Methods for detecting an analyte

Abstract

To provide methods that enable quantitative detection of multiple analytes in a sample using a single solid phase, in view of the fact that requirements for producing separate solid phase components specific to each analyte assayed renders multi-analyte assays imprecise, expensive, technically complex, and time-consuming.SOLUTION: The present disclosure provides methods for the detection of multiple analytes using a single solid phase. The present disclosure also relates to preparation of solid phases that include receptacles having antibodies affixed thereto and directed to at least two different analytes. The methods of the present disclosure can be used, for example, for quantitative detection of analytes in a sample and the measurement thereof.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 62 / 468,114, filed March 7, 2017, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to a method for detecting and measuring an analyte in a sample. The present disclosure also relates to making a solid phase comprising a vessel with immobilized antibodies directed to at least two different analytes. The method of the present disclosure can be used, for example, for detecting an analyte in a sample and for measuring the analyte. [Background technology]

[0003] Current quantitative methods and assays, such as immunoassays, for determining the presence and amount of a specific analyte in a sample are limited to the recognition of a single antigen (analyte) per solid phase. In general, such quantitative methods are limited to the use of solid phases such as plates containing microwells or membranes with only one specific antibody immobilized thereon. See U.S. Pat. No. 5,399,433 and U.S. Pat. No. 5,499,443. Generally, the antibodies used in such assays are labeled (e.g., fluorescent, radioactive, luminescent, secondary antibody) so that they become detectable (or undetectable) upon binding to the antigen in the sample. Thus, to quantitatively detect multiple analytes in a sample, assays currently employ the use of separate solid phase components, each of which has a single antibody specific for one particular analyte immobilized thereon.

[0004] Previous attempts to utilize a single solid phase to detect multiple analytes have been directed towards the use of lateral flow assays (see, for example, US Pat. No. 5,399,623, US Pat. No. 5,499,633, US Pat. No. 5,499,663, and US Pat. No. 5,523,063). Lateral flow assays require a test sample to flow in a chromatographic manner along a bibulous or non-bibulous porous solid phase, such as a membrane. In a typical lateral flow assay, the sample is applied to the solid phase at a first location (i.e., application zone), and the sample passes through the solid phase until it reaches a second separate location (first test zone) that has a first antibody specific for the first analyte. The first test zone is then analyzed to determine the presence or amount of the first analyte in the sample. The sample must then flow to a third separate location (second test zone) on the solid phase that contains another antibody specific for another analyte. In a lateral flow assay, the second test zone is then analyzed to determine the presence and amount of the analyte in the sample. In lateral flow assays, determining the presence of an analyte requires a detectable signal at each discrete location (test zone) on the solid phase, which is read by an instrument such as a fluorometer. However, separate labels such as different wavelengths of light, different fluorescent dye labels, or differently labeled secondary antibodies are typically used to decipher the amount of each analyte detected in each zone. As such, lateral flow detection assays are expensive, technically complex, and prone to operator-based error.

[0005] Solid-phase analyte immunoassays are often capable of "detecting" the presence of multiple analytes in a sample, but only in a qualitative manner. In some instances, solid-phase analyte immunoassays include a solid phase with multiple analytes immobilized thereon and can be quantitative, but they are limited to competitive assays. For example, in solid-phase analyte immunoassays, the analyte, rather than the antibody, binds to the solid phase. The bound analyte competes with the analyte present in the sample for binding to a labeled antibody specific for the analyte of interest. In these assays, the sample (and its bound antibody) is removed in a washing step, resulting in a loss of signal compared to the signal of a solid phase containing immobilized analytes all bound to a detectable antibody (i.e., the control). The presence of the analyte in the sample is therefore inferred from a reduced signal compared to the control. As mentioned above, detection of multiple analytes using a single solid phase requires differentially labeled antibodies, and application and reapplication of the sample and specific antibodies to the solid phase. The application of multiple labeled antibodies and subsequent washing steps required by such applications renders competitive solid-phase assays inefficient, labor-intensive and expensive. Furthermore, competitive assays are susceptible to unconjugated labeled antibodies binding to "immunoreactive" polypeptide species present in the sample instead of the analyte of interest, and are therefore prone to false positives.

[0006] Solid-phase immunoassays for detecting multiple analytes by using a solid phase with multiple antibodies attached thereto are usually qualitative. For example, when a sample is provided to a solid phase incorporating multiple antibodies, each antibody specific for a different antigen of interest contacts the sample and a signal is generated if the sample contains any of the antigens of interest. However, without different distinct labels and a means to detect each distinct label, the assay is unable to decipher the multiple antigens of interest present in the sample. Moreover, the signal obtained in such an assay is a composite of the signals generated by all the different bound antigens. Thus, current methods are unable to quantitatively determine how much of a particular antigen of interest is present in a sample without using a separate solid phase for each analyte of interest.

[0007] In view of the above, the requirement to produce separate solid phase components specific to each analyte being analyzed renders multi-analyte assays imprecise, expensive, technically complex and time consuming. Therefore, there is a need for a method that allows for the quantitative detection of multiple analytes in a sample using a single solid phase. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US Patent Application Publication No. 2016 / 0289308 [Patent Document 2] US Patent Application Publication No. 2016 / 0297893 [Patent Document 3] US Patent Application Publication No. 2010 / 061377 Summary of the Invention [Means for solving the problem]

[0009] The methods provided herein use a single solid phase that contains multiple receptacles. Each receptacle has at least two antibodies (capture antibodies) immobilized thereon. Each capture antibody recognizes an antigen (analyte), and the at least two capture antibodies recognize at least two different antigens (analytes) of interest. Therefore, the methods provided herein use a single solid phase and facilitate the detection of multiple analytes. The use of a single solid phase to detect multiple analytes eliminates the costs associated with manufacturing separate solid phases for detecting each specific analyte of interest. The use of a single solid phase to detect multiple analytes in a sample requires fewer consumables (e.g., reagents and buffers) and more efficiently uses internal space within an automated assay instrument. The methods of the present disclosure also improve efficiency by simplifying known experimental procedures and reducing the amount of components required to detect multiple analytes in a sample, thereby limiting the potential for user error. In addition, the methods use labeled detection antibodies with the same detectable label. This further simplifies the detection of multiple analytes by reducing the need for different detectable labels and means to quantitatively detect multiple signals.

[0010] In a first aspect, the disclosure provides a method for detecting the presence of an analyte of interest in a sample. The method includes providing a solid phase comprising containers each coated with an antibody that binds (captures) at least two different analytes of interest, if present, in the sample. Each container contains two or more different capture antibodies immobilized on its surface, such that each of the at least two immobilized antibodies recognizes a different analyte (i.e., the at least two immobilized antibodies recognize at least two different analytes).

[0011] In some embodiments, the analyte is any polypeptide that contains an epitope or amino acid sequence of interest. In some embodiments, the polypeptide (protein) analyte can be isolated from a cell, synthetically produced, or recombinantly produced. In one embodiment, the analyte is a protein, or a fragment thereof, produced by a cell. In one embodiment, the analyte is a protein present on the outermost surface of a cell membrane. In one embodiment, a protein present on the outermost surface of a cell membrane has an antigen or epitope accessible to an antibody. In yet other embodiments, the analyte is a protein, or a fragment thereof, secreted by a cell.

[0012] In one exemplary embodiment, the analyte is insulin-like growth factor binding protein 7 (IGFBP7), a derivative, analog, or homolog thereof. In another exemplary embodiment, the analyte is a matrix metalloproteinase inhibitor, a derivative, analog, or homolog thereof. In one embodiment, the analyte is a tissue inhibitor of metalloproteinases (TIMPs).

[0013] In some embodiments, an analyte in the present disclosure comprises at least two, at least three, at least four, at least five, at least six or more different proteins or polypeptides. In certain embodiments, an analyte in the present disclosure comprises two different polypeptides or proteins. In one embodiment, the method of the present disclosure is employed to detect the presence and / or amount of TIMP2 and IGFP7 in a sample.

[0014] In some embodiments, the antibodies immobilized on the solid phase containers recognize at least two different antigens or epitopes (analytes). In certain embodiments of the method, the solid phase containers contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different antibodies immobilized on their surface. In certain embodiments, the capture antibodies bind to two different analytes of interest. In other embodiments, the capture antibodies immobilized on each solid phase container are specific for TIMP2 and IGFP7. In some embodiments, the antibodies are monoclonal antibodies specific for TIMP2 and IGFBP7. In certain embodiments, the capture antibodies immobilized on each solid phase container are mouse monoclonal antibodies specific for TIMP2 and IGFBP7. In certain embodiments, the capture antibody specific for TIMP2 is mouse monoclonal antibody 6E2.1 and the capture antibody specific for IGFBP7 is mouse monoclonal antibody 1D6.

[0015] In certain embodiments, the capture antibody can be directly or indirectly immobilized on the surface of the container. For example, the capture antibody can be directly covalently bound to the solid phase via a chemical bond between a portion of the capture antibody and a functional group on the surface of the solid phase. Alternatively, the capture antibody can be indirectly covalently bound to the solid phase by covalently binding the antibody to a linker and covalently binding the linker to the solid phase. In some embodiments, the capture antibody is non-covalently bound directly to the solid phase through non-covalent association or adsorption of the antibody to the container.

[0016] In some embodiments, the surface of the solid phase container can be coated to facilitate attachment of the capture antibody. In other embodiments, the container contains functional groups that are incorporated into the container material. In some embodiments, the container surface is coated with avidin and the capture antibody is conjugated to biotin, or vice versa. In some embodiments, after coating with polystyrene, the capture antibody is immobilized on the container surface by adsorption.

[0017] In some embodiments, the solid phase used in the method is composed of polystyrene. In certain embodiments, the container of the solid phase is composed of polystyrene. In certain embodiments, the container of the solid phase is composed of white polystyrene.

[0018] The solid phase can take a variety of forms, including, for example, a membrane, a tip, a straw, a sleeve, a slide, a column, a hollow, a solid, a semi-solid, a gel, a fiber, and a matrix.

[0019] In some embodiments, the solid phase comprises a container in two or more sealed straws or sleeves. Non-limiting examples of containers include cups, wells, tubes, capillaries, vials, and any other vessel, groove, or recess capable of holding a solution, a sample, or a portion of a sample. The container can be included in the solid phase as part of a straw, sleeve, strip, plate, slide, etc., that includes at least two containers. In some embodiments, the solid phase comprises two or more containers in a sealed straw or sleeve. In one embodiment, the solid phase for use in the method comprises a container with vertical sidewalls, the sidewalls tapering from top to bottom, and the width of the bottom of the container is smaller than the width of the top of the container.

