilane) can be attached to the substrate so that the polypeptide can form a chemical bond with those linking groups to be fixed to the substrate. In some embodiments, the capture agent can be copolymerized into a solid support by first adding a suitable chemical group (such as an acrylic acid group) to the capture agent. In some embodiments, click chemistry can be used to attach the capture agent. In some embodiments, the capture agent is non-covalently bound to a carrier (such as a nitrocellulose membrane) after being deposited on the carrier.

[0057] In some cases, the polypeptide can be immobilized on the surface using methods such as the hydrophilic self-assembled monolayer method, the hydrophilic polymer brush method, the zwitterionic polymer brush method, and the nitrile coating method.

[0058] In some embodiments, the surface can also be coated with streptavidin at a known concentration, followed by attachment of a biotinylated binding agent. In other embodiments, the capture agent can be immobilized using other bridging molecules that form non-covalent or covalent complexes (such as Spytag and Spycatcher complexes), where one component of the system can be present on the capture agent and another component can be present on a solid support.

[0059] Migration of the detector-analyte complex

[0060] Various parameters may affect the migration rate of the detection agent-analyte complex through a channel or compartment. Such parameters include the pH of the solution, the density of the capture agent immobilized on the solid support, the channel size (e.g., diameter and length), the flow rate through the channel, or other applied forces, such as the strength of the electric field.

[0061] In some embodiments, the capture agents (eg, Fab) are immobilized on the solid support at high density, for example, at 100 μm 2 Surface area at least 10,000 molecules or per μm 2 Surface area of ​​at least 20,000 molecules, or per μm 2 The density of the capture agent is about 50,000 molecules or more per surface area. In some embodiments, 1 μg to 1 mg of capture agent is used to prepare the resin. As will be appreciated by those skilled in the art, the density of the capture agent is adjusted to provide sufficient instantaneous interaction of the analyte-detector complex with the solid support so that the complex is resolved in time and space from the nonspecific interaction of the unbound detector with the same solid support.

[0062] In some embodiments, the diameter of the channel is about 20 μm to about 500 μm, or in some embodiments, about 50 μm to about 200 μm. In some embodiments, the diameter of the channel is about 100 μm or less, for example, a diameter of about 1 μm to about 10 μm, or to about 100 μm. In some embodiments, the length of the channel is proportional to the volume of the sample, for example, about 20 times, about 50 times, or about 100 times the volume of the sample.

[0063] In some embodiments, the channel is a channel of a microfluidic device. Thus, in some embodiments, an initial eluate flowing through a channel can be diverted to another channel, thereby providing a solution comprising a detection agent / analyte complex that is free of unbound detection agent.

[0064] In some embodiments, the migration rate through a channel or compartment is regulated by manipulating various parameters that affect the affinity of the capture agent to the target analyte present in the detector-analyte complex. In some embodiments, an electric field, pressure or centrifugal force can be applied to regulate the migration rate through a channel. In some embodiments, as mentioned above, the temperature, pH and / or ion composition of the solution containing the detector-analyte complex are manipulated to regulate the flow rate of the complex through a channel or compartment. In some embodiments, one or more washing steps can be used to further remove or resolve free detectors from the detector present in the analyte-detector complex. Those skilled in the art know how to adjust such parameters to obtain the desired flow rate through a channel or compartment.

[0065] In some embodiments, the detector / analyte complex in the eluate is concentrated using beads, magnetic beads with complementary nucleic acids, chromatography resins (e.g., IEX, hydrophobic interactions), filters, or membrane concentrators, or another method known in the art for concentrating proteins and / or nucleic acids. In some embodiments, the detector / analyte complex is recovered from the polymer support by applying an electric field, pressure, or other force in an orthogonal direction. In some embodiments, the detector / analyte complex is recovered from the polymer support by excising an area containing the detector / analyte complex.

[0066] The eluate obtained containing the detector / analyte complex is substantially free of unbound detector after migration through the channel or compartment. A blank sample containing no analyte is used to subtract the background signal from the uncomplexed detector to determine the sensitivity limit. The sample containing the analyte preferably exhibits a signal-to-noise ratio of >3 as the threshold for the detection limit.

