EP2856104A1 - Vorrichtungen und verfahren zur durchführung von bindungsassays - Google Patents
Vorrichtungen und verfahren zur durchführung von bindungsassaysInfo
- Publication number
- EP2856104A1 EP2856104A1 EP13794009.4A EP13794009A EP2856104A1 EP 2856104 A1 EP2856104 A1 EP 2856104A1 EP 13794009 A EP13794009 A EP 13794009A EP 2856104 A1 EP2856104 A1 EP 2856104A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- well
- insert
- amount
- detectable label
- mixture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0609—Holders integrated in container to position an object
- B01L2300/0618—Holders integrated in container to position an object for removable separation walls
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0636—Integrated biosensor, microarrays
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0829—Multi-well plates; Microtitration plates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0848—Specific forms of parts of containers
- B01L2300/0854—Double walls
Definitions
- 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.
- LISA linked immunosorbent assay
- 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).
- 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.
- 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.
- the invention provides apparatuses and methods that would permit more robust binding assays with higher throughout and efficiency.
- compositions, methods, apparatuses, and kits that provide such advantages and related benefits.
- the present invention provides an insert for insertion into a well of a multi-well device.
- 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.
- 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.
- 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.
- the present invention provides arrays of inserts for insertion into a plurality of wells of a multi-well device.
- 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.
- 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.
- 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.
- 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.
- 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.
- the multi-well device has 6, 12, 24, 48, 96, 384, 1536, or 3456 wells.
- the present invention also provides methods of measuring an analyte present in a liquid sample in a well.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
- 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
- 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.
- the detectable label may be a radioactive probe, an optically detectable label, or a fluorescent dye.
- the analyte is a secreted protein.
- 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.
- the plate reader comprises a processor programmed to generate signals corresponding to the detected label.
- 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.
- the reader is capable of being configured to detect the label from a confined volume substantially exclusive of the one or more inserts.
- the reader is capable of detecting a radioactive label, an optically detectable label, a label which comprises a fluorescent dye, or a luminescent label.
- the present invention provides kits for measuring an analyte present in a liquid sample in one or more wells of a multi-well device.
- 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.
- the kit further comprises a labeled tracer reagent comprising a detectable label.
- the labeled tracer reagent is capable of forming a complex with either the analyte or the binding agent.
- the binding agent is capable of forming a complex with the analyte at a first binding site.
- the labeled tracer reagent is capable of forming a complex with the analyte at a second binding site.
- FIG. 1A and IB depicts a schematic diagram of exemplary embodiments of an insert, shaped to comprise a plurality of lumens.
- 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.
- FIG. 3A depicts a schematic diagram of an exemplary direct competitive binding assay utilizing the inserts of the present invention.
- FIG. 3B depicts a schematic diagram of an alternative embodiment of a direct competitive binding assay utilizing the inserts of the present invention.
- FIG. 4A depicts a schematic diagram of an exemplary sandwich binding assay utilizing the inserts of the present invention.
- FIG. 4B depicts a schematic diagram of an alternative embodiment of a sandwich binding assay utilizing the inserts of the present invention.
- FIG. 5 depicts an exemplary apparatus for conducting binding assays.
- FIG. 6 depicts a photograph of an exemplary embodiment of the inserts of the present invention in a 96-well multi-well plate.
- 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.
- 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
- the invention provides compositions, methods, and kits for conducting binding assays in multi-well devices for the detection of analytes of interest.
- the present invention provides an insert which can be inserted into a well of a multi-well device.
- the insert is dimensioned to permit insertion and optionally removal from a well of a multi-well device.
- the insert comprises an outer surface dimensioned to be smaller than the well to permit insertion and removal without distorting or breaking the well.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the polyhedral shape may have 3, 4, 5, 6, 7, or 8 or more sides.
- the polyhedral shape is a triangular, square, rectangular, pentagonal, hexahedral, heptahedral, octahedral, dodecahedral.
- the insert is shaped to provide a plurality of lumens.
- the term "lumen” refers to the inside space of a hollow structure.
- the lumens may be arranged in a variety of configurations.
- 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.
- 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.