[0020] In some embodiments of the method, the solid phase comprises a plurality of vessels. In certain embodiments, the solid phase comprises at least 2 vessels, at least 3 vessels, at least 4 vessels, at least 5 vessels, at least 6 vessels, at least 7 vessels, at least 8 vessels, at least 9 vessels, at least 10 vessels, at least 15 vessels, at least 20 vessels, at least 25 or more vessels. In embodiments, the solid phase used in the method comprises at least 2 vessels. In other embodiments, the solid phase comprises at least 2 tapered vessels.

[0021] The method further includes providing a sample or a portion of a sample to each container, whereby the sample contacts the capture antibody immobilized in each container. If the sample contains an analyte (e.g., a protein of interest or a fragment thereof) that is recognized by the immobilized capture antibody, the analyte in the sample will bind to the specific antibody present in the container.

[0022] In some embodiments, the sample may be obtained from a subject or from other materials. In some instances, the sample is generated for the purpose of determining the presence of an analyte therein. In certain embodiments, the sample for use in the present methods is a bodily fluid sample obtained from a subject, e.g., a patient. In some embodiments, samples of the present disclosure include blood, tears, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusion. In certain embodiments, the sample is a urine sample obtained from a subject, e.g., a human. In some instances, the present methods use multiple portions of a single sample.

[0023] In some embodiments, the sample is urine and the amount of urine provided to each container is between 5 μL and 100 μL, between 10 μL and 100 μL, between 20 μL and 100 μL, between 30 μL and 100 μL, or between 40 μL and 100 μL. In other embodiments, the amount of urine provided to each container is between 20 μL and 80 μL, between 25 μL and 80 μL, between 30 μL and 80 μL, between 35 μL and 80 μL, or between 40 μL and 80 μL. In yet other embodiments, the amount of urine sample provided to each container is about 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, or more. In some embodiments, the amount of urine sample provided in each container is exactly 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, or 80 μL. In certain embodiments, the amount of urine sample provided in each container is 20 μL or 35 μL. In certain embodiments, the amount of urine sample provided in each container is 80 μL.

[0024] The administration of the sample or a portion thereof to the container can be performed by an individual or by an automated device, such as an automated immunodiagnostic device. In embodiments in which the method is performed in whole or in part by an automated immunodiagnostic device, the sample is first provided to a designated reservoir and then a portion of the sample is dispensed into a solid phase container or containers provided on the automated immunodiagnostic device. In one embodiment, the method of the present disclosure is performed on one of the following automated immunodiagnostic devices: Ortho Clinical Diagnostics Vitros® ECiQ, Ortho Clinical Diagnostics Vitros® 3600, Ortho Clinical Diagnostics Vitros® 5600.

[0025] In some embodiments, a sample is provided in each container and incubated for a period of time to promote binding of the analyte of interest present in the sample to the corresponding capture antibody present in each container on the solid phase.

[0026] The disclosed method also includes providing each container with an antibody that includes a detectable element (detection antibody). The detection antibody provided in each container is specific for one of the analytes of interest. More specifically, the method includes providing an amount of a detection antibody that recognizes a first analyte of interest in a first container on the solid phase, and separately providing an amount of another detection antibody that recognizes a different analyte of interest in another container on the solid phase. The detection antibodies provided in each well all generate the same detectable signal. This aspect of the method simplifies the detection of multiple analytes by reducing the need for users to obtain detection antibodies with different detectable labels, as well as the need for a means to detect multiple different signals.

[0027] In some embodiments of the method, the detection antibodies are monoclonal antibodies, polyclonal antibodies, fragments thereof, or any combination thereof. In certain embodiments, the detection antibodies of the present disclosure are all monoclonal antibodies, each recognizing a different analyte of interest. In other embodiments, the detection antibodies of the present disclosure are all polyclonal antibodies, each recognizing a different analyte of interest. In yet other embodiments, the detection antibodies of the present disclosure are a combination of monoclonal and polyclonal antibodies, each specific for a different analyte of interest.

[0028] In some embodiments of the method, the detection antibodies are specific for TIMP2 and IGFP7, respectively. In certain embodiments, the detection antibodies of the present disclosure are monoclonal antibodies specific for TIMP2 and IGFBP7, respectively. In a specific embodiment, the detection antibody specific for TIMP2 is rabbit monoclonal antibody 40H2-40K3, and the detection antibody specific for IGFBP7 is mouse monoclonal antibody 6D2.1.

[0029] In one embodiment, the detectable label attached to the detection antibody is directly detectable, hi certain embodiments, the directly detectable label is a fluorescent moiety (dye), an electrochemical label, an electrochemiluminescent label, a metal chelate, or a colloidal metal particle.

[0030] In other embodiments, the detectable label is an indirectly detectable label, such as a molecule that becomes detectable after undergoing a chemical or enzymatic reaction, or after being bound by a molecule that itself provides a detectable signal, hi some embodiments, the detectable label is an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase.

[0031] In some embodiments, the detectable label is attached to the detection antibody. In certain embodiments, the detectable label is conjugated to the detection antibody. In certain embodiments, the detectable label is a dye or an enzyme conjugated to the respective detection antibody. In one embodiment of the method, horseradish peroxidase is used as a conjugate with each specific detection antibody.

[0032] The detection antibody may be dispensed directly into the container, or may be pre-mixed into a solution containing the desired concentration of detection antibody and provided as an aliquot of such pre-mixed solution.

[0033] In some embodiments, the amount of detection antibody provided in each container is about 0.02 μg to about 1.2 μg. In one embodiment, the amount of detection antibody provided in each container is 0.02 μg to 1.2 μg. In a specific embodiment, the amount of detection antibody provided in each container is 0.075 μg or 1.2 μg.

[0034] The administration of the detection antibody or a solution containing the detection antibody can be performed by an individual or by an automated device, such as an automated immunodiagnostic device. Methods for dispensing the detection antibody and contacting the solid phase can include placing a desired amount of the detection antibody in a manual pipette and / or a robotic or automated dispensing mechanism or container. In embodiments in which the method is performed in whole or in part by an automated immunodiagnostic device, the detection antibody is first provided in a designated reservoir, followed by dispensing an aliquot of the detection antibody into the container or containers of the solid phase provided to the automated immunodiagnostic device.

[0035] In one embodiment, the methods of the present disclosure are performed on one of the following automated immunodiagnostic devices: Ortho Clinical Diagnostics VITROS® ECiQ, Ortho Clinical Diagnostics VITROS® 3600, Ortho Clinical Diagnostics VITROS® 5600.

[0036] In some embodiments, the detection antibody is dispensed into the vessel and incubated for up to 40 minutes. In certain embodiments, the detection antibody is dispensed into the vessel and incubated for about 4, 5, 6, 7, 8, 9, or 10 minutes. In other embodiments, the detection antibody is incubated in the vessel for 8 minutes.

[0037] In certain embodiments, the sample is pre-mixed with the detection antibody prior to administration of the mixture of sample and detection antibody to the container. In one embodiment, the mixture of sample and detection antibody is incubated for 1-60 minutes, 1-50 minutes, 1-40 minutes, 1-30 minutes, 1-20 minutes, or 1-10 minutes inclusive. In another embodiment, the mixture of sample and detection antibody is incubated for 1-8 minutes inclusive.

[0038] A detectable signal is generated by the detection antibody present in each reservoir of the solid phase, which can be generated, for example, by a fluorometer that uses an excitation light source transducer that is spatially separated from the solid phase and directs excitation light into each well being analyzed to generate light of a detectable wavelength within the well that can be measured by an optical detector.

[0039] In yet other embodiments, an antibody-based biosensor can be employed to determine the presence or amount of an analyte bound to a detection antibody present in a container.

[0040] In certain embodiments, each detection antibody is conjugated to the same detectable label, such as horseradish peroxidase, and a detectable signal is generated by providing a substrate, such as a luminescent substrate, to each well, e.g., such that the HRP enzyme oxidizes the luminescent substrate to release a detectable signal (light).

[0041] In certain embodiments, the luminescent substrate (e.g., luminol, its derivatives, and persalts) is provided with an electron transfer agent (enhancer), such as a substituted acetanilide, to amplify the light signal emitted by the substrate and prolong the emission of the signal from the vessel. In some embodiments, the luminescent substrate is provided in a solution that includes an enhancer solution. In other embodiments, the luminescent substrate is provided to the vessel in a first solution and the enhancer is provided to the vessel in a second solution.

[0042] In some embodiments, the luminescent substrate and the electron transfer agent are provided to the vessel and incubated for about 1-20 minutes, 1-15 minutes, 1-10 minutes, 1-9 minutes, 1-8 minutes, 1-7 minutes, 1-6 minutes, 1-5 minutes, 1-4 minutes, 1-3 minutes, 1-2 minutes, or less than 1 minute. In certain embodiments, the luminescent substrate and the electron transfer agent are provided to the vessel and incubated for 4-5 minutes.

[0043] The methods provided herein further include detecting the presence of each analyte of interest. Generally, the detecting step is performed by measuring the amount of signal generated from each vessel into which a sample is provided.

[0044] The method can be developed for the simultaneous or sequential detection of two or more different analytes using a single solid phase with high sensitivity and minimal interference from other analytes. Generally, after applying the sample to the solid phase, the signal generated directly or indirectly from the detection antibody can be detected visually or obtained by a device (analytical instrument) such as a reflectometer, fluorometer or transmittance photometer.

[0045] In some embodiments, once a signal is generated and detected (indicating the presence of the analyte in the sample), the signal is then measured and quantified. In certain embodiments, the amount of signal measured in each vessel is quantified and provided as a single numerical value. In other embodiments, the amount of signal measured in each vessel correlates with the amount of analyte present in the sample.

[0046] In some embodiments, the detected signal or signals can be compared to signals generated after the use of a control sample in the method. Such a comparison facilitates quantification of the amount of analyte detected in the sample. In one example, a modified 4 or 5 parameter log-logistic software program, such as an Ortho Clinical Diagnostics Assay Data Disk (included in the Vitros® 3600 Immunodiagnostic System, Vitros® 5600 Integrated System), or a magnetic card (on the Vitros® ECiQ instrument) is used to fit a calibration curve for the quantification measurement.

[0047] In some embodiments, a single value can be provided that quantifies the total amount of all analytes of interest present in the sample. In one embodiment, the amount of a first analyte detected in a first container is multiplied by the amount of a second analyte detected in a second container, and then the total is divided by 1000.

[0048] In certain aspects of the present disclosure, kits are provided for carrying out the methods described herein. Suitable kits include sufficient reagents to carry out the methods of the present disclosure, together with instructions for carrying out the described methods. Additional optional elements that may be provided as part of the assay kit are described herein.