[0067] Lateral flow assay

[0068] The lateral flow type configuration that allows reagents to flow sequentially through the wicking matrix is ​​known, for example, U.S. Patents 10,688,487, 10,883,987 and 10,591,477. In the embodiment of the assay method of the present invention using lateral flow, the matrix itself with the bound capture agent (e.g., Fab) will provide separation, wherein the lateral flow is used to generate the flow path and control the delivery of the solution. The reagent is provided to the matrix in a sequential manner (see, for example, U.S. Patent 10,883,987). The surface used in the lateral flow type assay method can be a membrane, such as a nitrocellulose membrane, for example, with a porosity of 0.1 μm, 0.2 μm, 0.45 μm, 1 μm, 3 μm, 5 μm, 8 μm or 10 μm. In some embodiments, the capture agent (e.g., fast dissociation rate Fab) is deposited into discrete areas and combined with it. The surface can then be blocked (e.g., with bovine serum albumin or an alternative blocking agent) and washed. In some embodiments, the lateral flow type surface can be glass fiber, cellulose or synthetic fiber, or a combination thereof. In some embodiments, the lateral flow type surface is polyvinylidene fluoride, nylon or polysulfone membrane. In such cases, the capture agent (e.g., fast dissociation rate Fab) is covalently and / or non-covalently attached to the surface using chemical / biochemical means or by linker molecules (such as streptavidin and biotin, etc.), as described in detail above in the "solid support" section.

[0069] The flow path in the membrane / wicking matrix can be delineated using a hydrophobic barrier or a physical barrier using wax, acrylic, etc., which can fill the pores of the matrix, forming a wall to define the flow path (see, e.g., U.S. Pat. No. 10,883,987). A commercial example of such a flow path formed in a nitrocellulose membrane is the Unisart® from Sartorius. Membrane Products. In preferred embodiments, the flow paths are microfluidic in size, or a few millimeters in width and depth and a few centimeters in length, to allow sufficiently large flow distances to provide the desired separation.

[0070] In some embodiments, the assay is designed to employ a Y or double Y flow path, for example, to minimize the amount of free detection agent (eg, oligonucleotide-conjugated high affinity antibody) that migrates into the final eluate. Figure 3 and Figure 4 A top view and a side view of one embodiment using lateral flow are provided. This illustrative embodiment contains a Y cleavage at the end and shows an area where Ag:Ab-oligo complexes can be captured.

[0071] Figure 5A double Y flow path is shown. Thus, for example, using this design, a sample can be applied to port 1, separated along the flow path to port 4 for a specified period of time to allow free detector (e.g., oligonucleotide-conjugated detection antibody (abbreviated as Ag:Ab-oligo complex for the purposes of this example)) to be washed out to port 4. The flow path can then be changed along flow paths 2 and 3 to achieve washing of the detector-analyte complex (e.g., Ag:Ab-oligo complex) to port 3 for further analysis, such as by PCR.

[0072] Other split flow path configurations that achieve the desired goals of background reduction, flow and analysis optimization can be envisioned, where the number and angle of arm positions are different, such as where the arms intersect the initial path at a 90 degree angle. Optional concentration steps can also be introduced prior to further analysis, e.g., binding to membranes, magnetic particles, etc.

[0073] sample

[0074] The sample can be any composition containing an analyte of interest, including, for example, a food, soil or water sample, or a biological sample obtained from an organism. In some embodiments, the analyte is a protein or multiple proteins. Such proteins include, but are not limited to, polypeptides, small peptides, glycoproteins, lipoprotein antibodies, enzymes, disease markers (such as polypeptide cancer antigens), cell surface receptors, hormone receptors, cytokines, chemokines, tissue-specific antigens, or fragments of any of the aforementioned proteins. In some embodiments, the sample can be a microorganism, such as a virus.

[0075] In some embodiments, the sample is a biological sample. Biological samples can be obtained from any organism, including, for example, animals, plants, fungi, bacteria, protozoa or viruses. In some embodiments, the sample is a body fluid or excreta, for example, blood, serum, plasma, urine, cerebrospinal fluid, cell secretions, saliva, sputum or fecal samples. In some embodiments, the biological sample is a cell or tissue, for example, a tissue sample, a biopsy sample, an explant from an organ, or a preserved or fixed tissue sample; or a cell sample, including cancer cells, cultured cells, or preserved or fixed cells. In some embodiments, the biological sample is a supernatant from a cell preparation (for example, a culture medium in which cells are cultured).

