WO2013176749A1

WO2013176749A1 - Apparatuses and methods for conducting binding assays

Info

Publication number: WO2013176749A1
Application number: PCT/US2013/031047
Authority: WO
Inventors: Ronghao Li
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-05-25
Filing date: 2013-03-13
Publication date: 2013-11-28
Anticipated expiration: 2014-11-25
Also published as: EP2856104A1; EP2856104A4; CN104520690A

Description

APPARATUSES AND METHODS FOR CONDUCTING BINDING ASSAYS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 61/652,102, filed May 25, 2012, which application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Binding assays are widely used in biological and clinical applications for detecting or measuring the amount of an analyte present in a sample. Binding assays generally require the use of a binding partner that binds to the analyte of interest and a detection reagent that binds to the binding partner. In direct binding assays, the binding partner is conjugated to the detection reagent, whereas indirect binding assays involve the addition of a separate detection reagent that interacts with the binding partner. A widely used form of binding assays is the linked immunosorbent assay (LISA), in which a binding agent is immobilized on a solid phase. Typically, the immunosorbent assay involves the capture of the analyte of interest onto the solid phase through the binding of the analyte with the binding agent. Addition of a detection reagent capable of binding to the analyte then results in subsequent capture of the detection reagent onto the solid phase. Unbound detection reagent is typically removed in a series of one or more washing steps prior to detection of the reagent, which indicates the presence or amount of the analyte of interest. The detection of the reagent may utilize an enzymatic reaction (ELISA) or fluorescence detection (FLISA).

[0003] ELISA' s and FLISA' s are commonly carried out in multi-well plates (such as 96-well reaction plates) for high-throughput screening purposes. Use of multi-well plates and devices enable the parallel processing of a plurality of samples for the analysis of one or more analytes. For example, ELISA's and FLISA's are commonly utilized for antibody screening of hybridoma cell lines. These screening assays generally involve the incubation of hybridoma cell culture media samples in 96-well plates coated with the antigen of interest. Following the incubation, a multi-step process involving additional incubations with secondary and/or tertiary detection reagents and washing steps must take place before the detection of the antibody of interest and identification of the positive hybridoma lines.

However, these multi-step assays are time-consuming, which limits the assay throughput. Devices and methods that reduce the steps and hence the amount of time required for these assays would greatly increase assay throughput and efficiency. SUMMARY OF THE INVENTION

[0004] Thus, the invention provides apparatuses and methods that would permit more robust binding assays with higher throughout and efficiency. Provided herein are compositions, methods, apparatuses, and kits that provide such advantages and related benefits. In one aspect, the present invention provides an insert for insertion into a well of a multi-well device. In some embodiments, the insert comprise an inner surface and an outer surface that are substantially free of micropores; wherein the inner surface and/or outer surface are immobilized thereon a binding agent; wherein the insert is dimensioned to be smaller than the well such that the insert may be inserted into the well.

[0005] In another embodiments, the insert comprises an inner surface and an outer surface, wherein the inner surface and/or outer surface are immobilized thereon a binding agent, wherein the outer surface has a diameter that is smaller than that of the well, such that the insert may be inserted into the well; wherein insertion of the insert into a well of a multi-well device renders the well optically isolated from an adjacent well of the device.

[0006] In practicing the invention, any of the inserts may be made of an opaque material, they may be cylindrical in shape, hollow, or may comprise a plurality of lumens.

[0007] In a related aspect, the present invention provides arrays of inserts for insertion into a plurality of wells of a multi-well device. In some embodiments, the array is configured such that each individual insert is mounted on a continuous solid framework, the framework being dimensioned to permit insertion and optionally removal of each individual insert into each individual well of the plurality of wells simultaneously, wherein the individual insert comprises an inner surface and an outer surface, either or both of which are immobilized thereon a binding agent, wherein the inner and/or outer surface are substantially free of micropores.

[0008] In alternative embodiments, the array is configured such that each individual insert is mounted on a continuous solid framework, the framework being dimensioned to permit insertion and optionally removal of each individual insert into each individual well of the plurality of wells simultaneously, wherein the individual insert comprises an inner surface and an outer surface, either or both of which are immobilized thereon a binding agent, wherein insertion of the individual insert into the individual well renders the well optically isolated from other adjacent wells on the device.

[0009] In practicing the invention, any of the arrays can be fashioned such that the framework is dimensioned to permit insertion and removal of the each individual insert into each individual well of the multi-well device simultaneously. [0010] In some embodiments, the binding agent of the inserts or arrays of the present invention comprises a protein, an antibody, a monoclonal antibody, an antigen, a ligand, a receptor, avidin, biotin, streptavidin, or a hapten.

[0011] In a related embodiment, the present invention provides a multi-well device with a plurality of wells, at least one of which contains an insert described herein, or which comprise an array of inserts described herein. In a further embodiment, the multi-well device has 6, 12, 24, 48, 96, 384, 1536, or 3456 wells.

[0012] Accordingly, the present invention also provides methods of measuring an analyte present in a liquid sample in a well. In some embodiments, the method comprises adding a labeled tracer comprising a detectable label to the well containing the liquid sample to form a mixture, wherein the well contains therein an insert of the present invention; allowing the analyte and the binding agent to compete for binding of the tracer for a sufficient period of time to effect formation of a complex that comprises the tracer and either the analyte or the binding agent in the mixture, wherein the competition results in a first amount of the detectable label remaining in the mixture and a second amount of the detectable label sequestered onto the inner surface and/or the outer surface of the insert; and measuring in the well the first amount of the detectable label remaining in the mixture, wherein the first amount is proportional to the amount of the analyte present in the mixture.

[0013] In other embodiments, the method comprises adding a labeled tracer to the well containing the liquid sample to form a mixture, wherein the well contains therein an insert of the present invention; allowing formation of a complex on the inner and/or outer surface of the insert, wherein the complex comprises (1) the labeled tracer bound to a first binding site on the analyte, and (2) the binding agent bound to a second binding site on the same analyte, wherein the formation results in a first amount of the detectable label remaining in the mixture and a second amount of the detectable label sequestered onto the inner surface and/or the outer surface of the insert; and measuring in the well the first amount of the detectable label remaining in the mixture, wherein the first amount is inversely proportional to the amount of the analyte present in the mixture.

[0014] In yet other embodiments, the method comprises adding a labeled tracer comprising a detectable label to the well containing the liquid sample to form a mixture, wherein the well contains therein an insert of the present invention; allowing the analyte and the binding agent to compete for binding of the tracer for a sufficient period of time to effect formation of a complex that comprises the tracer and either the analyte or the binding agent in the mixture, wherein the competition results in a first amount of the detectable label remaining in the mixture and a second amount of the detectable label sequestered onto the inner surface and/or the outer surface of the insert; and measuring the second amount of the detectable label sequestered onto the inner surface and/or the outer surface of the insert, wherein the second amount is inversely proportional to the amount of the analyte present in the mixture.

[0015] In yet other embodiments, the method comprises adding a labeled tracer comprising a detectable label to the well containing the liquid sample to form a mixture, wherein the well contains therein an insert of the present invention; allowing formation of a complex on the inner and/or outer surface of the insert, wherein the complex comprises (1) the labeled tracer bound to a first binding site on the analyte, and (2) the binding agent bound to a second binding site on the same analyte, the formation results in a first amount of the detectable label remaining in the mixture and a second amount of the detectable label sequestered onto the inner surface and/or the outer surface of the insert; and measuring the second amount of detectable label sequestered onto the inner surface and/or the outer surface of the insert wherein the second amount is proportional to the amount of the analyte present in the mixture.

[0016] In related embodiments, the present invention provides a method of conducting a binding assay in a mixture of an analyte and a labeled tracer comprising a detectable label in a multi-well device, the method comprising: adding the labeled tracer to a plurality of inserts of an array of the present invention; allowing the analyte and the binding agent to compete for binding of the tracer for a sufficient period of time to effect formation of a complex that comprises the tracer and either the analyte or the binding agent in the mixture, wherein the competition results in a first amount of the detectable label remaining in the mixture and a second amount of the detectable label sequestered onto the inner surface and/or the outer surface of the insert; and measuring in the plurality of wells the first amount of the detectable label remaining in the mixture, wherein the first amount is proportional to the amount of the analyte present in the mixture.

[0017] In alternative embodiments, the present invention provides method of conducting a binding assay in a mixture of an analyte and a labeled tracer comprising a detectable label in a multi-well device, the method comprising: adding the labeled tracer to a plurality of inserts of an array of the present invention; allowing formation of a complex on the inner and/or outer surface of the insert, wherein the complex comprises (1) the labeled tracer bound to a first binding site on the analyte, and (2) the binding agent bound to a second binding site on the same analyte, the formation results in a first amount of the detectable label remaining in the mixture and a second amount of the detectable label sequestered onto the inner surface and/or the outer surface of the insert; and measuring in the plurality of wells the first amount of the detectable label remaining in the mixture, wherein the first amount is inversely proportional to the amount of the analyte present in the mixture.

[0018] In yet another embodiment, the present invention provides a method of identifying a cell expressing a secreted heterologous polypeptide, comprising: providing a multi-well device comprising a plurality of wells, wherein each well of the plurality is inserted therein with an insert of the present invention, and wherein each well of the plurality contains a cell culture medium in which a cell suspected for expressing the polypeptide has been cultured; adding a labeled tracer comprising a detectable label to the plurality of wells containing the cell culture medium to form a mixture; allowing the polypeptide and the binding agent to compete for binding of the tracer for a sufficient period of time to effect formation of a complex that comprises the tracer and either the polypeptide or the binding agent in the mixture, wherein the competition results in a first amount of the detectable label remaining in the medium and a second amount of the detectable label sequestered onto the inner surface and/or the outer surface of the insert; and measuring in the well the first amount of the detectable label remaining in the mixture, wherein the first amount is proportional to the amount of the polypeptide present in the cell culture medium, thereby identifying the cell expressing the secreted heterologous polypeptide.