[0049] In certain embodiments, reagents for carrying out the subject methods are provided in a kit, including one or more of the following: a solid phase, an antibody, a buffer solution, a luminescent substrate, an electron transfer agent, and instructions for carrying out the disclosed method.

[0050] The kit comprises a solid phase having a plurality of containers coated with at least two capture antibodies, in certain embodiments, each container of the solid phase provided in the kit has at least two different capture antibodies immobilized thereon, whereby each of the at least two different capture antibodies recognizes a different antigen or epitope (analyte).

[0051] In some embodiments, the solid phase provided in the kit comprises a plurality of tapered vessels, such as VITROS® microwells. In another embodiment, a solid phase is provided comprising a straw or sleeve of at least 20 VITROS® microwells. In another embodiment, a solid phase is provided comprising a straw or sleeve of at least 25 VITROS® microwells. In a particular embodiment, the solid phase comprises 25 VITROS® microwells within a sealed straw.

[0052] In one embodiment, the kit of the present disclosure includes four solid phases, each of which includes at least 10 tapered vessel straws or sleeves. In another embodiment, the kit of the present disclosure includes four solid phases, each of which includes at least 20 tapered vessel straws or sleeves. In yet another embodiment, the kit of the present disclosure includes four solid phases, each of which includes at least 25 tapered vessel straws or sleeves. In one embodiment, the kit of the present disclosure includes four solid phases, each of which includes 25 Vitros® microwell straws or sleeves.

[0053] In some embodiments, the kit comprises at least two different detection antibodies, whereby each of the at least two different detection antibodies recognizes a different analyte of interest. In some embodiments, each of the at least two different detection antibodies comprises a detectable label that is the same or that generates the same detectable signal.

[0054] In some embodiments, the detection antibodies are monoclonal antibodies, polyclonal antibodies, fragments thereof, or any combination thereof. In certain embodiments, the detection antibodies are all monoclonal antibodies, each recognizing a different analyte of interest. In other embodiments, the detection antibodies are all polyclonal antibodies, each recognizing a different analyte of interest. In yet other embodiments, the detection antibodies provided include a combination of monoclonal and polyclonal antibodies, each specific for a different analyte of interest.

[0055] In some embodiments, the kit comprises an antibody specific for TIMP2 and IGFP7, respectively. In certain embodiments, the detection antibodies of the present disclosure are monoclonal antibodies specific for TIMP2 and IGFBP7, respectively. In a particular embodiment, the detection antibody specific for TIMP2 is monoclonal antibody 40H2-40K3, and the detection antibody specific for IGFBP7 is monoclonal antibody 6D2.1.

[0056] In certain embodiments, the kit comprises at least two different detection antibodies, each of which recognizes a different analyte of interest and is conjugated to a detectable label. In one embodiment, the kit comprises at least two different detection antibodies, each of which recognizes a different analyte of interest and is conjugated to horseradish peroxidase (HRP).

[0057] In some embodiments of the present disclosure, the kit includes a substrate, such as a luminescent substrate. The luminescent substrate used in the present method and kit is known to those skilled in the art as an enhancer thereof. Therefore, the specific combination or combinations of enzyme, substrate and enhancer are not intended to be limiting. In some embodiments, the luminescent substrate provided in the kit is luminol or its derivative and a persalt salt. In some embodiments, the kit includes an electron transfer agent (enhancer), such as a substituted acetanilide, which can amplify the signal generated from the detectable label.

[0058] In some embodiments, the kits of the present disclosure include a reference solution (calibration solution) containing a known amount of a particular analyte of interest. In one embodiment, the reference solution can contain a known amount, or a plurality of known amounts, of at least two analytes of interest. In another embodiment, a reference solution can be provided for each corresponding analyte to be detected by such a kit.

[0059] In certain embodiments, the kits of the present disclosure include one or more solutions or buffers. For example, the kits may contain one or more of the following: phosphate buffer, detection antibody solution (e.g., TIMP2 detection antibody conjugate solution, IGFBP7 detection antibody conjugate solution), and water (e.g., deionized or sterile). [Brief description of the drawings]

[0060] [Figure 1] 1 is a schematic diagram of an exemplary solid phase container of the present disclosure. The solid phase shown includes a container with two different capture antibodies immobilized (bound) to the solid phase. Each capture antibody is specific for a different analyte, or antigen (analyte 1, analyte 2), either of which may or may not be present in the sample. The schematic shows the ability of the capture antibodies to bind to a particular antibody present in a sample when the sample is provided to the container, and further shows the formation of a capture antibody-analyte-detection antibody complex after further introducing a detection antibody of the present disclosure using the method. [Diagram 2] Schematic diagram of an exemplary embodiment of the present method for detecting multiple analytes in a sample. The schematic diagram shows a method for detecting a first and a second analyte in a sample, which includes providing a solid phase holding at least two containers, each having two different capture antibodies immobilized on the surface of each container. The method shown shows that when a sample is provided to each container, a specific detection antibody is also provided, which has a detectable label attached thereto. Although the labels attached to each detection antibody shown are the same, the detection antibody provided in container 1 recognizes only the first analyte, while the detection antibody provided in container 2 recognizes a second analyte different from the first analyte. The amount of each analyte present in the sample is detected by measuring a detectable signal generated by the detection antibody present in each well. This allows the measured amount of each of the two analytes of interest in the sample to be quantified. Here, the amount of the first analyte present in the sample is directly proportional to the amount of signal detected in container 1, and the amount of the second analyte present in the sample is directly proportional to the amount of signal detected in the second container. [Diagram 3]Schematic diagram of a second exemplary embodiment of the present method for detecting multiple analytes in a sample. The method shown illustrates the detection of two analytes (TIMP2, IGFBP7) using a solid phase with tapered vessels (vessel 1, vessel 2), each vessel having a capture antibody specific for TIMP2 and a capture antibody specific for IGFBP7 immobilized on its inner surface. The sample is poured into vessel 1, and a TIMP2-horseradish peroxidase (HRP)-conjugated detection antibody is poured into vessel 1. The sample is poured into vessel 2, and an IGFBP7-HRP-conjugated detection antibody is poured into vessel 2. The contents of vessels 1 and 2 are incubated at 37° C., and each vessel is washed. The schematic diagram also illustrates an embodiment of the present method, whereby after the formation of a capture antibody-analyte-detection antibody complex in each vessel, a signal reagent is provided to each vessel to promote the release of a detectable signal in the well. Here, a signal reagent containing a luminescent substrate and a signal enhancer is added to each vessel, light emission (luminescence) is detected from each vessel, and the amount of light emission from each vessel is measured using a luminometer. The amount of light signal detected in each vessel is directly proportional to the concentration of TIMP2 and IGFBP7 present in the sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The methods provided herein use a single solid phase that includes multiple containers, each of which has at least two antibodies immobilized thereon. The antibodies, i.e., capture antibodies, recognize at least two different analytes of interest. Thus, the methods provided herein facilitate the detection and measurement of multiple analytes using a single solid phase. The use of a single solid phase to detect multiple analytes eliminates the costs associated with producing separate solid phases to quantitatively detect each specific analyte of interest. The methods of the present disclosure also improve efficiency by simplifying the assay and reducing the amount of components required to detect multiple analytes in a sample, thereby limiting the potential for user error. In addition, the methods use labeled detection antibodies with the same detectable signal. This further simplifies the quantitative detection of multiple analytes by reducing the need for different detectable labels and means to detect multiple signals.

[0062] In a first aspect, the disclosure provides a method for detecting the presence of an analyte of interest in a sample. The method includes providing a solid phase including a plurality of containers, each coated with an antibody, such that when contacted with a sample or a portion of a sample, the antibody present in the sample binds (captures) the analyte of interest. Each container includes at least two different antibodies immobilized on its surface, whereby the at least two immobilized antibodies recognize different analytes. A sample is provided to each container, such that the sample or a portion of the sample comes into contact with the capture antibody immobilized in each container. If the sample includes an analyte recognized by the immobilized antibody (e.g., a protein of interest, or a fragment thereof, or a receptor of interest), the analyte in the sample will bind to the corresponding capture antibody present in the container. The method of the disclosure also includes providing an antibody that includes a detectable label (detection antibody) to each container. The detection antibody provided in each container is specific for one of the analytes of interest. However, each specific detection antibody provided generates the same detectable signal. The method provided herein also includes detecting the presence of each analyte of interest.

[0063] The term "analyte" or "analytes" as used herein is any molecule (e.g., an antigen, another antibody, or a portion thereof) that can be recognized by an antibody. In some embodiments, the analyte is a polypeptide. A "polypeptide," as also used herein, is a single polymeric chain of amino acids that are bound together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The term "protein" includes polypeptides. The term "protein" may also be used to describe polypeptides that have multiple domains, such as beta sheets, linkers, and alpha helices. Thus, the term "protein" is also meant to include polypeptides with quaternary structure, tertiary structure, and other complex macromolecules composed of at least one polypeptide. When a protein is composed of multiple polypeptides that are physically associated with each other, the term "protein" as used herein refers to multiple polypeptides that are physically associated and function together as a separate unit. In embodiments where the analyte is another antibody, the analyte-antibody is any antibody that is different from the capture antibody and is capable of binding to the capture antibody.

[0064] In embodiments of the method, the analyte is any polypeptide that contains an epitope or amino acid sequence of interest. Such polypeptides can be present in a bodily fluid or solid sample. In some examples, the analyte is a polypeptide or antibody present in, present within, or produced by an organism (e.g., a mammal, a bacterium, a virus). In some embodiments, the analyte is a polypeptide or antibody present in, present within, or produced by a cell. In yet other embodiments, the analyte is a synthetically or recombinantly produced polypeptide or antibody using methods known to those of skill in the art.

[0065] For example, the analytes may be prepared using solid phase synthesis techniques for the production of polypeptides, first described by Merrifield, J. Am. Chem. Soc. (1963) 85 pp. 2149-2154. Other polypeptide synthesis techniques can be found in M. Bodanszky, et al. Peptide Synthesis, John Wiley & Sons, 2d Ed., (1976), and other references readily available to those skilled in the art. A summary of polypeptide synthesis techniques can also be found in J. Stuart and JD Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, Ill., (1984). Analytes can also be synthesized by solution methods, as described in The Proteins, Vol. II. 3d Ed., Neurath, H. et al., Eds., pp. 105-237, Academic Press, New York, NY (1976). Suitable protecting groups for use in the synthesis of different peptides are described in the above texts as well as in J. F. W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, NY (1973). The analytes of the present disclosure can be prepared by chemical or enzymatic cleavage from a larger portion of the protein or polypeptide.