- the multi-well device is a multi-well plate, such as a tissue culture plate or multi-well assay plate.
- 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.
- the inserts may be shaped for insertion and optional removal from other devices for conducting binding assays, such as a single cuvette.
- micropore generally refer to pores in the submillimeter range.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
- non-microporous materials that may be used to make the inserts include acrylonitrile-acryloid-styrene, acrylonitrile-chlorizate ethylene-styrene, and acrylonitrile- butadiene-styrene.
- 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.
- the inserts are made from plastic tubes of 6mm OD and 4 mm ID, purchased from Graingers (cat. #2vdn6 and 2vdp4).
- 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.
- 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.
- 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.
- 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.
- 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.
- each individual insert of the array is mounted onto a continuous solid framework.
- the framework is an essentially flat framework with outer dimensions that are substantially the same as the outer dimensions of the multi-well device.
- the framework comprises uniform holes that are substantially dimensioned to match the dimensions of the wells of the multi-well device.
- the uniform holes of the framework are aligned to the wells of the multi-well device when the framework sits directly atop the device.
- the inserts are dimensioned to stably fit into the holes of the framework.
- 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.
- the individual inserts can be simultaneously inserted and optionally removed from the wells.
- 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.
- 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).
- 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.
- the analyte is a secreted protein.
- 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, thy
- 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 ⁇ , 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.
- the secreted protein is a secreted antibody.
- the secreted antibody is a monoclonal antibody.
- 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.
- 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
- the binding agent comprises avidin, biotin, or streptavidin.
- the binding agent is a secreted protein. Non-limiting examples of secreted proteins are provided herein.
- 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.
- 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).
- 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.
- any suitable immobilization method may be used for immobilizing binding agents to the inserts of the invention.
- 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
- the binding agent is immobilized onto the insert via a crosslinker.
- the crosslinker is a molecule with at least two reactive ends to connect the binding agent to the solid support material.
- 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.
- 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.
- NHS esters n-hydroxysuccinimide esters
- imidoesters maleimides
- haloacetyls pyridyl disulfides
- pentafluorphenyl esters pentafluorphenyl esters
- hydroxymethylphosphine thiosulfonate
- vinylsulfonate vinylsulfonate
- hydrazide alkoxyamine
- aryl azide isocyanate
- the binding agent is a glycoprotein
- glycoproteins i.e. -proteins that comprise carbohydrate side-chains
- lectins are known and used in the art to immobilize carbohydrate groups to solid supports.
- the inserts are made from a lectin-modified nonmicroporous material, for the immobilization of a glycoprotein binding agent.
- the binding agent comprises an affinity binding tag.
- 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.
- the binding agent is immobilized to the inserts by incubating the inserts with a coating buffer and the binding agent.
- the coating buffer comprises an agent with affinity to the crosslinker or the binding tag.
- the affinity binding tag comprises GST
- the agent in the coating buffer comprises a GST-binding reagent, e.g., anti- GST antibody or glutathione.
- the agent in the coating buffer is biotin.
- 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.
- the coating buffer comprises an agent that binds to the crosslinker.
- the crosslinker comprises a sulfhydryl group
- the coating buffer comprises activated maleimide which forms stable thioester bonds with sulfhydryl groups.
- 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.
- 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.
- the carbonate buffer is a sodium carbonate buffer, a carbonate-bicarbonate buffer, or sodium bicarbonate buffer.
- 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.
- the coating buffer has a pH of about 7.5-9, about 8-9.5, or about 9-10.
- the coating buffer is a sodium carbonate buffer with a pH of about 9-10.
- 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.
- 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
- the inserts are used to conduct binding assays in wells containing a liquid sample.
- the liquid sample can be any liquid suspected of containing the analyte of interest.
- 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.
- 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
- labeled tracer comprising a detectable label.
- 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.
- the binding assay is a competitive assay
- the labeled tracer is capable of binding to either of the analyte or to the binding agent.
- the labeled tracer is capable of binding to the analyte or to the binding agent with similar affinity.
- the tracer comprises a labeled antigen capable of binding the antibody.
- the tracer comprises a labeled antibody capable of binding the antigen.