[0019] In an alternative embodiment, the present invention provides a method of identifying a cell expressing a secreted heterologous polypeptide, the method comprising: providing a multi-well device comprising a plurality of wells, wherein each well of the plurality is inserted therein with an insert of the present invention, and wherein each well of the plurality contains a cell culture medium in which a cell suspected for expressing the polypeptide has been cultured; adding a labeled tracer comprising a detectable label to the plurality of wells containing the cell culture medium to form a mixture; allowing formation of a complex on the inner and/or outer surface of the insert, wherein the complex comprises (1) the labeled tracer bound to a first binding site on the polypeptide, and (2) the binding agent bound to a second binding site on the same polypeptide, the formation results in a first amount of the detectable label remaining in the medium and a second amount of the detectable label sequestered onto the inner surface and/or the outer surface of the insert; and measuring in the well the first amount of the detectable label remaining in the medium, wherein the first amount is inversely proportional to the amount of the polypeptide present in the medium; thereby identifying the cell expressing the heterologous polypeptide. [0020] There are a number of embodiments in practicing any of the methods of the present invention. In practicing any of the methods described herein, the first amount of the detectable label remaining in the mixture may be measured without transferring the mixture out of the well or adjusting contents of the mixture, or may be measured after the insert is removed from the well, or may be measured by directing light to an observation volume in the well, wherein the observation volume is occupied substantially by the first amount of the detectable label but substantially exclusive of the second amount of the detectable label.

[0021] In practicing any of the methods of the present invention, the detectable label may be a radioactive probe, an optically detectable label, or a fluorescent dye. In further

embodiments of any of the methods of the present invention, the analyte is a secreted protein.

[0022] In another aspect, the invention provides an apparatus for measuring an analyte present in a liquid sample in a well, the apparatus comprising: a multi-well device containing (i) one or more wells inserted therein one or more inserts of the present invention, wherein the well contains a mixture comprising a labeled tracer that comprises a detectable label; a plate reader capable of (i) detecting the label from the one or more wells. Where desired, the plate reader comprises a processor programmed to generate signals corresponding to the detected label. In some embodiments, the plate reader is operably linked to a computer configured to (i) receive the signals from the reader; (ii) generate quantitative measurement data from the signals; and optionally (iii) relay the quantitative measurement data to a cloud server. In some embodiments, the reader is capable of being configured to detect the label from a confined volume substantially exclusive of the one or more inserts. In some embodiments, the reader is capable of detecting a radioactive label, an optically detectable label, a label which comprises a fluorescent dye, or a luminescent label.

[0023] In another aspect, the present invention provides kits for measuring an analyte present in a liquid sample in one or more wells of a multi-well device. In some embodiments, the kit comprises one or more inserts of the present invention, and instructions for use of the one or more inserts for conducting a binding assay to measure the analyte. In some embodiments, the kit further comprises a labeled tracer reagent comprising a detectable label. In some embodiments, the labeled tracer reagent is capable of forming a complex with either the analyte or the binding agent. In other embodiments, the binding agent is capable of forming a complex with the analyte at a first binding site. In yet other embodiments, the labeled tracer reagent is capable of forming a complex with the analyte at a second binding site. INCORPORATION BY REFERENCE

[0024] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0026] FIG. 1A and IB depicts a schematic diagram of exemplary embodiments of an insert, shaped to comprise a plurality of lumens.

[0027] FIG. 2 depicts a schematic diagram of exemplary embodiments of an insert in a well of a multi-well plate, showing a top-down view into the plate and well.

[0028] FIG. 3A depicts a schematic diagram of an exemplary direct competitive binding assay utilizing the inserts of the present invention.

[0029] FIG. 3B depicts a schematic diagram of an alternative embodiment of a direct competitive binding assay utilizing the inserts of the present invention.

[0030] FIG. 4A depicts a schematic diagram of an exemplary sandwich binding assay utilizing the inserts of the present invention.

[0031] FIG. 4B depicts a schematic diagram of an alternative embodiment of a sandwich binding assay utilizing the inserts of the present invention.

[0032] FIG. 5 depicts an exemplary apparatus for conducting binding assays.

[0033] FIG. 6 depicts a photograph of an exemplary embodiment of the inserts of the present invention in a 96-well multi-well plate.

[0034] FIG. 7 depicts results of a competitive binding assay utilizing inserts of the invention. Depicted is a plot of fluorescence intensity measurements over human gamma globulin concentration, comparing wells utilizing the inserts of the present invention vs. wells without the inserts.

[0035] FIG. 8 depicts results of a sandwich binding assay utilizing inserts of the invention. Depicted is a plot of fluorescence intensity measurements over human gamma globulin concentration. DETAILED DESCRIPTION OF THE INVENTION

[0036] In general, the invention provides compositions, methods, and kits for conducting binding assays in multi-well devices for the detection of analytes of interest.

Inserts

[0037] In one aspect, the present invention provides an insert which can be inserted into a well of a multi-well device. In some embodiments of the present invention, the insert is dimensioned to permit insertion and optionally removal from a well of a multi-well device. In particular embodiments, the insert comprises an outer surface dimensioned to be smaller than the well to permit insertion and removal without distorting or breaking the well. In yet more particular embodiments, the outer surface of the insert has a dimension that is about 99.9% or less, about 99.5% or less, about 99% or less, about 95% or less, about 90% or less, about 85%) or less, about 80%> or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or about 50% or less than the dimension of the well. In yet even more particular embodiments, the outer surface of the insert has a dimension that is about 50%-60%, 55%-75%, 60%-90%, 75%-95%, or 80%-99.9% of the dimension of the well.

[0038] In another embodiment, the insert is shaped to have a bottom portion with an outer surface dimensioned to be smaller than the well to permit insertion of the bottom portion of the insert into the well, and additionally an upper lip portion with an outer dimension that is larger than the dimension of the well, to allow the insert to sit inside the well without contacting the bottom surface of the well. In particular embodiments, the bottom portion of the insert has an outer surface with a dimension that is about 99.9% or less, about 99.5% or less, about 99% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or about 50% or less than the dimension of the well. In yet even more particular embodiments, the outer surface of the bottom portion of the insert has a dimension that is about 50%-60%, 55%-75%, 60%-90%, 75%-95%, or 80%-99.9% of the dimension of the well. In some embodiments, the upper lip portion of the insert has an outer dimension that is about 100.1%) or more, about 100.5%) or more, about 101% or more, about 102% or more, about 103%) or more, about 104% or more, about 105% or more, about 1 10%) or more, about 1 15%) or more, about 120% or more, about 125%) or more, about 130%) or more, about 135%) or more, about 140% or more, about 145% or more, or about 150% of the diameter of the well. In particular embodiments, the upper lip portion of the insert has a dimension that is about 100.1-1 10%, about 101%-120%, about 105%-130%, about 1 10%-150% of the diameter of the well. Preferably, the upper lip portions of the inserts are dimensioned to permit a plurality of inserts to sit in adjacent wells without substantial overlap of their upper lip portions.

[0039] In some embodiments, the inserts have a hollow shape comprising an inner and an outer surface, which provide both a sufficient volume for binding assays to be carried out in liquid solution and allow the unhindered passage of light vertically from the well of a multi- well device to a detector. In some embodiments, the inner surface of the insert has a dimension that is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%), about 99.5%), about 99.9% of the dimension of the outer surface of the insert. The insert can take a variety of shapes including but not limited to cylindrical, square, elliptical, oval, and polyhedral. In some embodiments, the polyhedral shape may have 3, 4, 5, 6, 7, or 8 or more sides. In some embodiments, the polyhedral shape is a triangular, square, rectangular, pentagonal, hexahedral, heptahedral, octahedral, dodecahedral.

[0040] In some embodiments, the insert is shaped to provide a plurality of lumens. For the purposes of this invention, the term "lumen" refers to the inside space of a hollow structure. The lumens may be arranged in a variety of configurations. For example, lumens within an insert can be divided by linear walls to form separate but adjacent compartments, or by circular walls to form inner and outer ring compartments. FIGS. 1A and IB depict a cross section of an exemplary insert with multiple lumens.

[0041] In some embodiments, the inserts may be inserted and optionally removed from a well of a multi-well device. Multi-well devices are well-known in the art, and it should be understood that the inserts may be configured for insertion and removal into any multi-well device. However, for the purposes of description only, provided herein are examples of multi-well device configurations that can be used with the inserts of the present invention. In some embodiments, the multi-well device is a multi-well plate, such as a tissue culture plate or multi-well assay plate. In some embodiments, the multi-well device comprises 6 wells, 12 wells, 24 wells, 48 wells, 96 wells, 384 wells, 1536 wells, or 3456 wells. Where desired, the individual wells of each plate can be uniformly dimensioned. In alternative embodiments, the inserts may be shaped for insertion and optional removal from other devices for conducting binding assays, such as a single cuvette.

Material of the inserts:

[0042] The terms "micropore" or "micropores" generally refer to pores in the submillimeter range. In general, porous or microporous inserts for use in multi-well devices may promote the formation of unwanted bubbles in the well upon the addition or removal of liquids and thus may have a deleterious impact on binding assays conducted in the well. For instance, these bubbles can interfere with the binding assay or provide some optical interference that hinders subsequent detection of a labeled binding agent. In some embodiments of the present invention, the inserts of the present invention are substantially free of micropores. In some embodiments, the inserts are substantially free of micropores having a diameter in the sub- millimeter range. In some embodiments, the inserts are substantially free of micropores having a diameter of about 100 microns or less, 90 microns or less, 80 microns or less, 70 microns or less, 60 microns or less, 50 microns or less, 40 microns or less, 30 microns or less, 20 microns or less, 10 microns or less, 5 microns or less, or 1 micron or less.

[0043] In particular embodiments, the inserts are substantially free of micropores having a diameter in the range of about 0.001-30 microns. In other particular embodiments, the inserts are substantially free of micropores having a diameter in the in the range of about 0.02-20 microns. In yet other embodiments, the inserts are substantially free of micropores having a diameter in the range of about 0.5-10 microns.