[0066] In some embodiments, the analyte of the method is a protein or antibody produced by a cell, or a fragment thereof. In some embodiments, the analyte is a protein present on the outermost surface of a cell membrane. In one embodiment, the protein present on the outermost surface of a cell membrane has an antigen or epitope accessible to an antibody. In yet another embodiment, the analyte is a protein or a fragment thereof secreted by a cell.

[0067] The format / approach disclosed herein by immobilizing multiple antibodies to capture and detect multiple antigens in a sample can be designed in a reverse format. Thus, in certain embodiments where the analyte to be detected in the sample is an antibody or a fragment thereof, a solid phase can be provided that includes containers each coated with multiple antigens (e.g., peptides) that, when contacted with the sample or a portion thereof, bind (capture) the antibody of interest (i.e., the analyte) in the sample, if present. For example, each container includes at least two different antigens or peptides bound to its surface, whereby each of the at least two immobilized antigens recognizes a different antibody analyte. A sample is provided to each container, such that the sample or a portion of the sample comes into contact with the antigens immobilized in each container. If the sample contains an analyte (e.g., an antibody of interest, or a fragment thereof) recognized by the immobilized antigen, the antibody in the sample binds to the corresponding capture agent (i.e., antigen) present in the container. Such a method also includes providing an antibody with a detectable label (detection antibody) to each container. The detection antibody provided in each container is specific for one of the analytes (antigens) of interest or a portion thereof, however, each specific detection antibody provided produces the same detectable signal.

[0068] Additionally, the peptides (analytes) of the present disclosure may be prepared by recombinant techniques known to those of skill in the art. See, for example, Current Protocols in Molecular Cloning Ausubel et al., 1995, John Wiley & Sons, New York), Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, New York, and Coligan et al. (1994) Current Protocols in Immunology, John Wiley & Sons Inc., New York, NY. Those of skill in the art will appreciate that any of a wide variety of expression systems can be used to provide the recombinant peptides. The precise host cell used is not critical to the method. However, by way of example, the analytes of the present disclosure can be produced in prokaryotic hosts (e.g., E. coli), in eukaryotic hosts (e.g., S. cerevisiae), or in mammalian cells such as COS1, CHO, NIH3T3 and JEG3 cells, or in arthropod cells such as S. frugiperda.

[0069] In certain exemplary embodiments, the analyte is insulin-like growth factor binding protein 7 (IGFBP7), a derivative, analog, or homolog thereof. In certain embodiments, IGFBP7 is any polypeptide derived from a precursor of insulin-like growth factor binding protein 7, e.g., as set forth in Accession Nos. NP_001240764.1, AAH66339, AAR89912, or AAP35300. In other embodiments, the analyte is any polypeptide fragment of the IGFBP7 protein that contains an epitope that can be recognized by an antibody.

[0070] In another exemplary embodiment, the analyte is an inhibitor of matrix metalloprotease, a derivative, analog, or homolog thereof. In one embodiment, the analyte is a tissue inhibitor of metalloprotease (TIMP). In one embodiment, the TIMP analyte is any polypeptide derived from metalloprotease inhibitor 2 (TIMP2), e.g., as set forth in Accession Nos. NP_003246.1, NP_035724.2, DAA18186.1, or NP_068824.1. In another embodiment, the analyte is any polypeptide fragment of the TIMP2 protein that contains an epitope recognizable by an antibody.

[0071] The term "homolog" refers to the corresponding polypeptide of another vertebrate species that is substantially homologous, for example, in human, rat, mouse, rabbit, bovine, canine, or avian amino acid sequence. The term "analog" is meant to encompass polypeptides that differ only by one or more amino acids, such as polypeptides that contain one or more amino acid substitutions, one or more additions, or one or more deletions that do not abolish the ability of an antibody to recognize the polypeptide.

[0072] In some embodiments, the analytes of the present disclosure are at least two, at least three, at least four, at least five, at least six or more different proteins or polypeptides. In certain embodiments, the analytes of the present disclosure include at least two different polypeptides or proteins. In one embodiment, the analytes of the present disclosure include exactly two different polypeptides or proteins. In another embodiment, the method of the present disclosure is employed to detect the presence or amount of TIMP2 and IGFBP7 (analytes) in a sample.

[0073] In other embodiments, the analyte to be detected by the method is selected from a non-limiting list of the following polypeptides, prostate specific antigen (PSA), molecules that form a complex with PSA (e.g., α2-antichymotrypsin, α1-protease inhibitor (API) or α2 macroglobulin), amyloid-β peptides (Aβ, βAP, AβP, or β / A4), β-amyloid precursor protein (APP), tau microtubule protein, phospho-tau (e.g., tau protein phosphorylated at amino acid 181), their isoforms, their homologs, their variants, or portions thereof. In some embodiments, the methods of the present disclosure are employed to detect the presence and amount of the following analyte pairs: PSA and complexed PSA, Aβ (or a variant or portion thereof) and tau, and tau and phospho-tau.

[0074] Once the analytes of interest are determined, antibodies specific for each analyte are selected for use in the methods of the present disclosure.

[0075] The term "antibody" as used herein refers to an immunoglobulin molecule encoded by one or more immunoglobulin genes or derivatives thereof, which has the ability to bind to a specific antigen or epitope. See, for example, Fundamental Immunology, 3rd Edition, WE Paul, ed., Raven Press, NY (1993); Wilson et al., J. Immunol. Methods (1994) 175:267-273. As an example, it has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody, i.e., an "antigen-binding fragment" or "antigen-binding portion", and therefore various regions of the heavy and light chains of an immunoglobulin molecule contain binding domains (antigen-binding portions) that interact with and bind to antigens. Examples of antigen-binding portions or antigen-binding fragments that may be included within the term "antibody" include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CHI domains, (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of the VH domain (see Ward et al., Nature (1989) 341 pp. 544-546), and (vi) an isolated complementarity determining region (CDR). As with an intact antibody molecule, an antigen-binding portion may be monospecific or polyspecific (e.g., bispecific). Single chain antibodies are also included within the term "antibody". The term antibody also includes monoclonal and polyclonal antibodies.

[0076] Thus, in some embodiments, the antibodies of the present disclosure can be monoclonal, polyclonal, fragments thereof, and any combination thereof. In certain embodiments, the antibodies of the present disclosure are all monoclonal antibodies, each recognizing an analyte of interest. In other embodiments, the antibodies of the present disclosure are all polyclonal antibodies, each recognizing an analyte of interest. In yet other embodiments, the antibodies of the present disclosure are a combination of monoclonal and polyclonal antibodies, each specific for a different analyte of interest.

[0077] In some embodiments, the antibodies of the disclosure comprise at least 2, at least 3, at least 4, at least 5, at least 6 or more different antibodies. In certain embodiments of the methods, the solid phase comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different antibodies that recognize different analytes. In some embodiments, the solid phase comprises two or more different antibodies specific for only two different analytes. In certain embodiments, the solid phase comprises two different antibodies, each recognizing two different antigens or epitopes (analytes).

[0078] In another embodiment, the antibody of the disclosure is specific for TIMP2 and IGFP7, respectively. In some embodiments, the antibody of the disclosure is a monoclonal antibody specific for TIMP2 and IGFBP7, respectively. In other embodiments, the antibody is a mouse monoclonal antibody, a human monoclonal antibody, or a rabbit monoclonal antibody. In some embodiments, the antibody is a mouse or rabbit monoclonal antibody specific for TIMP2 and IGFBP7. In a particular embodiment, the antibody specific for TIMP2 is the mouse monoclonal antibody 6E2.1, and the antibody specific for IGFB7 is the mouse monoclonal antibody 1D6.

[0079] In other embodiments, the antibody used in the method is any of the following antibodies: any monoclonal or polyclonal antibody that specifically recognizes tau (e.g., HT7 and AT270 monoclonal antibodies, antibodies that recognize normally and abnormally phosphorylated tau (e.g., Alz50 (Ghanbari et al., 1990), HT7 (Mercken et al., 1992), and AT120 (Vandermeeren et al., 1993), any antibody that recognizes PSA or its binding partner (e.g., 2E9, 2H11 and 5A10, which are described in U.S. Pat. No. 5,501,983, the entire contents of which are incorporated herein by reference), any antibody that binds to Aβ or a portion thereof, which are described in U.S. Pat. No. 7,700,309, the entire contents of which are incorporated herein by reference, and other antibodies known to those skilled in the art, for example, which are disclosed in U.S. Pat. No. 9,174,097, the entire contents of which are incorporated herein by reference.

[0080] In general, the antibodies used in the present method are divided into two categories: (i) capture antibodies and (ii) detection antibodies, regardless of the analyte they recognize. As used herein, the term "capture antibody" or "capture antibodies" refers to an antibody that can be attached (immobilized) to a portion or surface of a solid-phase container and can bind (capture) the analyte of interest when it comes into contact with a corresponding epitope present on or in the analyte. For example, a detection antibody is attached to a solid-phase container, such that the antigen-binding portion of the antibody is presented in the container in such a way that it can bind to the antigen, if present.

[0081] The term "detection antibody" or "detection antibodies" as used herein refers to an antibody that is capable of binding an analyte of interest when contacted with an epitope present on or in the analyte and to which a detectable label is attached (e.g., conjugated or linked). "Detectable label" or "detection element" are used interchangeably herein and refer to any molecule or molecules that are directly detectable (e.g., fluorescent moieties, electrochemical labels, electrochemiluminescent labels, metal chelates, colloidal metal particles) and any molecule or molecules that can be indirectly detected by the production of a detectable reaction product (e.g., an enzyme such as horseradish peroxidase, alkaline phosphatase, etc.), a molecule or molecules that can be detected by recognition of a molecule that specifically binds to the detection antibody, such as a labeled antibody that binds to the detection antibody, biotin, digoxigenin, maltose, oligohistidine, 2,4-dinitrobenzene, phenylarsenate, nucleic acids (e.g., ssDNA, dsDNA), etc. In a particular example, the detectable label is horseradish peroxidase.

[0082] The capture antibody used in the method is provided on the surface of a solid-phase container. In one embodiment, a first portion of the capture antibody of the present disclosure is attached to the surface of the container, such that the antigen-binding portion of the capture antibody is positioned such that it can contact the antigen presented to the antibody.