[0076] In some embodiments, the assay is performed as a multiplex assay to detect more than one analyte in a sample; and / or multiple samples are evaluated simultaneously. The skilled artisan understands that the detection agents for different target analytes and / or samples can be labeled with detectable markers that can be distinguished from each other (e.g., barcoded oligonucleotides or different fluorescent markers).

[0077] Reagent test kit

[0078] In some embodiments, the solid carrier with the low affinity Fab population of the target analyte is packaged in the test kit. In some embodiments, the solid carrier includes 96 holes, 384 holes or more holes in the form of a plate, so that the signal associated with the detection agent is processed automatically, and aligned with the method for generating and measuring the signal associated with the detection agent. In some embodiments, the solid carrier on which Fab is fixed is a microbead, for example, and these microbeads can be accommodated in a channel or a container. In some embodiments, low affinity Fab is fixed to a film (for example, nitrocellulose). In some embodiments, the test kit also includes a high affinity detection agent; and / or reagents, such as oligonucleotides, primers, probes or other reagents generate signals from the detection component of the detection agent.

[0079] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and those skilled in the art will propose various modifications or changes based thereon, and these modifications or changes will be included within the spirit and scope of the present application and within the scope of the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method for quantifying a target analyte, the method comprising incubating (i) a solution comprising a complex comprising a target analyte bound to a detection agent, wherein the detection agent binds to the target analyte with high affinity and is labeled with a detectable label; and (ii) a population of capture agents immobilized on a solid support in a channel or compartment, or immobilized in discrete regions on a solid support; wherein the capture agents specifically bind to the analyte and have a high off-rate for binding to the target analyte, and the analyte:detector complex then migrates through the channel or compartment, or through the discrete regions; spatially resolving the complex from other molecules in the solution based on a migration rate of the complex relative to unbound detection agent across the solid support to provide an eluate comprising the complex that is substantially free of unbound detection agent; as well as The amount of detection agent complexed with the analyte is quantified, thereby determining the amount of the target analyte present in the solution.

2. The method of claim 1, wherein the capture agent is a Fab or an aptamer.

3. The method of claim 2, wherein the capture agent is Fab.

4. The method of any one of claims 1 to 3, wherein the high affinity detection agent is an aptamer, an antibody or a ligand.

5. The method of claim 4, wherein the high affinity detection agent is an antibody.

6. The method of any one of claims 1 to 5, wherein the detectable label is specific for the analyte.

7. The method of any one of claims 1 to 6, wherein the detectable label is an oligonucleotide.

8. The method of claim 7, wherein the oligonucleotide comprises a primer binding site, or comprises a sequence that targets a primer binding site of a detection oligonucleotide that is detected in an amplification reaction.

9. The method of claim 7 or 8, wherein the oligonucleotide comprises an analyte identification region and / or a sample identification region.

10. The method of any one of claims 7 to 9, wherein quantification comprises amplifying a target region of the oligonucleotide to obtain an amplicon. The method of claim 10 , wherein amplification comprises PCR.

12. The method of claim 11, wherein the PCR is quantitative PCR.

13. The method of any one of claims 7 to 10, wherein quantification comprises sequencing a region of the oligonucleotide that is specific for the analyte to determine the amount of oligonucleotide present in the detector-analyte complex.

14. The method of any one of claims 1 to 13, wherein the complex comprises a fluorescent marker.

15. The method of claim 14, wherein quantifying comprises detecting the level of fluorescent signal generated by the fluorescent marker.

16. The method of any one of claims 1 to 15, wherein the channel is a capillary.

17. The method of any one of claims 1 to 15, wherein the solid support is a plurality of beads present in the channel or compartment.

18. The method of any one of claims 1 to 15, wherein the channel or compartment contains a solid support composed of a polymer.

19. The method of any one of claims 1 to 17, wherein pressure or an electric field is applied to the solution as it flows through the channel.

20. The method of any one of claims 1 to 19, wherein the channel is a channel of a microfluidic device.

21. The method of any one of claims 1 to 19, wherein the channels are capillaries present in microwells.

22. The method of any one of claims 1 to 15, wherein the solid support is a membrane or a wicking matrix and the solution flows through the membrane or wicking matrix by lateral flow.

23. The method of claim 22, wherein the solid support is nitrocellulose.

24. A kit comprising a capture agent bound to a solid support, and a high affinity antibody.

25. The kit of claim 24, wherein the solid support is a microbead or microparticle.

26. The kit of claim 24, wherein the solid support is a membrane or a wicking matrix.

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