[0044] In general, inserts that are substantially free of micropores are made from non- microporous materials. Non-microporous materials are well known to those skilled in the art, and it is understood that any non-microporous materials may be used to make the inserts of the present invention. Non-limiting examples of non-microporous materials include plastic polymers, glass, cellulose or nitrocellulose, metal, and semi-conducting materials such as, e.g., silicon or germanium. Non-limiting examples of plastic polymers that may be used to make the inserts of the present invention include polypropylene, polystyrene, polycarbonate, polyurethane, polydimethylsiloxanes, polyvinylchloride, polysulfone,

polymethylmethacrylate, polyimide, polyamide, polyacrylonitrile, polybutadiene,

polybutylene, polycapro lactam, polychlorotrifluoroethylene, polyethylene

polytetrafluoroethylene, polyethylene terephathalate, polyvinylidene chloride,

polyisobutylene, polyolefme, polymeric polyisocyanate, polyvinyl fluoride. Other non- limiting examples of non-microporous materials that may be used to make the inserts include acrylonitrile-acryloid-styrene, acrylonitrile-chlorizate ethylene-styrene, and acrylonitrile- butadiene-styrene. In some cases, the material used for making the inserts may be modified to comprise cross-linking or other linker moieties for the immobilization of a binding agent. In a particular embodiment, the inserts are made from plastic tubes of 6mm OD and 4 mm ID, purchased from Graingers (cat. #2vdn6 and 2vdp4). [0045] Analysis of binding assays in multi-well devices often involves detection of signals (e.g. -light) from multiple or adjacent wells. Prevention of crosstalk between wells would improve the accuracy of the binding assay measurement. In some embodiments of the invention, the inserts substantially prevent the transmission of light through the material of the inserts. In some embodiments, the inserts substantially prevent the transmission of visible light. In other embodiments, the inserts substantially prevent the transmission of ultraviolet light. In yet other embodiments, the inserts substantially prevent the transmission of infrared light. In more particular embodiments, the inserts substantially prevent the transmission of visible, ultraviolet, and infrared light. Where desired, in some embodiments, the inserts are opaque.

[0046] In some embodiments, the inserts are opaque due to the incorporation of one or more pigments into the material of the inserts. The one or more pigments may be incorporated into the material of the inserts by any known methods of attaching a pigment to the insert. The attachment of the pigment can be via a covalent bond, a non-covalent interaction, or by deposition of the one or more pigments onto the inner and/or outer surface of the insert. In some embodiments, the pigment is a dye. Opaque pigments and dyes are known to those of skill in the art commercially available through a number of vendors, such as, e.g., Epo light 7276F Visible Opaqe Dye by Epolin, Inc. Other non-limiting examples of commonly used pigments include titanium dioxide, which provides a white pigmentation, and carbon black, charcoal black, ebony, ivory black, and onyx, which provide black pigmentation.

Array of inserts

[0047] Multi-well devices are often used for the parallel processing of multiple binding assays on a single sample, or a single binding assay on a plurality of samples, or multiple assays on a plurality of samples. In another aspect, the present invention provides an array of inserts described herein, which may be inserted and optionally removed from a plurality of wells in a multi-well device simultaneously. In some embodiments, each individual insert of the array is mounted onto a continuous solid framework. In some embodiments, the framework is an essentially flat framework with outer dimensions that are substantially the same as the outer dimensions of the multi-well device. In some embodiments, the framework comprises uniform holes that are substantially dimensioned to match the dimensions of the wells of the multi-well device. In some cases, the uniform holes of the framework are aligned to the wells of the multi-well device when the framework sits directly atop the device. In some cases, the inserts are dimensioned to stably fit into the holes of the framework. For example, an individual insert would have a bottom portion that is dimensioned to fit through the hole of the framework and to be inserted into a well of the multi-well device, and would also have an upper lip portion that that is dimensioned to be larger than the dimension of a hole of the framework, to enable the insert to sit inside the framework. In this exemplary configuration, the individual inserts can be simultaneously inserted and optionally removed from the wells. In some cases, the inserts are securely attached to the framework so that the inserts and framework act as one unit that can be inserted and optionally removed from the multi-well device.

Analytes

[0048] In another aspect of the invention, the inserts have immobilized thereon a binding agent used for the detection of an analyte in a liquid sample. The analyte may be, without limitation, a protein, a glycoprotein, a polypeptide (encompassing a fragment thereof), a polysaccharide, a lipid, a nucleic acid, a biological particle, a cell compartment, a cell, alloy metal or other elements, or any combination thereof. The analyte can also be, for example, a drug, prodrug, pharmaceutical agent, drug metabolite, a biomarker (e.g., an expressed protein or cell marker, an antibody or antibody fragment, a serum protein, a cell surface receptor, a receptor ligand, or functional motif thereof). In some embodiments, the analyte is a secreted protein. Non-limiting examples of secreted proteins include hormones, growth factors, neurotransmitters, antibodies, secreted enzymes, secreted toxins, or any secreted proteins comprising a signal sequence that targets the protein to the endoplasmic reticulum. Non- limiting examples of hormones include melatonin, thyroid hormone, epinephrine, antimullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensin, antidiuretic hormone (vasopressin), atrial-natriuretic peptide, calcitonin, cholecystokinin, cortocotropin- releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone, human chorionic gonadotropin, growth hormone, human placental lactogen, inhibin, insulin, insulin-like growth factor, leptin, leutenizing hormone, melanocyte stimulating hormone, orexin, oxytocin, parathyroid hormone, prolactin, relaxin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, Cortisol, aldosterone, testosterone,

dehydroepiandrosterone, dihydrotestosterone, estrogen, progesterone, prostaglandins, leukotrienes, endothelin, renin. Non-limiting examples of growth factors include brain- derived neurotrophic factor, epidermal growth factor, fibroblast growth factor, glial cell line- derived neurotrophic factor, hepatocyte growth factor, nerve growth factor, platelet-derived growth factor, transforming growth factor a or β, tumor necrosis factor-alpha, vascular endothelial growth factor, placental growth factor, interleukins 1-7, among others. Non- limiting examples of secreted enzymes include digestive enzymes, such as proteases, peptidases, lipases, carbohydrases, nucleases, Type I secreted proteins, Type II secreted proteins, Type III secreted proteins, Type IV secreted proteins, Type V secreted proteins, Type VI secreted proteins, plant-secreted proteins, among others. Non-limiting examples of secreted toxins include heat-stable enterotoxins, hemolysins, cholesterol-dependent cytolysins, RTX toxins, diphtheria toxin, cholera toxin, pertussis toxin, hyaluronidase, collagenase, among others. In particular embodiments, the secreted protein is a secreted antibody. In yet more particular embodiments, the secreted antibody is a monoclonal antibody.

Binding agents

[0049] A wide variety of binding agents may be utilized in the present invention. The choice of binding agent can depend on the analyte being assayed. The binding agent may be, without limitation, a protein, a glycoprotein, a polypeptide, a protein or peptide fragment, a polysaccharide, a lipid, a nucleic acid, a biological particle, a cell compartment, a cell, or any combination thereof. The binding agent can also be, for example, a drug, prodrug, pharmaceutical agent, drug metabolite, a biomarker (.e.g., an expressed protein or cell marker, an antibody or antibody fragment, a serum protein, a cell surface receptor, a receptor ligand, or functional motif thereof), a ligand, a receptor, or hapten. In some cases, the binding agent comprises avidin, biotin, or streptavidin. In some embodiments, the binding agent is a secreted protein. Non-limiting examples of secreted proteins are provided herein.

[0050] Generally, the selection of the binding agent will depend on the needs of the user and the type of binding assay to be utilized. A common type of binding assay is the sandwich assay, in which a binding agent binds to an analyte and the analyte further binds to a labeled tracer. In some embodiments, the binding agent is selected based on its ability to bind selectively to the analyte being investigated. For example, if the analyte is an antibody, then the binding agent comprises an antigen capable of selective interaction with the antibody. For another example, if the analyte is a secreted protein (e.g., insulin), then the binding agent is an antibody capable of binding to the secreted protein (e.g., an anti-insulin antibody).

[0051] Another common type of binding assay is the competitive assay, in which the binding agent competes with the analyte for binding to a labeled tracer. Therefore, in some embodiments, the binding agent is selected to comprise a motif, a molecule, biological particle, substance, or other structure with similar binding capability to the labeled tracer as the analyte. In other embodiments, the tracer is utilized as a reference to guide the selection of compounds that bind better than the reference compound. In particular embodiments, the binding agent is the same substance as the analyte. For example, if the analyte is an antibody, then the antigen comprises the same antibody. In other embodiments, the binding agent is a different substance than the analyte but has a similar binding affinity to the labeled tracer as the analyte.

Immobilization of the binding agent to the insert

[0052] Methods for immobilizing binding agents on solid surfaces are known to those skilled in the art. Generally, any suitable immobilization method may be used for immobilizing binding agents to the inserts of the invention. For example, many techniques are known for immobilizing nucleic acids and/or peptides to a solid surface, or to a linker moiety, or to an immobilized moiety by covalent bonds, non-covalent bonds, or adsorption. Such techniques are described in, e.g., Beier et al, Nucleic Acids Res. 27: 1970-1-977 (1999), Joos et al, Anal. Chem. 247:96-101 (1997), Guschin et al, Anal. Biochem. 250:203-211 (1997), Bhatia et al. 1998, Analytical Biochemistry, 178 408-13,Tedeschi et al. 2003, Biosensors and

Bioelectronics, (19) 85-93, Methods Mol. Biol. Vol. 20 (1993), Tischer and Wedekind., Topics in Current Chemistry 200:95-126 (1999), all of which are incorporated herein by reference.

[0053] In some embodiments, the binding agent is immobilized onto the insert via a crosslinker. In some embodiments, the crosslinker is a molecule with at least two reactive ends to connect the binding agent to the solid support material. In general, the selection of an appropriate crosslinker is informed by matching the reactive ends of the cross-linker to the functional groups of the solid support material and to the functional groups of the binding agent. In some cases, the crosslinkers contain reactive groups specific toward functional groups common on proteins, e.g., carboxyls, amines, sulfhydryls, or hydroxyls. These reactive groups can be used to crosslink proteinaceous binding agents to a solid support, such as an insert of the present invention. Non-limiting examples of crosslinker reactive groups include n-hydroxysuccinimide esters (NHS esters), imidoesters, maleimides, haloacetyls, pyridyl disulfides, pentafluorphenyl esters, hydroxymethylphosphine, thiosulfonate, vinylsulfonate, hydrazide, alkoxyamine, aryl azide, isocyanate, disuccinimidyl suberate, and diazirines.