[0083] The capture antibody can be attached to the container present on or within the solid phase in many ways known to those of skill in the art. For example, the capture antibody can be covalently or non-covalently bound to the container. In some instances, the capture antibody can be attached to the surface of the container either directly or indirectly. For example, the capture antibody can be covalently bound directly to the container through a chemical bond between a portion of the capture antibody and a functional group on the surface of the solid phase container. Alternatively, the capture antibody can be covalently bound indirectly to the solid phase container by covalently binding the antibody to a linker, which in turn is covalently bound to the solid phase container. In some instances, the capture antibody is non-covalently bound directly to the solid phase container through non-covalent association or adsorption of the antibody to the solid phase container. In other instances, the capture antibody is non-covalently bound indirectly to the solid phase container by covalently binding the capture antibody to a linker or other intermediate agent, which forms a non-covalent bond with the solid phase container. In all cases, the association of the capture antibody with the container should expose the antigen-binding portion of the antibody and immobilize the capture antibody to the solid phase container or portion thereof in a manner that does not affect or limit the specificity of the capture antibody, i.e., does not reduce the ability of the capture antibody to bind the analyte of interest when presented to the antibody.

[0084] A variety of chemical reactions useful for covalently attaching an antibody to a solid phase container are well known to those skilled in the art. Illustrative examples of functional groups useful for covalently attaching a capture antibody to a container include alkyl, Si-OH, carboxy, carbonyl, hydroxyl, amide, amine, amino, ether, ester, epoxide, cyanate, isocyanate, thiocyanate, sulfhydryl, disulfide, oxide, diazo, iodo, sulfone, or similar groups with chemical or potential chemical reactivity. In certain embodiments, the capture antibody of the present disclosure is directly immobilized to the container by adsorption.

[0085] For example, the antibody can be non-covalently associated with the solid phase container by adsorption or coating to the container, or by covalent or non-covalent association to a linker or binder that is itself non-covalently bound or immobilized to the container. Illustrative examples of linkers or binders useful for binding antibodies to solid phases include proteins, organic polymers (e.g., PEG and its derivatives), and small molecules. More specific examples of linkers useful for immobilizing the antibodies of the present disclosure to solid phases include human serum albumin (HAS), bovine serum albumin (BSA), streptavidin, avidin, biotin, PEG, and antibodies or antibody fragments.

[0086] In one non-limiting example, the capture antibody can be covalently conjugated to a binding agent such as HSA or BSA, and the resulting conjugate can be used to coat the container. In another embodiment, the antibody can be covalently conjugated to one of streptavidin, biotin, or avidin, and the conjugated antibody can be bound to a different streptavidin, biotin, or avidin molecule immobilized on the container.

[0087] In some embodiments, the surface of the solid phase container can be modified to facilitate stable attachment (immobilization) of the capture antibody to the container. In general, one skilled in the art can use conventional methods to modify the container in such a way as to facilitate the immobilization of the antibody to the container surface. The following are non-limiting examples of applicable modifications:

[0088] To facilitate attachment of the antibody, the surface of the solid phase vessel can be coated. Generally, the coating is complementary to a portion of the antibody. The vessel surface can be amidated, for example, by silylation of the surface with trialkoxyaminosilanes. Silane-treated vessels can also be derivatized with homobifunctional and heterobifunctional linkers. The vessel surface can be derivatized so that the vessel contains hydroxyl, amino (e.g., alkylamine), carboxyl, N-hydroxy-succinimidyl ester, photoactivatable groups, sulfhydryl, ketone, or other functional groups available for reaction. Examples of molecules useful for non-covalent attachment of antibodies to solid phase vessels include, but are not limited to, agents capable of binding antibodies, such as Staphylococcus aureus protein A or protein G.

[0089] In other embodiments, the solid phase container comprises functional groups that are incorporated into the material of the container. Illustrative functional groups useful for covalently attaching antibodies to the container include alkyl, Si-OH, carboxy, carbonyl, hydroxyl, amide, amine, amino, ether, ester, epoxide, cyanate, isocyanate, thiocyanate, sulfhydryl, disulfide, oxide, diazo, iodo, sulfone, or similar groups that are chemically reactive or potentially chemically reactive.

[0090] In an exemplary embodiment, as shown in Figure 1, the capture antibody is immobilized by adsorption onto the surface of a solid phase reservoir. More specifically, a solid phase having multiple reservoirs is each coated with polystyrene and irradiated.

[0091] The appropriate amount of irradiation can be determined by one of skill in the art such that the amount of radiation provided to the solid phase maximizes the adsorption of the capture antibody. In certain embodiments, the container of the solid phase can be irradiated to between 1.0 MRad and 3.5 MRad inclusive. In other embodiments, the container is irradiated to about 1.0 MRad, about 1.5 MRad, about 2.0 MRad, about 2.5 MRad, about 3.0 MRad, or about 3.5 MRad. In certain embodiments, the container was irradiated to about 1 MRad. Containers irradiated to about 1 MRad demonstrated improved ability to immobilize monoclonal capture antibodies to irradiated surfaces.

[0092] The capture antibody coating solution, which may include, for example, phosphate, sodium chloride, and a buffer, at an optimized pH (e.g., a pH of about 5.5-9.0, more specifically a pH of about 6.0-7.0) to prevent denaturation of the antibody, and at least two different capture antibodies are dispensed into each of the containers of the solid phase such that the inner surface of each container is in contact with the antibody coating solution. The solid phase containers are then incubated to promote adsorption of the capture antibodies in the solution. The solid phase containers are then washed to remove unattached antibodies from the containers. Washing can be performed one, two, three, four or more times. However, minimizing the amount of washing reduces the time and cost of producing the solid phase. In some embodiments, two washes of the solid phase are sufficient to remove all excess antibodies from the containers. For example, a post-coating solution including Tris / HCL, sucrose, NaCl, and serum albumin is dispensed into each container such that the post-coating solution contacts the antibody-coated surface of each container. After incubation with the post-coating solution, the post-coating solution is removed, such as by aspiration, and the container is dried and stored for future use. As shown in Figure 1, the above process results in multiple capture antibodies being immobilized on the surface of the solid-phase container, such that the antibodies are oriented with their antigen-binding regions exposed.

[0093] The term "solid phase" as used herein refers to any solid or semi-solid material into which two or more containers can be incorporated. By way of example, the solid phase provides materials to which the containers can be attached and dispensed from. Suitable solid phase materials are known in the art. The solid phase can be composed of a single material or a variety of materials, including, but not limited to, natural or synthetic polymers, resins, metals, silicates, or combinations thereof, so long as the material or combination of materials within the solid phase does not inhibit the attachment or incorporation of the containers or interfere with the steps of the methods provided herein.

[0094] A non-exhaustive list of materials suitable as the solid phase includes agarose, celluloses such as carboxymethylcellulose, dextran such as Sephadex®, polyacrylamides, polystyrenes, polyethylene glycols, resins, silicates, divinylbenzenes, methacrylates, polymethacrylates, glass, ceramics, paper, metals, metalloids, polyacryloylmorpholidse, polyamides, poly(tetrafluoroethylene), polyethylene, polypropylene, poly(4-methylbutene), poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene fluoride (PVDF), silicones, polyformaldehyde, cellulose acetate, nitrocellulose, or a combination of two or more of any of the foregoing.

[0095] In some embodiments, the solid phase used in the method is composed of polystyrene.

[0096] The solid phase can have a variety of shapes and can include, for example, membranes, chips, plates, straws, sleeves, slides, columns, hollow, solid, semi-solid, holes or depressions containing particles such as beads, gels, fibers containing fiber optic material, and matrices. In some embodiments, the solid phase comprises a chip, plate, straw, sleeve, slide, column, or matrix that contains multiple reservoirs. In some embodiments, the solid phase comprises a straw or sleeve that contains at least two reservoirs.

[0097] The term "receptacle" or "receptacles" is used to define a subset of a solid phase that can receive and contain a large volume of a solution, sample, or other material. Non-limiting examples of receptacles include cups, wells, tubes, capillaries, vials, and other vessels, channels, or depressions that can receive a solution, sample, or a portion thereof. A receptacle can be contained within a solid phase, such as part of a straw, sleeve, strip, plate, slide, column, matrix, etc. Specifically, a plurality of receptacles can be contained within or on a solid phase, such as a straw or strip containing a cup or well, a multi-well plate, a microwell plate, etc., that can be used in an automated immunodiagnostic device.

[0098] In one embodiment, the solid phase used in the method comprises a vessel with vertical side walls tapering from the apex to the bottom, such that the bottom of the vessel has a width narrower than the width of the top of the vessel. For example, the tapered vessel is cone-shaped or cup-shaped. Tapered vessels, such as those shown in FIG. 2, improve the efficiency of the method, for example, by reducing the incubation period and the amount of materials (e.g., solutions, antibodies, samples) used throughout the method. In certain embodiments, the solid phase used in the method comprises a plurality of Vitros® microwells.

[0099] In some embodiments, the solid phase comprises a plurality of vessels. In certain embodiments, the solid phase comprises at least 2 vessels, at least 3 vessels, at least 4 vessels, at least 5 vessels, at least 6 vessels, at least 7 vessels, at least 8 vessels, at least 9 vessels, at least 10 vessels, at least 15 vessels, at least 20 vessels, at least 25 or more vessels. In embodiments, the solid phase used in the methods comprises at least two vessels. In embodiments, the solid phase comprises at least two tapered vessels. In yet another embodiment, the solid phase comprises at least two Vitros® microwell straws or sleeves.

[0100] In certain embodiments of the method, the solid phase comprises between 2-100 vessels, between 2-90 vessels, between 2-80 vessels, between 2-70 vessels, between 2-50 vessels, between 2-60 vessels, between 2-50 vessels, between 2-40 vessels, between 2-30 vessels, between 2-20 vessels, between 2-10 vessels (inclusive). In some embodiments, the solid phase comprises between 2-100, 2-75, 2-50, 2-25, 2-15, 2-10, or 2-5 vessels. In other embodiments, the solid phase is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, The solid phase may comprise 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 vessels. In one embodiment, the solid phase comprises 25 tapered vessels. In a particular embodiment, the solid phase comprises 25 vessels within a straw or sleeve. In a particular embodiment, the solid phase comprises 25 VITROS® microwells within a sealed straw.

[0101] As described herein, depending on the number of analytes to be detected by the method, two or more capture antibodies are bound to each container surface of the solid phase carrier. In some embodiments, each container of the solid phase has at least two, at least three, at least four, at least five, at least six or more different capture antibodies immobilized thereon. In some embodiments of the method, each container of the solid phase has 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different capture antibodies immobilized thereon. In certain embodiments, each container of the solid phase has at least two different capture antibodies immobilized thereon, whereby the at least two different capture antibodies recognize different antigens or epitopes (analytes). In an embodiment, as illustrated in Figures 1 and 2, each container of the solid phase has two exactly different types of capture antibodies immobilized on its surface, whereby each type of capture antibody recognizes a different analyte.