- the binding assay is a sandwich assay
- the labeled tracer is selected such that it can bind to the analyte at a second site.
- the labeled tracer is capable of binding to the analyte at a second site and has little or no affinity for the binding agent.
- the analyte is an antigen
- the binding agent is an antibody capable of binding the antigen at a specific epitope
- the labeled tracer is a second antibody capable of binding the antigen at a different epitope.
- 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).
- the labeled tracer is a secreted protein. Examples of secreted proteins are described herein.
- the labeled tracer is a molecule conjugated to a detectable label.
- 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.
- the detectable label comprises a fluorescent dye.
- the fluorescent dye comprises a fluorophore which absorbs light energy of a first wavelength range and re-emits light within a second wavelength range.
- the fluorescent dye is a xanthenes derivative, such as, e.g., fluorescein, rhodamine, TRITC, X- rhodamine, Lissamine rhodamine B, Oregon green, or Texas Red.
- the fluorescent dye is a cyanine derivative, such as, e.g., cyanine, indocarbocyanine,
- the fluorescent dye is a naphthalene derivative, such as, e.g., a danysl or drodan derivative.
- the fluorescent dye is a coumarin derivative.
- the fluorescent dye is an oxadiazole derivative, such as, e.g., pryidyloxazole,
- the fluorescent dye is an oxazine derivative, such as, e.g., Nile red, Nile blue, or oxazine 170.
- fluorescent dyes include, e.g., CF dye (Biotium— see, e.g., US Patent Nos.
- BioMedicals Quasar and Cal Fluor dyes (Biosearch Technologies), Pacific Blue, Pacific Orange, Lucifer Yellow, or allophycocyanin.
- 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.
- 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.
- 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
- 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
- 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.
- HRP horseradish peroxidase
- SP soybean peroxidase
- alkaline phosphatase alkaline phosphatase
- luciferase luciferase.
- 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.
- 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.
- 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.
- labels are attached by indirect means.
- 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.
- signal systems include, e.g., a detectable enzyme or a chemiluminescent compound.
- conjugation of the detectable label to the molecule involves molecular cloning, e.g., recombinant DNA technology.
- 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.
- 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).
- the method comprises adding a labeled tracer 310 comprising a detectable label to a well containing a liquid sample 320.
- the method comprises adding labeled tracer 310 comprising a detectable label to a plurality of wells containing a plurality of liquid samples 320.
- the liquid sample is suspected of containing an analyte 330, depicted as light gray circles.
- the well contains an insert 340 with a binding agent 350 immobilized thereon.
- the plurality of wells contain an array of inserts 340 with binding agents 350 immobilized thereon.
- the labeled tracer is capable of binding to the analyte and is additionally capable of binding to the binding agent.
- the labeled tracer is capable to binding to the analyte and the binding agent with similar affinity.
- 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.
- the analyte itself has no significant binding affinity to the binding agent.
- adding the labeled tracer to the liquid sample forms a mixture which comprises the liquid sample 320, analyte 330, and labeled tracer 310.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the method further comprises measuring in the well the amount of detectable label remaining in the mixture.
- the amount of detectable label remaining in the mixture is proportional to the amount of analyte present in the liquid sample.
- 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.
- measuring the amount of label sequestered onto the insert is obviated by measuring in a detection region 380 that is exclusive of the insert.
- measuring the amount of label sequestered onto the insert is obviated by removal of the insert prior to the measuring step.
- 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.
- 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).
- the method comprises adding a labeled tracer comprising a detectable label 410 to a well containing a liquid sample 420.
- the method comprises adding labeled tracer 410 comprising a detectable label to a plurality of wells containing a plurality of liquid samples 420.
- the liquid sample is suspected of containing an analyte 430, depicted as light gray crescents.
- 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.
- adding the labeled tracer to the liquid sample forms a mixture which comprises the liquid sample 420, analyte 430, and labeled tracer 410.
- the labeled tracer 410 is capable of binding to the analyte 430 at a first site.
- the binding agent 450 is capable of binding to the analyte 430 at a second site.
- the labeled tracer has no significant binding affinity to the binding agent 450.