[0054] In other embodiments, if the binding agent is a glycoprotein, methods for

immobilizing glycoproteins (i.e. -proteins that comprise carbohydrate side-chains) onto the inserts utilizing the carbohydrate groups for immobilization purposes may be used. For example, lectins are known and used in the art to immobilize carbohydrate groups to solid supports. In one embodiment, the inserts are made from a lectin-modified nonmicroporous material, for the immobilization of a glycoprotein binding agent.

[0055] In yet other embodiments, the binding agent comprises an affinity binding tag. Non- limiting examples of affinity tags include IgG binding domains, histidine tags, arginine tags, cellulose-binding domains, streptavidin, streptavidin binding domains, biotin, glutathione-S- transferase (GST) tags, and others.

[0056] In other embodiments, the binding agent is immobilized to the inserts by incubating the inserts with a coating buffer and the binding agent. In some cases where the binding agent comprises a crosslinker or affinity binding tag, the coating buffer comprises an agent with affinity to the crosslinker or the binding tag. For example, if the affinity binding tag comprises GST, the agent in the coating buffer comprises a GST-binding reagent, e.g., anti- GST antibody or glutathione. In some cases where the affinity binding tag comprises streptavidin, the agent in the coating buffer is biotin. In other cases where the affinity binding tag is a histidine tag, the agent in the coating buffer is a metal that interacts with histidine, e.g., copper or nickel. For another example, if the binding agent comprises a crosslinker, the coating buffer comprises an agent that binds to the crosslinker. For example, in some cases where the crosslinker comprises a sulfhydryl group, the coating buffer comprises activated maleimide which forms stable thioester bonds with sulfhydryl groups.

[0057] Non specific coating buffers for immobilizing binding agents to a solid surface are also known in the art, and include, for example, alkaline carbonate buffers, alkaline phosphate buffers, or alkaline tris-based buffers. In some embodiments, the coating buffer is a carbonate buffer with a molarity of about 0.05M, 0.06M, 0.07M, 0.08M, 0.09M, 0.1M, or 0.2M sodium carbonate. In some embodiments, the carbonate buffer is a sodium carbonate buffer, a carbonate-bicarbonate buffer, or sodium bicarbonate buffer. In some embodiments the coating buffer has a pH of about 7.5 or greater, about 8.0 or greater, about 8.5 or greater, or about 9.0 or greater. In other embodiments, the coating buffer has a pH of about 7.5-9, about 8-9.5, or about 9-10. In a particular embodiment, the coating buffer is a sodium carbonate buffer with a pH of about 9-10. In other embodiments, the coating buffer is a phosphate buffer, a phosphate-buffered saline, a tris-based buffer, or a tris-buffered saline. Non-limiting examples of coating buffers include 0.2 M sodium bicarbonate, pH 9.4, PBS-50 mM Phosphate, pH 8.0, 0.15 M NaCl, Carbonate-bicarbonate, Phosphate Buffer with 1.7 mM NaH2P04, 98 mM Na2HP04.7H20, 0.1% NaN3, pH 8.5, TBS - 50 mM TRIS, pH 8.0, 0.15 M NaCl. [0058] FIG. 2 depicts a schematic diagram of a particular embodiment of the inserts for insertion into wells of a multi-well device (200). The inserts can be utilized for conducting binding assays. The insert 210 is dimensioned for insertion and optional removal from a well (220), and further has immobilized on the inner and outer surface binding agents (230). Liquid Samples

[0059] In another aspect of the invention, the inserts are used to conduct binding assays in wells containing a liquid sample. In general, the liquid sample can be any liquid suspected of containing the analyte of interest. In some embodiments, the liquid sample is a bodily fluid sample, e.g., blood, serum, plasma, saliva, urine, amniotic fluid, aqueous humour, vitreous humour, bile, breast milk, cerebrospinal fluid, cerumen, endolymph, perilymph, sperm, female ejaculate, gastric juice, mucus, peritoneal fluid, pleural fluid, sebum, sweat, tears, vaginal secretion, or vomit. In other embodiments, the liquid sample is a protein sample, or a DNA sample. In other embodiments, the liquid sample is a lysed cell sample. In a particular embodiment, the liquid sample is a cell culture medium suspected of harboring the analyte. Labeled Tracers

[0060] General methods of conducting binding assays utilize a labeled tracer comprising a detectable label. For the purposes of the invention, the term "labeled tracer" is defined as an entity that can be used in a binding assay for the detection of an analyte, wherein the tracer additionally comprises a detectable label. Detection and measurement of the label can be used to determine the presence, absence, or quantity of the analyte. In some embodiments where the binding assay is a competitive assay, the labeled tracer is capable of binding to either of the analyte or to the binding agent. In particular embodiments, the labeled tracer is capable of binding to the analyte or to the binding agent with similar affinity. By way of example only, if the analyte and binding agent is a particular antibody, then the tracer comprises a labeled antigen capable of binding the antibody. By way of another example, if the analyte and binding agent are a particular antigen, then the tracer comprises a labeled antibody capable of binding the antigen.

[0061] In some embodiments, where the binding assay is a sandwich assay, if the binding agent is capable of binding the analyte at a first site, then the labeled tracer is selected such that it can bind to the analyte at a second site. In other embodiments, the labeled tracer is capable of binding to the analyte at a second site and has little or no affinity for the binding agent. By way of example only, if the analyte is an antigen, and the binding agent is an antibody capable of binding the antigen at a specific epitope, then the labeled tracer is a second antibody capable of binding the antigen at a different epitope. In either case, the labeled tracer can comprise, without limitation, a protein, a glycoprotein, a polypeptide, a polysaccharide, a lipid, a nucleic acid, or any combination thereof. The labeled tracer can also comprise, for example, a drug, prodrug, pharmaceutical agent, drug metabolite, a biomarker (.e.g., an expressed protein or cell marker, an antibody or antibody fragment, a serum protein, a cell surface receptor, a receptor ligand, or functional motif thereof). In some embodiments, the labeled tracer is a secreted protein. Examples of secreted proteins are described herein.

[0062] In some embodiments, the labeled tracer is a molecule conjugated to a detectable label. A variety of labels can be used for the instant invention, with the choice of label depending on the sensitivity required, ease of conjugation to, stability requirements, available instrumentation and detection methods, and disposal provisions.

[0063] In some embodiments, the detectable label comprises a fluorescent dye. In general, the fluorescent dye comprises a fluorophore which absorbs light energy of a first wavelength range and re-emits light within a second wavelength range. In some embodiments, the fluorescent dye is a xanthenes derivative, such as, e.g., fluorescein, rhodamine, TRITC, X- rhodamine, Lissamine rhodamine B, Oregon green, or Texas Red. In other embodiments, the fluorescent dye is a cyanine derivative, such as, e.g., cyanine, indocarbocyanine,

oxacarbocyanine, thiacarbocyanine, or merocyanine. In yet other embodiments, the fluorescent dye is a naphthalene derivative, such as, e.g., a danysl or drodan derivative. In other embodiments, the fluorescent dye is a coumarin derivative. In other embodiments, the fluorescent dye is an oxadiazole derivative, such as, e.g., pryidyloxazole,

nitrobenzooxadiazole, or benzoxadiazole. In other embodiments, the fluorescent dye is an oxazine derivative, such as, e.g., Nile red, Nile blue, or oxazine 170. Other examples of fluorescent dyes include, e.g., CF dye (Biotium— see, e.g., US Patent Nos. 8148518, 8092784, 7803943, 7601498, 7776567, all of which are incorporated by reference), BODIPY (Invitrogen), DyLight Fluor (Thermo scientific), Atto and Tracy (Sigma Aldrich), FluoProbes (Interchim), DY and MegaStokes Dyes (Dyomics), Seatau and Square Dyes (SETA

BioMedicals), Quasar and Cal Fluor dyes (Biosearch Technologies), Pacific Blue, Pacific Orange, Lucifer Yellow, or allophycocyanin.

[0064] In yet other embodiments, the fluorescent dye comprises an indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance. In more particular embodiments, the fluorescent dye is an Alexa Fluor dye, such as, e.g., Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor. 700, Alexa Fluor 750, or Alexa Fluor 790.

[0065] In other embodiments, the detectable label comprises a fluorescent protein.

Fluorescent proteins are well known in the art. Non-limiting examples of fluorescent proteins include Y66H, Y66F, EGFP, EGFP2, Azurite, GFP, T-Sapphire, Cerulean, CFP, TagCFP, TurboCFP, ECFP, CyPet, Y66W, m eima-Red, AmCyanl, TFP1, Midori-ishi Cyan, GFP, Turbo GFP, TagGFP, EGFP, S65C, , S65L, Emerald, S65T, Azami Green, ZsGreenl, YFP, TagYFP, EYFP, Turbo YFP, Topaz, Venus, mCitrine, mOrange, RFP, TagRFP, TurboRFP, DsRed, mStrawberry, R-phycoerytrin, B-phycoerythrin, mCherry, Peridinin Chlorophyll (PerCP), mPlum, mOrange, or mRaspberry.

[0066] In other embodiments, the detectable label comprises a luminescent or

chemiluminescent tag. Common luminescent/chemiluminescent tags include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. These protein tags can catalyze chemiluminescent reactions given the appropriate substrates (e.g., an oxidizing reagent plus a chemiluminescent compound. A number of compound families are known to provide chemiluminescence under a variety of conditions. Non-limiting examples of chemiluminescent compound families include 2,3-dihydro-l,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz analog. These compounds can luminesce in the presence of alkaline hydrogen peroxide or calcium hypochlorite and base. Other examples of chemiluminescent compound families include, e.g., 2,4,5-triphenylimidazoles, para-dimethylamino and - methoxy substituents, oxalates such as oxalyl active esters, p-nitrophenyl, N-alkyl acridinum esters, luciferins, lucigenins, or acridinium esters. Many substrates for chemiluminescent protein tags are well known in the art and commercially available, and it is understood that any suitable substrate for chemiluminescent reactions may be used in the present invention.