[0102] In other embodiments, the capture antibody used in the method is any of the following antibodies: any monoclonal or polyclonal antibody that specifically recognizes tau (e.g., HT7 and AT270 monoclonal antibodies, antibodies that recognize normally and abnormally phosphorylated tau (e.g., Alz50 (Ghanbari et al., 1990), HT7 (Mercken et al., 1992), and AT120 (Vandermeeren et al., 1993), any antibody that recognizes PSA or its binding partner (e.g., 2E9, 2H11 and 5A10, which are described in U.S. Pat. No. 5,501,983, the entire contents of which are incorporated herein by reference), any antibody that binds to Aβ or a portion thereof, which are described in U.S. Pat. No. 7,700,309, the entire contents of which are incorporated herein by reference, and other antibodies known to those skilled in the art, for example, which are disclosed in U.S. Pat. No. 9,174,097, the entire contents of which are incorporated herein by reference.

[0103] In some embodiments, at least one capture antibody immobilized on a solid phase is a monoclonal antibody, a polyclonal antibody, or a fragment thereof. In some embodiments, the capture antibody immobilized on a solid phase is a polyclonal antibody or a fragment thereof. In other embodiments, the capture antibody is a monoclonal antibody.

[0104] In an exemplary embodiment shown in FIG. 1, each container has a plurality of capture antibodies that recognize a first analyte and a plurality of capture antibodies that recognize a second analyte. In a particular embodiment as shown in FIG. 3, each container has at least one capture antibody that recognizes a TIMP2 analyte and at least one capture antibody that binds an IGFBP7 analyte, which are immobilized on the surface of the container. In one embodiment, the TIMP2 and IGFBP7 capture antibodies are monoclonal antibodies or fragments thereof. In one embodiment, the capture antibodies immobilized on each container of the solid phase are mouse monoclonal antibodies specific for TIMP2 and IGFBP7. In a particular embodiment, the capture antibody specific for TIMP2 is mouse monoclonal antibody 6E2.1 and the capture antibody specific for IGFBP7 is mouse monoclonal antibody 1D6.

[0105] In one embodiment of the method, the sample or a portion thereof is provided to a solid phase comprising a plurality of reservoirs therein, each reservoir of the solid phase comprising at least two capture antibodies each capable of binding to a different analyte.

[0106] Regardless of the number of analytes or the method for obtaining the analytes of interest, the analytes are provided in or contained within a sample and can be easily applied to a solid phase. In some embodiments, the sample may be obtained from a subject or from other materials. As used herein, the term "subject" refers to a human or non-human organism. Thus, the methods described herein are applicable to both humans and various fields. Furthermore, while the subject is preferably a living organism, the methods described herein may also be used for post-mortem analysis. A human subject can be a "patient," and as used herein, a patient refers to a living human who is or may be receiving medical care for a disease or condition.

[0107] In some instances, samples are created for the purpose of determining the presence of a particular analyte therein. For example, samples may be obtained from cell cultures, body fluids, or tissues known to contain or not contain the analyte of interest. In other instances, samples are created by adding synthetic or recombinantly produced peptides to a solution that is easy to store and dispense.

[0108] In certain embodiments, the sample used in the method is a bodily fluid sample obtained from a subject, such as a patient. In some embodiments, samples of the present disclosure include blood, tears, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusion. Those skilled in the art will appreciate that some samples are easily analyzed after processing, such as fractionation or purification, such as fractionation of whole blood obtained from a subject into serum and / or plasma components. Thus, the sample can be used as is or can be processed to provide a final sample for detecting the analyte. For example, the sample can be liquefied, concentrated, dried, diluted, lyophilized, extracted, fractionated, chromatographically processed, purified, acidified, reduced, digested, enzymatically treated, or processed in a manner known to those skilled in the art to release the analyte of interest. If desired, the sample can be a combination (pool) of samples, such as from an individual or a manufacturing process.

[0109] The sample can be in various physical states, such as liquid, solid, emulsion or gel. The sample can be treated with usual care to preserve the integrity of the sample. Treatment can include the use of appropriate buffers and / or inhibitors, such as inhibitors of certain biological enzymes. The skilled artisan can determine the appropriate conditions, taking into account the analyte of interest and the nature of the sample.

[0110] In certain embodiments, the sample analyzed is a urine sample obtained from a subject. In certain embodiments, the sample analyzed is a human urine sample obtained from a subject.

[0111] In some embodiments, the methods use multiple portions of a single sample. For example, a sample (e.g., blood, urine, or other bodily fluid) is obtained from a subject as an initial volume. The initial sample volume can then be separated into one or more aliquots, whereby each separate aliquot can be processed, treated, stored, and / or analyzed using the methods disclosed herein.

[0112] Regardless of the type of sample used in the method, an amount of sample is provided to each container of the solid phase so that the sample comes into contact with the capture antibody immobilized on the surface of each container. As shown in FIG. 2, solid phase containers 1 and 2 contain a first capture antibody and a second capture antibody attached (immobilized) to each container. The method illustrated in FIG. 2 also shows that each capture antibody recognizes and binds a different analyte of interest when compared to the other capture antibodies (e.g., analyte 1 and analyte 2). FIG. 2 then shows that a portion of the sample is administered to each container, so that the sample, with (or without) the analyte of interest, comes into contact with the corresponding capture antibody present on the surface of each container, and the antigen (analyte) present in the sample binds to the specific capture antibody.

[0113] One of skill in the art can readily determine the appropriate amount of sample to provide in each container. In certain embodiments, the amount of sample dispensed into each container can be different or the same. The amount of sample placed in each container will depend on a variety of factors, such as the type of sample, the type of analyte of interest, the type and shape of the container, the detection method employed and / or the type of antibody used.

[0114] For example, the amount of liquid sample provided to the containers is 1-10 mL, 1-5 mL, 1-4 mL, 1-3 mL, 1-2 mL, or less than 2 mL of sample. In some embodiments, the amount of liquid sample is 1-100 μL, 1-50 μL, 1-40 μL, 1-30 μL, 1-20 μL, 1-10 μL, 1-5 μL or less of sample. In some embodiments, the amount of sample provided to each container is 5 μL-100 μL, 10 μL-100 μL, 20 μL-100 μL, 30 μL-100 μL, or 40 μL-100 μL. In other embodiments, the amount of sample provided to each container is 10 μL-80 μL, 20 μL-80 μL, 25 μL-80 μL, 30 μL-80 μL, 35 μL-80 μL, or 40 μL-80 μL. In certain embodiments, the amount of sample provided in the container is between 10 μL and 80 μL, inclusive.

[0115] In yet other embodiments, the amount of liquid sample provided in each container is about 10 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL or more. In some embodiments, the amount of sample provided in each container is exactly 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, or 80 μL. In certain embodiments, the amount of liquid sample provided in each container is 20 μL or 35 μL. In one embodiment, the amount of sample provided in each container is 80 μL.

[0116] In some embodiments, the amount of liquid sample provided in each container is about 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, 25 μL, 26 μL, 27 μL, 28 μL, 29 μL, 30 μL, 31 μL, 32 μL, 33 μL, 34 μL, 35 μL, 36 μL, 37 μL, 38 μL, 39 μL, 40 μL, 41 μL, 42 μL, 43 μL, 44 μL, 45 μL, 46 μL, 47 μL, 48 μL, 49 μL, 50 μL, 51 μL, 52 μL, 53 μL, 54 μL, 55 μL, 56 μL, 57 μL, 58 μL, 59 μL, 60 μL, 61 μL, 62 μL, 63 μL, 64 μL, 65 μL, 66 μL, 67 μL, 68 μL, 69 μL, 70 μL, 71 μL, 72 μL, 73 μL, 74 μL, 75 μL, 76 μL, 77 μL, 78 μL, 79 μL, 80 μL, 81 μL, 82 μL, 83 μL, 84 μL, μL, 40μL, 41μL, 42μL, 43μL, 44μL, 45μL, 46μL, 47μL, 48μL, 49μL, 50μL, 51μL, 52μL, 53μL, 54μL, 55μL, 56μL, 57μL, 58μL, 59μL, 60μL, 61 μL, 62 μL, 63 μL, 64 μL, 65 μL, 66 μL, 67 μL, 68 μL, 69 μL, 70 μL, 71 μL, 72 μL, 73 μL, 74 μL, 75 μL, 76 μL, 77 μL, 78 μL, 79 μL, 80 μL or more.

[0117] In certain embodiments, the sample is urine (e.g., human urine) and the amount of urine provided to each receptacle is between 5 μL and 100 μL, between 10 μL and 100 μL, between 20 μL and 100 μL, between 30 μL and 100 μL, or between 40 μL and 100 μL. In other embodiments, the amount of urine provided to each receptacle is between 20 μL and 80 μL, between 25 μL and 80 μL, between 30 μL and 80 μL, between 35 μL and 80 μL, or between 40 μL and 80 μL. In yet other embodiments, the amount of urine sample provided to each receptacle is about 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, 80 μL, or more. In some embodiments, the amount of urine sample provided in each container is exactly 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL, 65 μL, 70 μL, 75 μL, or 80 μL. In certain embodiments, the amount of urine sample provided in each container is 20 μL or 35 μL. In one embodiment, the amount of urine sample provided in each container is 80 μL.

[0118] The sample or a portion of the sample can be administered to the container by any means known to one of skill in the art. Non-limiting examples of methods of administering a sample include dispensing, for example, by injecting, spraying, or pouring the sample or a portion of the sample into the container. Methods for dispensing and contacting the sample with the solid phase include manual pipetting, washing, robotic or automated dispensing mechanisms, or other methods known to one of skill in the art. Usual care in methods of administering a sample, such as the use of sterile techniques or other methods to preserve the integrity of the sample, is understood by one of skill in the art.

[0119] Administration of the sample or a portion of the sample can be performed by an individual or by an automated device, such as an automated immunodiagnostic device. In embodiments in which the method is performed in whole or in part by an automated immunodiagnostic device, the sample is first provided in a designated reservoir, and then a portion of the sample is dispensed into a container or containers of a solid phase that has been provided to the automated immunodiagnostic device. Automated immunodiagnostic devices suitable for use in the method are known in the art. For example, certain automated immunodiagnostic devices are described in U.S. Pat. Nos. 7,312,084, 6,143,576, 6,113,855, 6,019,944, 5,985,579, 5,947,124, 5,939,272, 5,922,615, 5,885,527, 5,851,776, 5,824,799, 5,679,526, 5,525,524, and 5,480,792, and in The Immunoassay Handbook, David Wild, ed. Stockton Press, New York, 1994, all of which are incorporated herein by reference in their entireties.