- adding the labeled tracer to the liquid sample forms a mixture which comprises the liquid sample 420, analyte 430, and labeled tracer 410.
- 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.
- incubating steps are described herein.
- the incubating results in an amount of uncomplexed labeled tracer 460 remaining in the mixture.
- the incubating results in the formation of one or more complexes 470 that comprises the labeled tracer 410, analyte 430, and binding agent 450.
- the competition results in an amount of uncomplexed labeled tracer 460 and one or more complexes 470.
- 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.
- the method further comprises measuring in the well the amount of detectable label remaining in the mixture.
- the amount of detectable label remaining in the mixture is inversely proportional to the amount of analyte present in the liquid sample.
- 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.
- measuring the amount of label sequestered onto the insert is obviated by measuring in a detection region 480 that is exclusive of the insert.
- measuring the amount of label sequestered onto the insert is obviated by removal of the insert prior to the measuring step.
- 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.
- measuring in the detection region 490 substantially obviates the amount of detectable label remaining in the mixture.
- the amount of detectable label sequestered onto the insert is directly proportional to the amount of analyte present in the liquid sample.
- 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.
- the methods disclosed herein are particularly suited for measuring a wide range of analyte concentrations with linear detection.
- the methods can measure between about 0-100 ⁇ g/ml of an analyte, about 0-90 ⁇ g/ml of an analyte, about 0-80 ⁇ g/ml of an analyte, about 0-70 ⁇ g/ml of an analyte, about 0-60 ⁇ g/ml of an analyte, about 0-50 ⁇ g/ml of an analyte, about 0-40 ⁇ g/ml of an analyte, about 0-30 ⁇ g/ml of an analyte, about 0- 20 ⁇ g/ml of an analyte, about 0-10 ⁇ g/ml of an analyte, about 0-5 ⁇ g/ml of an analyte, or about 0-2.5 ⁇ g/ml of an analy
- 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.
- the relative concentration of labeled tracer is adjusted relative to the binding capacity of the binding agent.
- 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.
- 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.
- the relative concentration of labeled tracer is adjusted relative to the binding capacity of the analyte.
- 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.
- 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.
- the relative concentration of the labeled tracer is about 50% of the relative binding capacity of the analyte.
- the relative concentration of labeled tracer is adjusted relative to the sum binding capacity of the analyte and binding agent.
- 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.
- 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.
- 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.
- the measuring step comprises use of a fluorescence detection method. Methods of fluorescence detection are well known to those of skill in the art.
- 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.
- excitation of the fluorophore results in the emission of response light.
- the response light is of a wavelength range with higher wavelengths than the excitation wavelength range.
- 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.
- Fluorescence polarization represents a method of fluorescence detection that can further distinguish bound or unbound labeled tracers.
- 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.
- the measuring step comprises utilization of fluorescence polarization methods.
- 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.
- the detectable label comprises a luminescent or chemiluminescent tag
- the measuring step comprises a luminescence detection method.
- the measuring step comprises addition of a chemiluminescent compound and activating agent, wherein the
- the measuring step comprises addition of an alkaline hydrogen peroxide or calcium hypochlorite and base, which induces the label to luminesce.
- the measuring step comprises addition of an alkaline buffer with an oxidizing reagent, which induces the compound to luminesce with a green light.
- the luminescent tag comprises luciferase
- the measuring step comprises addition of a luciferin to induce luminescence.
- the chemiluminescent compound is of the acridium ester family
- the measuring step comprises addition of a superoxide anion under neutral-alkaline conditions.
- 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).
- amount of detectable label is measured without adjusting the contents of the mixture or transferring the mixture out of the well.
- separating the measurement of label remaining in the mixture from the label sequestered onto the insert involves removing the insert to a new well.
- 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).
- the methods utilizing arrays of inserts described herein can be used for a wide variety of high-throughput, multiplexed assays.
- 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.
- the array of inserts is used for high-throughput cell screening purposes.
- the array of inserts is used to identify a cell expressing a secreted polypeptide, utilizing the methods described above.
- the secreted polypeptide is a heterologous polypeptide.