[0067] Methods for conjugating or coupling the detectable label to the molecule are well known to those skilled in the art, and may be used to prepare the labeled tracer. In some embodiments, labels are attached by indirect means. In particular embodiments, a label is attached by the following steps: (1) a ligand molecule is covalently bound to a polymer. (2) The ligand binds to an anti-ligand molecule which can be either inherently detectable or covalently bound to a signal system. Many ligand/anti- ligand combinations are known and available in the art, for example, biotin/streptavidin, thyroxine/thyroxine-binding globulin, hapten/antibody or antigen/antibody. Examples of signal systems include, e.g., a detectable enzyme or a chemiluminescent compound. In other embodiments, conjugation of the detectable label to the molecule involves molecular cloning, e.g., recombinant DNA technology.

Methods of conducting binding assays

[0068] In another aspect, the invention provides methods of using the individual inserts or arrays of inserts described herein to measure an analyte present in a liquid sample in a well or plurality of wells in multi-well devices. In one aspect, the invention provides a method of using the inserts or insert arrays in a competitive binding assay. FIG. 3A provides a schematic diagram of an exemplary competitive binding assay utilizing the inserts or insert arrays of the present invention (300). In some embodiments, the method comprises adding a labeled tracer 310 comprising a detectable label to a well containing a liquid sample 320. In some embodiments utilizing insert arrays, the method comprises adding labeled tracer 310 comprising a detectable label to a plurality of wells containing a plurality of liquid samples 320. In some embodiments, the liquid sample is suspected of containing an analyte 330, depicted as light gray circles. In some embodiments, the well contains an insert 340 with a binding agent 350 immobilized thereon. In some embodiments utilizing insert arrays, the plurality of wells contain an array of inserts 340 with binding agents 350 immobilized thereon. In some embodiments, the labeled tracer is capable of binding to the analyte and is additionally capable of binding to the binding agent. In particular embodiments, the labeled tracer is capable to binding to the analyte and the binding agent with similar affinity. In particular embodiments, the binding agent is the same type of molecule as the analyte, therefore the labeled tracer is capable of binding to both with substantially equal affinity. In more particular embodiments, the analyte itself has no significant binding affinity to the binding agent. In some embodiments, adding the labeled tracer to the liquid sample forms a mixture which comprises the liquid sample 320, analyte 330, and labeled tracer 310.

[0069] In some embodiments, the method further comprises incubating the mixture for a sufficient period of time as to allow the analyte and binding agent to compete for binding of the labeled tracer. In some embodiments, the incubation step comprises incubating for about 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours, or more. Where desired, the incubating step comprises incubating for about 24 hours or more. The incubating step can also be performed for less than about 24 hours, less than about 20 hours, less than about 16 hours, less than about 12 hours, less than about 8 hours, less than about 4 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than about 0.5 hours. In yet other embodiments, the incubating step comprises incubating for about 0.5-2 hours, about 1-4 hours, about 2-8 hours, about 4-12 hours, about 8-24 hours, or more. Incubation can be carried out at any suitable temperature for a given assay, such as at about 37 degrees Celsius, at about room temperature, or at about 20-25 degrees Celsius. In yet other embodiments, the incubating step comprises incubating at about 4 degrees Celsius. In some embodiments, the incubating step comprises shaking and/or rocking. In other embodiments, the incubating step does not require shaking or rocking. In a particular embodiment, the incubating step comprises incubating for 0.5-2 hours at about 37 degrees Celsius, with or without shaking and/or rocking. In another particular embodiment, the incubating step comprises incubating for about 1-12 hours at room temperature (e.g., 20-25 degrees Celsius), with or without shaking and/or rocking. In yet another particular embodiment, the incubating step comprises incubating for about 8-24 hours or more at about 4 degrees Celsius, with or without shaking and/or rocking. In some embodiments, the competition results in the formation of one or more complexes 360 that comprises the labeled tracer and analyte. In other embodiments, the competition results in the formation of one or more complexes 370 that comprises the labeled tracer and binding agent. In yet other embodiments, the competition results in the formation of one or more complexes 360 and one or more complexes 370. In some embodiments, the one or more complexes 370 are sequestered or bound onto the inner and/or outer surfaces of the insert, while the one or more complexes 360 remain in the mixture. For the purposes of this invention, the phrases "remain in the mixture" or "remaining in the mixture" refer to remaining essentially free-floating in the mixture and unbound to the insert.

[0070] In some embodiments, the method further comprises measuring in the well the amount of detectable label remaining in the mixture. In some cases, the amount of detectable label remaining in the mixture is proportional to the amount of analyte present in the liquid sample. In particular embodiments, the measuring of the amount of detectable label remaining in the mixture substantially obviates measuring an amount of detectable label sequestered or bound to the inner and/or outer surface of the insert. In more particular embodiments, measuring the amount of label sequestered onto the insert is obviated by measuring in a detection region 380 that is exclusive of the insert. In yet other particular embodiments, measuring the amount of label sequestered onto the insert is obviated by removal of the insert prior to the measuring step.

[0071] In an alternative embodiment 301 depicted in FIG. 3B, the methods are substantially identical to those described as above, except that instead of measuring the amount of detectable label remaining in the solution, the method comprises measuring in the well the amount of detectable label sequestered onto the insert by measuring in a detection region 390 that comprises the insert. In a particular alternative embodiment, measuring in the detection region 390 substantially obviates the amount of detectable label remaining in the mixture. In these alternative embodiments, the amount of detectable label sequestered onto the insert is inversely proportional to the amount of analyte present in the liquid sample. In some embodiments, after the incubating step, the device comprising the well is stored at about 4 degrees Celsius for any suitable period of time, before measurement of the analyte is carried out.

[0072] In another aspect, the invention provides a method of using the individual inserts or insert arrays in a sandwich-type assay. FIG. 4 A provides a schematic diagram of an exemplary sandwich-type binding assay utilizing the inserts or insert arrays of the present invention (400). In some embodiments, the method comprises adding a labeled tracer comprising a detectable label 410 to a well containing a liquid sample 420. In embodiments utilizing insert arrays, the method comprises adding labeled tracer 410 comprising a detectable label to a plurality of wells containing a plurality of liquid samples 420. In some embodiments, the liquid sample is suspected of containing an analyte 430, depicted as light gray crescents. In some embodiments, the well contains an insert 440 with a binding agent 450 immobilized thereon. In some embodiments utilizing insert arrays, the plurality of wells contain an array of inserts 440 with binding agents 450 immobilized thereon. In some embodiments, adding the labeled tracer to the liquid sample forms a mixture which comprises the liquid sample 420, analyte 430, and labeled tracer 410. In some embodiments, the labeled tracer 410 is capable of binding to the analyte 430 at a first site. In some embodiments, the binding agent 450 is capable of binding to the analyte 430 at a second site. In particular embodiments, the labeled tracer has no significant binding affinity to the binding agent 450. In some embodiments, adding the labeled tracer to the liquid sample forms a mixture which comprises the liquid sample 420, analyte 430, and labeled tracer 410.

[0073] In some embodiments, the method further comprises incubating the mixture for a sufficient period of time as to allow the formation of a complex comprising the labeled tracer, the analyte, and the binding agent, wherein the labeled tracer is bound to the analyte at a first site and the binding agent is bound to the analyte at a second site. Various embodiments of incubating steps are described herein. In some embodiments, the incubating results in an amount of uncomplexed labeled tracer 460 remaining in the mixture. In other embodiments, the incubating results in the formation of one or more complexes 470 that comprises the labeled tracer 410, analyte 430, and binding agent 450. In yet other embodiments, the competition results in an amount of uncomplexed labeled tracer 460 and one or more complexes 470. In some embodiments, the one or more complexes 470 are sequestered or bound onto the inner and/or outer surfaces of the insert, while the uncomplexed labeled tracer 460 remains in the mixture.

[0074] In some embodiments, the method further comprises measuring in the well the amount of detectable label remaining in the mixture. In some cases, the amount of detectable label remaining in the mixture is inversely proportional to the amount of analyte present in the liquid sample. In particular embodiments, the measuring of the amount of detectable label remaining in the mixture substantially obviates measuring an amount of detectable label sequestered or bound to the inner and/or outer surface of the insert. In more particular embodiments, measuring the amount of label sequestered onto the insert is obviated by measuring in a detection region 480 that is exclusive of the insert. In yet other particular embodiments, measuring the amount of label sequestered onto the insert is obviated by removal of the insert prior to the measuring step. In an alternative embodiment 401 depicted in FIG. 4B, the methods are substantially identical to those described as above, except that instead of measuring the amount of detectable label remaining in the solution, the method comprises measuring in the well the amount of detectable label sequestered onto the insert by measuring in a detection region 490 that comprises the insert. In a particular alternative embodiment, measuring in the detection region 490 substantially obviates the amount of detectable label remaining in the mixture. In these alternative embodiments, the amount of detectable label sequestered onto the insert is directly proportional to the amount of analyte present in the liquid sample. In some embodiments, the device comprising the well can be stored at 4 degrees Celsius for an indefinite period of time following the incubating step, prior to measuring.

[0075] The methods disclosed herein are particularly suited for measuring a wide range of analyte concentrations with linear detection. In some embodiments, the methods can measure between about 0-100 μg/ml of an analyte, about 0-90 μg/ml of an analyte, about 0-80 μg/ml of an analyte, about 0-70 μg/ml of an analyte, about 0-60 μg/ml of an analyte, about 0-50 μg/ml of an analyte, about 0-40 μg/ml of an analyte, about 0-30 μg/ml of an analyte, about 0- 20 μg/ml of an analyte, about 0-10 μg/ml of an analyte, about 0-5 μg/ml of an analyte, or about 0-2.5 μg/ml of an analyte.