[0120] In certain embodiments, the automated immunodiagnostic device is an Ortho Clinical Diagnostics VITROS® ECiQ, an Ortho Clinical Diagnostics VITROS® 3600, an Ortho Clinical Diagnostics VITROS® 5600, an Ortho Clinical Diagnostics ACCESS®, an Abbott AXSYM®, a Roche ELECSYS®, or a Dade Behring STRATUS® device.

[0121] In one embodiment, the methods of the present disclosure can be performed on one of the following automated immunodiagnostic devices: Ortho Clinical Diagnostics VITROS® ECiQ, Ortho Clinical Diagnostics VITROS® 3600, or Ortho Clinical Diagnostics VITROS® 5600.

[0122] The sample may be provided in the solid phase container alone or in a mixture that includes the detection antibody.

[0123] Regardless of the type, form, amount, or manner in which the sample is provided to the vessels, the sample, or a portion of the sample, is incubated in the vessels to allow the capture antibodies immobilized on the vessel surfaces to bind to any corresponding analytes that may be present in the sample. Specifically, as shown in FIG. 2, once a sample containing an analyte of interest is provided to each vessel, each vessel contains a first capture antibody bound to a first analyte of interest (analyte 1) and a second capture antibody bound to a different analyte of interest (analyte 2), generating multiple capture antibody-analyte complexes attached to each vessel. In an example, if only one type of analyte of interest is present in the provided sample, the capture antibody for that particular analyte will not bind to the sample.

[0124] In some embodiments, a sample is provided to the containers and incubated for a period of time to promote binding of any analytes of interest present in the sample to the corresponding capture antibodies present in each container. The incubation time can be readily determined by one of skill in the art and can vary based on a variety of factors, such as the type of sample, the type of analyte of interest, the type and shape of the container, the detection method employed and / or the type of antibody used.

[0125] In certain embodiments, the sample is incubated in the container in the absence of detection antibody for about 1-60 minutes, 1-50 minutes, 1-40 minutes, 1-30 minutes, 1-20 minutes, 1-15 minutes, 1-10 minutes, 1-5 minutes, 1-4 minutes, 1-3 minutes, 1-2 minutes, or less than 1 minute. In one embodiment, the sample is dispensed into the container in the absence of detection antibody and incubated for 1-5 minutes inclusive. In other embodiments, the sample is incubated in the vessel for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 minutes. In another embodiment, the sample is dispensed into the vessel in the absence of detection antibody and incubated for 1, 2, 3, 4, or 5 minutes.

[0126] In certain embodiments, the sample is dispensed into a vessel along with the detection antibody and the mixture is incubated for about 1-60 minutes, 1-50 minutes, 1-40 minutes, 1-30 minutes, 1-20 minutes, 1-15 minutes, 1-10 minutes, 1-8 minutes, 1-5 minutes, 1-4 minutes, 1-3 minutes, 1-2 minutes, or less than 1 minute. In one embodiment, the mixture of the sample and the detection antibody is dispensed into a vessel and incubated for 1-10 minutes, inclusive. In another embodiment, the mixture of the sample and the detection antibody is dispensed into a vessel and incubated for 1-8 minutes, inclusive. In other embodiments, the sample mixed with the detection antibody is incubated in the vessel for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 minutes. In yet other embodiments, the mixture of sample and detection antibody is dispensed into the vessel and incubated for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In certain embodiments, the mixture of sample and detection antibody dispensed into the vessel is incubated for 8 minutes. do.

[0127] The disclosed method also includes providing a separate detection antibody to each container of the solid phase. More specifically, the method includes providing a quantity of a detection antibody that recognizes a first analyte of interest to a first container of the solid phase and providing a quantity of another detection antibody that recognizes a different analyte of interest to another container of the solid phase. The detection antibodies provided to each container of the solid phase each correspond to only one of the analytes of interest that may or may not be present in the sample and each include a detectable label that generates the same detectable signal.

[0128] As shown in FIG. 2, the solid phase containers 1 and 2 contain a first capture antibody and a second capture antibody attached (immobilized) to each container. Each capture antibody recognizes and binds a different analyte of interest when compared to the other capture antibodies (e.g., analyte 1 and analyte 2). When a sample is administered to each container, it comes into contact with the capture antibodies present on the surface of each container, and if an antigen (analyte) is present in the sample, it binds to its specific corresponding capture antibody. This generates multiple capture antibody-analyte complexes immobilized on each container. In the example where only one type of analyte of interest is present in the provided sample, the capture antibody for the absent but analyte of interest does not bind to the sample. Then, each container is provided with a certain amount of detection antibody specific for only one of the analytes of interest, i.e., either analyte 1 or analyte 2. This generates multiple antigen-analyte-detection antibody complexes immobilized on each container, thereby causing only one specific analyte to be detectably labeled in each well (e.g., analyte 1 or analyte 2). For example, as shown in FIG. 2, container 1 contains only capture antibody-analyte 1-detection antibody complexes and a second container contains only capture antibody-analyte 2-detection antibody complexes.

[0129] In some embodiments of the method, the detection antibodies are monoclonal antibodies, polyclonal antibodies, fragments thereof, or any combination thereof. In certain embodiments, the detection antibodies of the present disclosure are all monoclonal antibodies each recognizing an analyte of interest. In other embodiments, the detection antibodies of the present disclosure are all polyclonal antibodies each recognizing an analyte of interest. In yet another embodiment, the detection antibodies of the present disclosure are a combination of monoclonal and polyclonal antibodies each specific for a different analyte of interest.

[0130] In some embodiments of the method, the detection antibodies are specific for TIMP2 and IGFP7, respectively. In certain embodiments, the detection antibodies of the present disclosure are monoclonal antibodies specific for TIMP2 and IGFP7, respectively. In certain embodiments, the detection antibody provided to a container of solid phase is a monoclonal antibody specific for TIMP2, such as rabbit monoclonal 40H2-40K3, and the detection antibody provided to another container of solid phase is a mouse monoclonal antibody specific for IGFBP7, such as 6D2.1.

[0131] In other embodiments, the detection antibodies for use in the present methods include the following detection antibodies: any monoclonal or polyclonal antibody that specifically recognizes tau (e.g., HT7 and AT270 monoclonal antibodies, antibodies that recognize normally and abnormally phosphorylated tau (e.g., Alz50 (Ghanbari et al., 1990), HT7 (Mercken et al., 1992), and AT120 (Vandermeeren et al., 1993), any antibody that recognizes PSA or its binding partner (e.g., 2E9, 2H11 and 5A10, which are described in U.S. Pat. No. 5,501,983, the entire contents of which are incorporated herein by reference), any antibody that binds to Aβ or a portion thereof, which are described in U.S. Pat. No. 7,700,309, the entire contents of which are incorporated herein by reference, and other antibodies known to those skilled in the art, for example, which are disclosed in U.S. Pat. No. 9,174,097, the entire contents of which are incorporated herein by reference.

[0132] As described herein, the detection antibodies used in the present methods contain the same detectable label, or detectable labels that generate the same detectable signal. This aspect of the present methods simplifies the detection of multiple analytes by reducing the need for the user to obtain detection antibodies with different detectable labels and a means to detect multiple different signals.

[0133] Detectable labels are known to those skilled in the art, as are means for conjugating such labels to antibodies. In some embodiments, the detectable label associated with the detection antibody is directly detectable. In certain embodiments, the directly detectable label is a fluorescent moiety (dye), an electrochemical label, an electrochemiluminescent label, a metal chelate, or a colloidal metal particle. In other embodiments, the detectable label is an indirectly detectable label, such as a molecule that becomes detectable after undergoing a chemical or enzymatic reaction, or after being bound by a molecule that itself provides a detectable signal. In some embodiments, the detectable label is an enzyme, such as horseradish peroxidase or alkaline phosphatase, which can be contacted with a substrate (e.g., a luminescent substrate such as a chemiluminescent substrate (5-amino-2,3-dihydrophthalazine-1,4-dione (luminol) or a chromogenic substrate (e.g., 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS))) and oxidize the substrate to provide a detectable signal.

[0134] In some embodiments, the detectable label is attached to the detection antibody. In certain embodiments, the detectable label is conjugated to the detection antibody. In certain embodiments, the detectable label is a dye or enzyme conjugated to each detection antibody. In one embodiment of the method, horseradish peroxidase (HRP) is used as a conjugate with each specific detection antibody. Preparation of such conjugates can be achieved using various known techniques. For example, the methods described by Yoshitake et al, Eur.J.Biochem., 101, 395, 1979, and in U.S. Patent No. 5,106,732 to Kondo et al, both of which are incorporated herein by reference in their entirety. As shown in FIG. 3, the detection antibodies of the present disclosure are specific for different analytes (TIMP2 and IGFP7), each of which is conjugated to a horseradish peroxidase enzyme (detectable label). The HRP enzyme can then be contacted in each well with a substrate, such as a luminescent substrate, which reacts (eg, oxidizes) with the substrate to provide a detectable signal (eg, light, luminescence).

[0135] Regardless of the detection antibodies used in the method, a fixed amount of each detection antibody is provided to each container of the solid phase such that the antigen-binding portion of the detection antibody comes into contact with the corresponding antigen (if present) bound by the corresponding capture antibody in the portion of the sample.

[0136] One of skill in the art can readily determine the appropriate amount of detection antibody to provide in each container. In certain embodiments, the amount of detection antibody dispensed into each container can be different or the same. Determining the amount of detection antibody to place in each container depends on a variety of factors, such as the type of sample, the type of analyte of interest, the type and shape of the container, the detection method employed and / or the type of detection antibody used.

[0137] The detection antibody may be dispensed directly into the container, or may be premixed in a solution containing the desired concentration of the detection antibody and provided as an aliquot of such a premixed solution. Methods for diluting stock solutions of antibodies are known to those of skill in the art.

[0138] In some embodiments, the amount of detection antibody provided in each container is about 0.02 μg to about 1.2 μg. In one embodiment, the amount of detection antibody provided in each container is 0.02 μg to 1.2 μg. In certain embodiments, the amount of detection antibody provided in each container is 0.02 μg, 0.025 μg, 0.03 μg, 0.035 μg, 0.04 μg, 0.045 μg, 0.05 μg, 0.055 μg, 0.06 μg, 0.065 μg, 0.07 μg, 0.075 μg, 0.08 μg, 0.085 μg, 0.09 μg, 0.095 μg, 0.1 μg, 0. In certain embodiments, the amount of detection antibody provided in each container is 0.075 μg or 1.2 μg.