- the liquid sample or samples comprise cell culture media in which a cell suspected for expressing the polypeptide has been cultured. Apparatuses
- the invention provides apparatuses suitable for use in conducting the binding assays described herein.
- 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.
- 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).
- 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.
- 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.
- the light source is configured to provide continuous illumination.
- 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.
- 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.
- 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.
- 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).
- a light pattern generator can be configured to create any regularly shaped beamlet array of any dimension.
- 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.
- the light pattern generator provides uniform light intensity across each of the target regions.
- 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.
- 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.
- 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.
- 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.
- 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.
- the plurality of light detectors detects response light from the plurality of wells simultaneously.
- 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).
- the apparatus can optionally comprise a processor that is adapted to communicate with and process data from the plate reader.
- 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.
- 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.
- the user selects a top read, as the bottom surface of the well may provide optical interference that reduces the accuracy of the measurement.
- 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.
- 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.
- 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.
- 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).
- 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 for measuring an analyte in present in liquid samples in wells of a multi-well device 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.
- kits include a labeled tracer.
- 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.
- the binding agent is selected such that it does not have appreciable binding affinity for the analyte.
- 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.
- 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.
- 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.
- the labeled tracer does not have appreciable binding affinity for the binding agent.
- 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.
- 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.
- kits further comprise standards, e.g., standards for use in the assays of the invention.
- 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.
- 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 NaHCO 3 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.
- 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.
- FIG. 7 depicts a plot of fluorescence intensity as a function of human gamma globulin concentration.
- nylon tubes of 1 ⁇ 4" OD were purchased from Graingers
- 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.
- 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.
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US201261652102P | 2012-05-25 | 2012-05-25 | |
PCT/US2013/031047 WO2013176749A1 (en) | 2012-05-25 | 2013-03-13 | Apparatuses and methods for conducting binding assays |
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EP2856104A4 EP2856104A4 (de) | 2016-02-24 |
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Publication number | Priority date | Publication date | Assignee | Title |
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US4111754A (en) * | 1976-11-29 | 1978-09-05 | Hydow Park | Immunological testing devices and methods |
FI54842C (fi) * | 1977-01-14 | 1979-03-12 | Suovaniemi Finnpipette | Formstycke i anordningar foer immunitetsbestaemningar och enzymreaktioner |
US4680275A (en) * | 1985-02-11 | 1987-07-14 | Becton, Dickinson And Company | Homogeneous fluorescence immunoassay using a light absorbing material |
IE850830L (en) * | 1985-04-01 | 1986-10-01 | Noctech Ltd | Enzyme immunoassay method |
GB2239947A (en) * | 1989-10-27 | 1991-07-17 | Raymond Edwards | Microtitre plate well inserts |
US5376313A (en) * | 1992-03-27 | 1994-12-27 | Abbott Laboratories | Injection molding a plastic assay cuvette having low birefringence |
DE19903576C2 (de) * | 1999-01-29 | 2001-02-22 | Bodenseewerk Perkin Elmer Co | Quantitative Bestimmung von Analyten in einem heterogenen System |
DE10015448A1 (de) * | 2000-03-29 | 2001-10-11 | November Ag Molekulare Medizin | Verfahren zum Nachweisen und/oder Quantifizieren des Bindens erster Moleküle an dazu affine zweite Moleküle |
WO2003022421A2 (en) * | 2001-09-07 | 2003-03-20 | Corning Incorporated | Microcolumn-platform based array for high-throughput analysis |
WO2003031980A1 (en) * | 2001-10-10 | 2003-04-17 | Tecra International Pty Ltd | System and apparatus for use in detecting microorganisms |
US20040043398A1 (en) * | 2002-04-15 | 2004-03-04 | Demetrio Sanchez-Martinez | Use of the multipin platform as anchor device |
US7981664B1 (en) * | 2008-05-22 | 2011-07-19 | Maven Technologies, Llc | Apparatus and method for performing ligand binding assays on microarrays in multiwell plates |
-
2013
- 2013-03-13 EP EP13794009.4A patent/EP2856104A4/de not_active Withdrawn
- 2013-03-13 CN CN201380039736.3A patent/CN104520690A/zh active Pending
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