[0076] In practicing the methods above, the relative amount of labeled tracer, binding agent, and/or analyte may be adjusted to increase the efficiency and/or accuracy of the binding assay. In some embodiments, the relative concentration of labeled tracer is adjusted relative to the binding capacity of the binding agent. In particular embodiments, the relative concentration of labeled tracer is about 100%, about 95%, about 90%>, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%), about 35%, about 30%>, about 25%, about 20%>, about 15%, about 10%>, or about 5% of the relative binding capacity of the binding agent. In some embodiments, the relative concentration of labeled tracer is about 10-90%), about 20-80%), about 30-70%), about 40%>- 60%, or about 45%-55% of the relative binding capacity of the binding agent. In a particular embodiment, the relative concentration of the labeled tracer is about 50% of the relative binding capacity of the binding agent.

[0077] In other embodiments, the relative concentration of labeled tracer is adjusted relative to the binding capacity of the analyte. In particular embodiments, the relative concentration of labeled tracer is about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%), about 30%), about 25%, about 20%, about 15%, about 10%, or about 5% of the relative binding capacity of the analyte. In some embodiments, the relative concentration of labeled tracer is about 10-90%, about 20-80%, about 30-70%, about 40%-60%, or about 45%-55% of the relative binding capacity of the analyte. In a particular embodiment, the relative concentration of the labeled tracer is about 50% of the relative binding capacity of the analyte.

[0078] In yet other embodiments, the relative concentration of labeled tracer is adjusted relative to the sum binding capacity of the analyte and binding agent. In particular embodiments, the relative concentration of labeled tracer is about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5% of the sum binding capacity of the analyte and binding agent. In some embodiments, the relative concentration of labeled tracer is about 10-90%, about 20- 80%, about 30-70%, about 40%-60%, or about 45%-55% of the sum binding capacity of the analyte and binding agent. In a particular embodiment, the relative concentration of the labeled tracer is about 50% of the sum binding capacity of the analyte and binding agent. Methods of Detection

[0079] Generally, the methods described above comprise a measuring step that involves detection of the label. Methods of detection are well known to those of skill in the art, and any suitable detection method may be utilized for the present invention. Those of skill in the art will understand that the choice of detection method depends on the needs of the user and the type of detectable label utilized. [0080] In some embodiments, where the detectable label is a fluorescent dye or fluorescent protein, the measuring step comprises use of a fluorescence detection method. Methods of fluorescence detection are well known to those of skill in the art. In some embodiments, fluorescence detection comprises excitation of the fluorescent dye or protein by illuminating a target region with a source light, the source light being of a wavelength range sufficient to excite a fluorophore in the dye or protein. Source light may be provided by a number of suitable light sources, including, e.g., lasers, laser diodes, and high intensity lamps, such as xenon arc or mercury vapor lamps. The wavelength range of the source light may be selected using, for example, optical filters or a monochromator systems.

[0081] Generally, excitation of the fluorophore results in the emission of response light. In some embodiments, the response light is of a wavelength range with higher wavelengths than the excitation wavelength range. In some embodiments, fluorescence detection comprises detection of the response light. Detection can be accomplished using, by way of example only, digital cameras, charge coupled devices (CCDs), photomultipliers tubes, phototubes, or CMOS image sensors.

[0082] Fluorescence polarization represents a method of fluorescence detection that can further distinguish bound or unbound labeled tracers. Generally, fluorescence polarization uses tumbling motion of molecules to qualify binding events. For example, if a small fluorescent dye or protein binds to a much larger molecule (e.g., in cases where the labeled tracer forms a complex with an analyte, a binding agent, or a binding agent and analyte), the tumbling speed of the fluorescent dye or protein will be reduced, compared to an unbound small fluorescent dye or protein (e.g., if the labeled tracer remains unbound). The tumbling speed of fluorescent dye or protein molecules may be determined by excitation of the fluorophore with plane polarized light. If the fluorophore has a lower tumbling speed (e.g., in cases where the labeled tracer has formed a complex with another binding partner), the emitted response light will tend to remain polarized in the same plane, by contrast, fluorophores with a higher tumbling speed (e.g., in cases where the labeled tracer remains unbound) will tend to lose polarization during the excitation step, and therefore emit response light with lower polarization relative to the excitation light plane. In particular embodiments, the measuring step comprises utilization of fluorescence polarization methods. In more particular embodiments, measuring the amount of detectable label remaining in the mixture utilizes fluorescence polarization methods, which enable the user to distinguish labeled tracer that has formed a complex with the analyte vs. unbound labeled tracer. [0083] In some embodiments, where the detectable label comprises a luminescent or chemiluminescent tag, the measuring step comprises a luminescence detection method.

Luminescent detection methods are known to those of skill in the art (see, e.g., Biomedical Chromatography 17:83-95 (2003), Complementary ImmunoAssays, Ch. 14, copyright 1988, Canadian Journal of Chemistry, 1987, 65(6): 1392-1396), US Patent RE39047, all of which are incorporated herein by reference). In some embodiments, the measuring step comprises addition of a chemiluminescent compound and activating agent, wherein the

chemiluminescent tag catalyzes the reaction. Chemiluminescent compounds are well known in the art, and are described in the above references. In some embodiments, where the chemiluminescent compound is selected from 2,3-dihydro-l,4-phthalazinedione luminol, 5- amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz analog, the measuring step comprises addition of an alkaline hydrogen peroxide or calcium hypochlorite and base, which induces the label to luminesce. In other embodiments, where the chemiluminescent compound is of the 2,4,5-triphenylimidazole family or oxalate family (e.g., oxalyl active esters), the measuring step comprises addition of an alkaline buffer with an oxidizing reagent, which induces the compound to luminesce with a green light. In yet other embodiments, where the luminescent tag comprises luciferase, the measuring step comprises addition of a luciferin to induce luminescence. In yet further embodiments, where the chemiluminescent compound is of the acridium ester family, the measuring step comprises addition of a superoxide anion under neutral-alkaline conditions.

[0084] In some embodiments of the methods described herein, separating measurement of detectable label remaining in the mixture versus detectable label sequestered onto the insert enables the user to proceed to the measuring step immediately following the incubating step, without requiring any intermediate adjustments to the mixture or transferring the mixture out of the well (e.g., without requiring intermediate wash steps). In some embodiments, amount of detectable label is measured without adjusting the contents of the mixture or transferring the mixture out of the well. In particular embodiments, separating the measurement of label remaining in the mixture from the label sequestered onto the insert involves removing the insert to a new well. In more particular embodiments, measurement of the amount of detectable label remaining in the mixture in the original well can be combined with the measurement of the amount of label sequestered onto the insert in the new well to indicate the quantity of the analyte in the liquid sample (e.g. -by ratiometric analysis).

[0085] The methods utilizing arrays of inserts described herein can be used for a wide variety of high-throughput, multiplexed assays. For example, the array of inserts can be used to measure an analyte of interest in a plurality of liquid samples, or to measure a plurality of analytes in a liquid sample divided into a plurality of wells, or the measure a plurality of analytes in a plurality of liquid samples. In particular embodiments, the array of inserts is used for high-throughput cell screening purposes. In yet more particular embodiments, the array of inserts is used to identify a cell expressing a secreted polypeptide, utilizing the methods described above. In yet even more particular embodiments, the secreted polypeptide is a heterologous polypeptide. In these embodiments, the liquid sample or samples comprise cell culture media in which a cell suspected for expressing the polypeptide has been cultured. Apparatuses

[0086] In another aspect, the invention provides apparatuses suitable for use in conducting the binding assays described herein. In some embodiments, the apparatus comprises a plate reader instrument capable of detecting and/or measuring signals from one or more wells of a multi-well plate containing the inserts of the present invention. In some embodiments, the plate reader instrument can be configured to detect and/or measure signals from a 6 well, a 12-well, a 24-well, a 48-well, a 96-well, a 384-well, or a 3456-well plate. Plate readers are well-known to those of skill in the art, and are readily commercially available. Examples of commercially available plate readers include, e.g., the Perkin-Elmer LS55 luminescence spectrometer, the LJL Biosystems Analyst Plate Reader (Molecular Devices, Sunnyvale, CA), the CytoFluor.TM. 2300 machine (Millipore).

[0087] In some embodiments, the plate reader instrument includes a light source, a detector, and an optical relay structure configured to direct light from the light source to target regions corresponding with wells of the multi-well device.

[0088] Suitable light sources include, e.g., high-intensity lamps, incandescent lamps, fluorescent lamps, electroluminescent devices, lasers, laser diodes, and light-emitting diodes (LEDs), among others. In some embodiments, the light source is configured to provide continuous illumination. In other embodiments, the light source is configured to provide time-varying illumination, e.g., a pulsed laser. The light source can be configured to produce coherent and/or incoherent light. The light source can also be configured to provide polarized and/or unpolarized light. Exemplary light sources include a Q-switched YAG laser, a Nd:V04 laser, a Nd:glass laser, a Nd:YAG laser, a nitrogen laser, a Q-switched argon laser, a Ti:sapphire laser, a fiber laser, or any suitable laser or monochromatic light source.

[0089] In some embodiments, the plate reader comprises a light director that directs source light to one or more target regions corresponding to one or more wells of the multi-well device. In particular embodiments, the light director is configured to provide source light to a single target region corresponding with a single well, and to move from well to well, and at each well, there is a dwell time during which the light director directs source light to the target region of the well. In these cases, the wells of the multi-well device would be illuminated one at a time.

[0090] In other embodiments, the light director comprises a light pattern generator capable of directing source light to a plurality of target regions corresponding with a plurality of wells of a multi-well device. The light pattern generator generally comprises any mechanism capable of manipulating light into a preselected light pattern. This pattern may be created using diffraction, refraction, reflection, and/or other mechanisms, or a combination thereof. The light pattern generator can be used to generate any desired pattern of light, including one- dimensional or two-dimensional patterns (or arrays). For example, a light pattern generator can be configured to create any regularly shaped beamlet array of any dimension. For example, the desired pattern may be an array of substantially equally target regions positioned to correspond to the spacing of one or more target wells of a multi-well device. In some cases, the light pattern generator provides uniform light intensity across each of the target regions. In other embodiments, the desired pattern corresponds to a plurality of target regions within a well of a multi-well device (e.g., corresponding to a plurality of lumens). In some embodiments, the desired pattern corresponds to regions of the well that substantially comprise the mixture volume but not the insert, or vice versa.

[0091] In some embodiments, the plate reader additionally comprises a detector apparatus that detects response light from a plurality of wells in a multi-well device, and generates signals related to the detectable label from the response light. In particular embodiments, the detector apparatus comprises a single detector configured to detect light from a single target region corresponding to a single well of a multi-well device. In more particular

embodiments, the single detector is moved from well to well and at each well, there is a dwell time during which the signal from the response light is digitized and stored into memory.

[0092] In other embodiments, the detector apparatus comprises a light relay structure that is configured to direct response light from a plurality of wells to a plurality of light detectors corresponding to individual wells of the plurality of wells. In particular embodiments, the plurality of light detectors detects response light from the plurality of wells simultaneously. In more particular embodiments, the detector apparatus detects response light from the plurality of wells simultaneously by taking a snapshot of the entire plate using a CCD camera or other imaging apparatus (e.g., a CMOS chip-based camera). [0093] In some embodiments, the apparatus can optionally comprise a processor that is adapted to communicate with and process data from the plate reader. In some embodiments, the plate reader communicates signals generated by the detector apparatus to the processor. The processor can convert the signals into measurements which indicate the presence or amount of the detectable label in the target region. In some embodiments, the processor is additionally configured to communicate user-defined experiment settings to the plate reader. For example, the user may set whether the plate reader conducts a top-read or bottom read of the plate, may choose the wells that he/she wishes to obtain data from, may set the type of assay (e.g., fluorescent vs. luminescent assay), may define excitation/emission settings for fluorescence assays, or may define a preselected light pattern for directing source light to specific target regions in specific wells. The processor described herein can be integrated to the plate reader or as a separate component embodied in a computer that is operably linked to the plate reader. In a preferred embodiment, the user selects a top read, as the bottom surface of the well may provide optical interference that reduces the accuracy of the measurement.

[0094] In some embodiments, the plate reader is capable of detecting signals from a plurality of defined target subregions (scan points) within a single well. In some embodiments, the plate reader is capable of detecting signals from the same plurality of defined target subregions from multiple wells of a multi-well plate. In other embodiments, the apparatus comprises software that is capable of displaying signals from each target subregion graphically to create a map for each well. In yet other embodiments, the apparatus comprises a processor that is capable of enabling the user to select or remove individual target subregions or entire sections of the well for analysis, thereby defining the detection region of the well. In some embodiments, the user selects a detection region that is substantially exclusive of the insert. In other embodiments, the user selects a detection region that is inclusive of the insert by substantially exclusive of the lumens of the insert. Plate readers that are capable of scanning wells in the manner described herein are known in the art and commercially available from a number of vendors, e.g., PHERAstar FS (BMG Labtech), EnSpire Multimode Plate Reader (PerkinElmer), Gemini EM Fluorescence Microplate Reader (Molecular Devices), among others.

[0095] In some embodiments, the computer additionally includes an interface that communicatively couples to a cloud, which can include one or more external servers for data storage and/or further processing.

[0096] FIG. 5 provides a schematic of an exemplary apparatus of the invention (500). The apparatus includes a multi-well device with one or more wells inserted thereto inserts of the present invention (510), a plate reader (520) capable of detecting signals related to a detectable label and communicating the signals to a computer (530), the computer capable of converting the signals into measurement data indicating the presence or amount of detectable label, and optionally capable of relaying the data to a cloud (540). Where desired, the plate reader itself or the operably linked computer contains reference or standard charts of a given analyte being detected to affect a quantitative analysis of the analyte in a test sample.

Kits

[0097] In another aspect, the invention provides kits for measuring an analyte in present in liquid samples in wells of a multi-well device. In some embodiments, kits include one or more of the inserts described herein, wherein the inserts are immobilized thereon a binding agent. In some embodiments, kits also include instructions for the use of the inserts in conducting a binding assay for measuring an analyte.

[0098] In additional embodiments, kits include a labeled tracer. In some embodiments, the labeled tracer and binding agent are selected such that the labeled tracer has similar binding affinities to the analyte and to the binding agent. In further embodiments, the binding agent is selected such that it does not have appreciable binding affinity for the analyte. In yet further embodiments, the instructions for use instruct the user to measure the amount of detectable label remaining in the mixture, wherein the amount is directly proportional to the amount of analyte in the liquid sample. In other embodiments, the instructions for use instruct the user to measure the amount of detectable label sequestered onto the one or more inserts, wherein the amount is inversely proportional to the amount of analyte in the liquid sample.

[0099] In alternative embodiments, the labeled tracer and binding agent are selected such that the binding agent is capable of binding to the analyte at a first site, and the labeled tracer is capable of binding to the analyte at a second site. In further embodiments, the labeled tracer does not have appreciable binding affinity for the binding agent. In yet further embodiments, the instructions for use instruct the user to measure the amount of detectable label remaining in the mixture, wherein the amount is inversely proportional to the amount of analyte in the liquid sample. In other embodiments, the instructions for use instruct the user to measure the amount of detectable label sequestered onto the one or more inserts, wherein the amount is directly proportional to the amount of analyte in the liquid sample.

[00100] In some embodiments, kits further comprise standards, e.g., standards for use in the assays of the invention. Examples of standards include, e.g., a stock solution comprising a known concentration of the analyte, wherein the stock solution may be serially diluted to produce a standard curve. [00101] Further exemplary embodiments of the inserts and methods of the present invention are provided herein.

EXAMPLE 1: competitive FLISA assay utilizing inserts of the invention

[00102] To prepare the inserts, plastic tubes of 6mm OD and 4 mm ID were purchased from Graingers (cat. #2vdn6 and 2vdp4) and cut into cylinders of approximately 6 mm height with a sharp razor blade. The cylinders were boiled in IN NaHCO³ solution for about 10 minutes, rinsed 5x with deionized water, and air dried. The cylinders were then immersed in 5 microgram/ml goat anti-human IgG(H+L) (Jackson Immuno Research Laboratories, Inc., Cat. #109-005-003) prepared in coating buffer (0.1 N NaHC03, pH 9.0) and incubated at about 4-9 °C overnight. The coated rings were rinsed 3X in PBS and were ready to use. The coated rinses could be air dried and stored for future use. The prepared immuno-absorbent inserts were inserted into wells of 96 well plates. FIG. 6 depicts an exemplary photograph of the inserts inserted into a 96-well plate.

[00103] Human gamma globulin (Jackson ImmunoResearch Laboratories, inc., cat. #

009-000-002) and Alexa Fluor® 488 conjugated ChromoPure human IgG (Jackson

ImmunoResearch Laboratories, Inc., cat.# 009-540-003) were diluted in FLISA buffer (PBS containing 0.1% Tween 20 and 1% BSA. A series of dilutions of human gamma globulin were prepared at a ratio of 1 :2 from starting concentration of 5 microgram/ml. The human gamma globulin solutions were added at 100 micro liter/well to a 96 well plate with the immune-absorbent inserts or to a 96 well plate that had been coated with 5 microgram ml goat anti-human IgG(H+L) in duplicates. Then, 50 microliters of 45 micrograms/ml Alexa Fluor® 488 conjugated ChromoPure human IgG were added to each well except for the blank control well, which received 150 microliters of FLISA buffer only. The plates were then mixed on a shaker briefly and incubated at 37°C for 2 hours or at about 4-9 °C overnight before reading with Perkin Elmer Envision plate reader. Excitation light of 493nm was used to illuminate the wells, and emission light of 519 nm was detected from each well. Data were plotted as the average of duplicates and analyzed by curve fitting. FIG. 7 depicts a plot of fluorescence intensity as a function of human gamma globulin concentration. Data demonstrates that in wells with the inserts (coated rings), fluorescence intensity linearly increased along with the increased concentrations of human gamma globulin, ranging from 0.08 to 2.5 micrograms/ml. On the contrary, the fluorescence intensity of the different human gamma globulin groups remained to be a flat line in the plate directly coated with antibody. EXAMPLE 2: sandwich FLISA assay utilizing inserts of the invention

[00104] To prepare the inserts, nylon tubes of ¼" OD were purchased from Graingers

(cat. #2vdn6 and 2v4) and cut into cylinders of approximately 3 mm height with a sharp razor blade. The cylinders were boiled in 1 M NaHC03 solution for 10 minutes, rinsed 5x with deionized water, and air dried. The cylinders were then immersed in 5 microgram/ml goat anti-human IgG kappa light chain (SouthernBiotech, Cat. No. 2060-01) prepared in coating buffer (0.1 N NaHC03, pH 9.0) and incubated at 4-9 °C overnight. The coated rings were rinsed 3X in PBS, blocked by incubating in PBS with 0.5% bovine albumin, and were ready to use. The prepared rings were inserted into wells of 96 well plates (E&K Scientific, EK- 25076).

[00105] Human gamma globulin (Jackson ImmunoResearch Laboratories, Inc., cat. #

009-000-002) was diluted in sample buffer (PBS containing 0.1% Tween 20 and 1 % BSA). A series of dilutions of human gamma globulin were prepared at a dilution factor of 2 starting from a concentration of 1 microgram/ml. The human gamma globulin solutions were added at 100 micro liter/well in duplicates to in wells of 96 well plates with the cylindrical inserts coated with anti-human kappa light chain antibody above. After 60 minutes incubation at room temperature, the plate was washed 3 times with 300 microliter/well PBS containing 0.1% Tween 20 with a plate washer. Then, Alexa Fluo® 488 conjugated AffiniPure anti- human IgG Fc specific antibody (Jackson ImmunoResearch Laboratories, inc., cat.# 109-545- 098), diluted in Probe Buffer (PBS containing 0.1% Tween 20 and 0.1 % BSA), was added (100 micro liters/well) at a concentration of 100 ng/ml except for the wells of blank control, which received 100 microliter of Probe Buffer only. The plates were incubated at room temperature with gently shaking for 45 minutes before reading by Perkin Elmer Envision Multipurpose Plate Reader (Excitation 493nm and Emission 519 nm). Data were plotted as average of duplicates and analyzed by curve fitting with Microsoft Excel. As showed in FIG. 8, the fluorescence intensity has decreased linearly along with the increasing concentration of human gamma globulin.

[00106] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. An insert for insertion into a well of a multi-well device, said insert comprising an inner surface and an outer surface that are substantially free of micropores; wherein the inner surface and/or outer surface are immobilized thereon a binding agent; wherein the insert is dimensioned to be smaller than the well such that said insert may be inserted into said well.

2. An insert for insertion into a well of a multi-well device, said insert comprising an inner surface and an outer surface, wherein the inner surface and/or outer surface are immobilized thereon a binding agent, wherein the outer surface having a diameter that is smaller than that of said well, such that said insert may be inserted into said well; wherein said insertion of said insert into a well of a multi-well device renders the well optically isolated from an adjacent well of said device.

3. The insert of claim 1 or 2, wherein the insert is made of an opaque material.

4. The insert of claim 1 or 2, wherein the insert is cylindrical in shape.

5. The insert of claim 1 or 2, wherein the insert is hollow.

6. The insert of claim 1 or 2, wherein the insert comprises a plurality of lumens.

7. The insert of claim 1 or 2, wherein the insert is dimensioned to be removable from said well.

8. The insert of claim 1 or 2, wherein the binding agent comprises an antibody.

9. The insert of claim 1 or 2, wherein the binding agent comprises an antigen.

10. An array of inserts for insertion into a plurality of wells of a multi-well device, wherein each individual insert is mounted on a continuous solid framework, said framework being dimensioned to permit insertion and optionally removal of each individual insert into each individual well of said plurality of wells simultaneously, wherein said individual insert comprises an inner surface and an outer surface, either or both of which are immobilized thereon a binding agent, wherein the inner and/or outer surface are substantially free of micropores.

11. An array of inserts for insertion into a plurality of wells of a multi-well device, wherein each individual insert is mounted on a continuous solid framework, said framework being dimensioned to permit insertion and optionally removal of each individual insert into each individual well of said plurality of wells simultaneously, wherein said individual insert comprises an inner surface and an outer surface, either or both of which are immobilized thereon a binding agent, wherein insertion of said individual insert into said individual well renders said well optically isolated from other adjacent wells on the device.

12. The array of inserts of claim 10 or 11, wherein the framework is dimensioned to permit insertion and removal of said each individual insert into each individual well of said multi-well device simultaneously.

13. A multi-well device comprising the array of inserts of claim 10 or 11.

14. The multi-well device of claim 13, wherein said multi-well device is a multi-well plate having 6, 12, 24, 96, 384, 1536, or 3456 wells

15. A multi-well device comprising a plurality of wells, at least one of which contains an insert of claim 1 or 2.

16. The multi-well device of claim 15, wherein said multi-well device is a multi-well plate having 6, 12, 24, 96, 384, 1536, or 3456 wells.

17. A method of measuring an analyte present in a liquid sample in a well, comprising:

(a) adding a labeled tracer comprising a detectable label to the well containing the liquid sample to form a mixture, wherein the well contains therein an insert of claim 1 or 2;

(b) allowing said analyte and said binding agent to compete for binding of said tracer for a sufficient period of time to effect formation of a complex that comprises the tracer and either the analyte or the binding agent in the mixture, wherein said competition results in a first amount of said detectable label remaining in said mixture and a second amount of said detectable label sequestered onto the inner surface and/or the outer surface of said insert; and

(c) measuring in said well said first amount of said detectable label remaining in said mixture, wherein said first amount is proportional to the amount of the analyte present in the mixture.

18. A method of measuring an analyte present in a liquid sample in a well, comprising:

(a) adding a labeled tracer to the well containing the liquid sample to form a mixture, wherein the well contains therein an insert of claim 1 or 2;

(b) allowing formation of a complex on the inner and/or outer surface of the insert, wherein the complex comprise (1) the labeled tracer bound to a first binding site on the analyte, and (2) the binding agent bound to a second binding site on the same analyte, said formation results in a first amount of said detectable label remaining in said mixture and a second amount of said detectable label sequestered onto the inner surface and/or the outer surface of said insert; and (c) measuring in said well said first amount of said detectable label remaining in said mixture, wherein said first amount is inversely proportional to the amount of the analyte present in the mixture.

19. The method of claim 17 or 18, wherein the first amount of said detectable label remaining in said mixture is measured without transferring said mixture out of the well or adjusting contents of said mixture.

20. The method of claim 17 or 18, wherein the first amount of said detectable label remaining in said mixture is measured after the insert is removed from the well.

21. The method of claim 17 or 18, wherein said method is capable of measuring said analyte with a linear detection range of 3.9 to 1000 ng/ml.

22. A method of measuring an analyte present in a liquid sample in a well, comprising:

(c) measuring the second amount of said detectable label sequestered onto the inner surface and/or the outer surface of said insert, wherein said second amount is inversely proportional to the amount of the analyte present in the mixture.

23. A method of measuring an analyte present in a liquid sample in a well, comprising:

(b) allowing formation of a complex on the inner and/or outer surface of the insert, wherein the complex comprise (1) the labeled tracer bound to a first binding site on the analyte, and (2) the binding agent bound to a second binding site on the same analyte, said formation results in a first amount of said detectable label remaining in said mixture and a second amount of said detectable label sequestered onto the inner surface and/or the outer surface of said insert; and

(c) measuring said second amount of detectable label sequestered onto the inner surface and/or the outer surface of said insert wherein said second amount is proportional to the amount of the analyte present in the mixture.

24. The method of claim 22 or 23, wherein second amount of said detectable label remaining in said mixture is measured without transferring said mixture out of the well or adjusting contents of said mixture.

25. The method of claim 22 or 23, wherein the second amount of said detectable label is measured after removing the insert out of the well.

26. A method of conducting a binding assay in a mixture of an analyte and a labeled tracer comprising a detectable label in a multi-well device, the method comprising:

(a) adding said labeled tracer to a plurality of inserts of the array of claim 10 or claim

11;

(c) measuring in said plurality of wells said first amount of said detectable label remaining in said mixture, wherein said first amount is proportional to the amount of the analyte present in the mixture.

27. A method of conducting a binding assay in a mixture of an analyte and a labeled tracer comprising a detectable label in a multi-well device, the method comprising:

11;

(c) measuring in said plurality of wells said first amount of said detectable label remaining in said mixture, wherein said first amount is inversely proportional to the amount of the analyte present in the mixture.

28. A method of identifying a cell expressing a secreted heterologous polypeptide, comprising:

(a) providing a multi-well device comprising a plurality of wells, wherein each well of said plurality is inserted therein with an insert of claim 1 or 2, and wherein each well of said plurality contains a cell culture medium in which a cell suspected for expressing said polypeptide has been cultured;

(b) adding a labeled tracer comprising a detectable label to said plurality of wells containing the cell culture medium to form a mixture;

(c) allowing said polypeptide and said binding agent to compete for binding of said tracer for a sufficient period of time to effect formation of a complex that comprises the tracer and either the polypeptide or the binding agent in the mixture, wherein said competition results in a first amount of said detectable label remaining in said medium and a second amount of said detectable label sequestered onto the inner surface and/or the outer surface of said insert; and

(d) measuring in said well said first amount of said detectable label remaining in said mixture, wherein said first amount is proportional to the amount of the polypeptide present in the cell culture medium, thereby identifying the cell expressing the secreted heterologous polypeptide.

29. A method of identifying a cell expressing a secreted heterologous polypeptide, method comprising:

(c) allowing formation of a complex on the inner and/or outer surface of the insert, wherein the complex comprises (1) the labeled tracer bound to a first binding site on the polypeptide, and (2) the binding agent bound to a second binding site on the same

polypeptide, said formation results in a first amount of said detectable label remaining in said medium and a second amount of said detectable label sequestered onto the inner surface and/or the outer surface of said insert; and

(d) measuring in said well said first amount of said detectable label remaining in said medium, wherein said first amount is inversely proportional to the amount of the polypeptide present in the medium; thereby identifying the cell expressing the heterologous polypeptide.

30. The method of any of the preceding claims, wherein the detectable label is a radioactive probe.

31. The method of any of the preceding claims, wherein the detectable label is an optically detectable label.

32. The method of any of the preceding claims, wherein the detectable label comprises a fluorescent dye.

33. The method of any of the preceding claims, wherein the analyte is a secreted protein.

34. The method of any of the preceding claims, wherein said measuring comprises directing light to an observation volume in said well, wherein said observation volume is occupied substantially by said first amount of said detectable label but substantially exclusive of said second amount of said detectable label.

35. An apparatus for measuring an analyte present in a liquid sample in a well, comprising:

(a) a multi-well device containing (i) one or more wells inserted into one or more inserts of claim 1 or 2, wherein said well contains a mixture comprising a labeled tracer that comprises a detectable label;

(b) a plate reader capable of (i) detecting said label from said one or more wells.

36. The apparatus of claim 35, wherein the plate reader comprises a processor programmed to generate signals corresponding to said detected label, and wherein said plate reader is operably linked to a computer configured to (i) receive said signals from said reader; and (ii) generate quantitative measurement data from said signals; and optionally (iii) relay said quantitative measurement data to a cloud server.

37. The apparatus of claim 35, wherein the reader is capable of being configured to detect said label from a confined volume substantially exclusive of the one or more inserts.

38. The apparatus of claim 35, wherein the reader is capable of detecting a radioactive label.

39. The apparatus of claim 35, wherein the reader is capable of detecting an optically detectable label.

40. The apparatus of claim 39, wherein the optically detectable label comprises a fluorescent dye.

41. The apparatus of claim 40, wherein the optically detectable label comprises a luminescent label.

42. A kit for measuring an analyte present in a liquid sample in one or more wells of a multi-well device, comprising:

(a) one or more inserts of claim 1 or 2. (b) instructions for use of said one or more inserts for conducting a binding assay to measure said analyte.

43. The kit of claim 42, further comprising a labeled tracer reagent comprising a detectable label, wherein said labeled tracer reagent is capable of forming a complex with either of said analyte or said binding agent.

44. The kit of claim 43, wherein said binding agent is capable of forming a complex with said analyte at a first binding site.

45. The kit of claim 43, further comprising a labeled tracer reagent comprising a detectable label, wherein said labeled tracer reagent is capable of forming a complex with said analyte at a second binding site.