[0139] In other embodiments, the amount of detection antibody provided to the container is 1-10 μg / mL, 1-5 μg / mL, 1-4 μg / mL, 1-3 μg / mL, 1-2 μg / mL or less of detection antibody per container. In certain embodiments, the amount of detection antibody is about 0.5 μg / mL to about 2.0 μg / mL. In specific embodiments, the amount of detection antibody is 0.5 μg / mL or 2.0 μg / mL per container.

[0140] The detection antibody can be administered to the container by any means known to one of skill in the art. Non-limiting examples of administering the detection antibody include dispensing, for example, by injecting, spraying, or pouring the detection antibody into the container. Usual care in administering the detection antibody, such as use of aseptic technique or other methods to prevent contamination, will be understood by one of skill in the art.

[0141] Administration of the detection antibody or a solution containing the detection antibody can be performed by an individual or by an automated device, such as an automated immunodiagnostic device. Methods for dispensing the detection antibody into contact with the solid phase can include by manual pipetting or placing the desired amount of detection antibody into a container, and / or by means of a robotic or automated dispensing mechanism.

[0142] In embodiments where the method is performed in whole or in part by an automated immunodiagnostic device, the detection antibody is first provided in a designated reservoir, and then an aliquot of the detection antibody is dispensed into the container or containers of the solid phase that have been provided in the automated immunodiagnostic device. Automated immunodiagnostic devices suitable for use in the method are known in the art. For example, certain automated immunodiagnostic devices are described in U.S. Pat. Nos. 7,312,084, 6,143,576, 6,113,855, 6,019,944, 5,985,579, 5,947,124, 5,939,272, 5,922,615, 5,885,527, 5,851,776, 5,824,799, 5,679,526, 5,525,524, and 5,480,792, and in The Immunoassay Handbook, David Wild, ed. Stockton Press, New York, 1994, all of which are incorporated herein by reference in their entireties.

[0143] In certain embodiments, the automated immunodiagnostic device is an Ortho Clinical Diagnostics VITROS® ECiQ, an Ortho Clinical Diagnostics VITROS® 3600, an Ortho Clinical Diagnostics VITROS® 5600, a Beckman ACCESS®, an Abbott AXSYM®, a Roche ELECSYS®, or a Dade Behring STRATUS® device.

[0144] In certain embodiments, the methods of the present disclosure are performed on one of the following automated immunodiagnostic devices: an Ortho Clinical Diagnostics Vitros® ECiQ, an Ortho Clinical Diagnostics Vitros® 3600, or an Ortho Clinical Diagnostics Vitros® 5600.

[0145] Regardless of the type, amount, or manner in which the detection antibody is provided to the container, the detection antibody is incubated in the container. The incubation allows the antigen-binding portion of the detection antibody to recognize and bind to the corresponding antigen (analyte) in the sample. The detection antibody binds to the analyte of interest in the sample, forming a capture antibody-analyte-detection antibody complex in the container. One skilled in the art can readily determine the incubation time, and it can vary based on a variety of factors, such as the type of sample, the type of antibody, the type of analyte of interest, the type and shape of the container, and / or the detection method employed.

[0146] Incubation can be for any period of time greater than 1 second. In one embodiment, the detection antibody is dispensed into the container and incubated for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes or more. In a particular embodiment, the detection antibody is provided to the container and incubated for at least 5 minutes. In a particular embodiment, the detection antibody is provided to the container and incubated for at least 8 minutes.

[0147] In certain embodiments, the detection antibody is provided to the container and incubated for about 1-40 minutes, 1-30 minutes, 1-20 minutes, 1-15 minutes, 1-10 minutes, 1-9 minutes, 1-8 minutes, 1-7 minutes, 1-6 minutes, 1-5 minutes, 1-4 minutes, 1-3 minutes, 1-2 minutes, or less than 1 minute. In some embodiments, the detection antibody is provided to the container and incubated for 5-10 minutes, 6-10 minutes, 7-10 minutes, 8-10 minutes, or 9-10 minutes. In other embodiments, the detection antibody is provided to the container and incubated for 4-9 minutes, 4-8 minutes, 4-7 minutes, 4-6 minutes, or 4-5 minutes. In other embodiments, the detection antibody is provided to the container and incubated for 5-9 minutes, 5-8 minutes, 5-7 minutes, or 5-6 minutes. In other embodiments, the detection antibody is provided to the container and incubated for 6-9 minutes, 6-8 minutes, or 6-7 minutes. In yet other embodiments, the detection antibody is provided to the vessel and incubated for 7-9 minutes, or 7-8 minutes.

[0148] In some embodiments, the detection antibody is dispensed as a solution into the container and incubated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 minutes. In certain embodiments, the detection antibody is dispensed into the container and incubated for 4, 5, 6, 7, 8, 9, or 10 minutes. In other embodiments, the detection antibody is incubated in the container for 8 minutes.

[0149] In certain embodiments, the sample is pre-mixed with the detection antibody prior to administering the mixture of sample and detection antibody to the container. In such embodiments, the sample and detection antibody are mixed and incubated for about 1-40 minutes, 1-30 minutes, 1-20 minutes, 1-15 minutes, 1-10 minutes, 1-8 minutes, 1-5 minutes, 1-4 minutes, 1-3 minutes, 1-2 minutes, or less than 1 minute before administering the mixture or a portion thereof to the container. In one embodiment, the mixture of sample and detection antibody is incubated for 1-10 minutes, inclusive. In another embodiment, the mixture of sample and detection antibody is incubated for 1-8 minutes, inclusive. In yet other embodiments, the mixture of sample and detection antibody is incubated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 minutes. The incubation period can be easily determined by one of skill in the art and can vary based on a variety of factors. As such, longer or shorter incubation periods are contemplated.

[0150] Generation of a detectable signal from a detectable label can be performed using a variety of optical, acoustic and electrochemical methods known in the art. Exemplary detection modes include fluorescence, radiochemical detection, reflectance, absorbance, amperometry, conductivity, impedance, interferometry, ellipsometry, and the like.

[0151] In some embodiments, the detectable signal is generated by a fluorometer that employs an excitation light source transducer that is spatially separated from the solid phase and directs excitation light into each well being analyzed to generate light within the well at a detectable wavelength that can be measured by an optical detector.

[0152] In yet another embodiment, an antibody-based biosensor may be employed to determine the presence or amount of analyte bound to a detection antibody present in the container.

[0153] In one embodiment, the detectable label conjugated to each detection antibody is an enzyme, such as a peroxidase enzyme. In one embodiment, the detectable label conjugated to each detection antibody is horseradish peroxidase (HRP) or a suitable isozyme thereof. Suitable isozymes of horseradish peroxidase include available types VI and IX, for example available from Sigma Chemical Co. The detectable signal is generated by providing a substrate, such as a luminescent substrate, to each well so that the HRP enzyme oxidizes the luminescent substrate and releases a detectable signal (light).

[0154] Luminescent substrates for use with the HRP enzyme are known to those of skill in the art as are its enhancers. Non-limiting examples of luminescent substrates for use in this embodiment include luminol (i.e., 2,3-dihydro-1,4-phthalazinedione) or substituted luminols and perborate in aqueous solvent, as described in U.S. Patent Nos. 5,846,756 and 5,705,357, the entire contents of which are incorporated herein by reference.

[0155] In another embodiment, alkaline phosphatase conjugated to a detection antibody and the AMPPD chemiluminescent substrate with emerald enhancer (Tropix) is used to generate a detectable signal, as described in C. Vigo-Pelfrey et al. J Neurochem (1994) 61:1965-1968, the entire contents of which are expressly incorporated herein by reference, and thus the particular combination of enzyme, substrate and enhancer is not intended to be limiting.

[0156] In certain embodiments, such as the exemplary embodiment shown in FIG. 3, a luminescent substrate (e.g., luminol, its derivatives and persalts) is provided with an electron transfer agent (enhancer), such as a substituted acetanilide, to amplify the light signal emitted from the substrate and prolong the emission of the signal from the vessel.

[0157] Any enhancer capable of facilitating the transfer of electrons from an enzyme (e.g., hydrogen peroxide (derived from perborate)) to luminol via peroxidase can be used in the present method. For example, when the reaction between luminol and hydrogen peroxide is caused by peroxidase in the presence of a suitable enhancer, the enhancer increases the rate of oxidation by the enzyme. Non-limiting examples of enhancers for use in the present method include those described in U.S. Pat. Nos. 4,842,997 and 5,279,940, the entire contents of each of which are incorporated herein by reference. Suitable enhancers include 4-iodophenol, 4-bromophenol, 4-chlorophenol, 4-phenylphenol, 2-chloro-4-phenylphenol, 6-hydroxybenzothiazole ... 1 -(2 1 -methyl)thiazolylphenol, 4-2 1 -(4 1 -methyl)thiazolylphenol, 4-(2 1 -benzothiazolylphenol, 3-(10-phenothiazyl)-n-propylsulfonate and 3-chloro,4-hydroxyacetanilide.

[0158] Incubation of the luminescent substrate and the electron transfer agent promotes oxidation of the substrate and amplification of the luminescent signal. Incubation can be performed for any period of time greater than 1 second. Incubation times can be readily determined by one of skill in the art and can vary based on a variety of factors. Thus, longer or shorter incubation periods are contemplated.

[0159] In some embodiments, the luminescent substrate and the electron transfer agent are dispensed into the vessel and incubated for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes or more. In certain embodiments, the luminescent substrate and the electron transfer agent are provided to the vessel and incubated for at least 4 minutes. In certain embodiments, the luminescent substrate and the electron transfer agent are provided to the vessel and incubated for at least 5 minutes.

[0160] In certain embodiments, the luminescent substrate and the electron transfer agent are provided to the container and incubated for about 1-20 minutes, 1-15 minutes, 1-10 minutes, 1-9 minutes, 1-8 minutes, 1-7 minutes, 1-6 minutes, 1-5 minutes, 1-4 minutes, 1-3 minutes, 1-2 minutes, or less than 1 minute. In other embodiments, the detection antibody is provided to the container and incubated for 4-9 minutes, 4-8 minutes, 4-7 minutes, 4-6 minutes, or 4-5 minutes. In some embodiments, the luminescent substrate and the electron transfer agent are provided to the container and incubated for 3-9 minutes, 3-8 minutes, 3-7 minutes, 3-6 minutes, or 3-5 minutes. In other embodiments, the luminescent substrate and the electron transfer agent are provided to the container and incubated for 2-6 minutes, 2-5 minutes, 2-4 minutes, or 2-3 minutes. In certain embodiments, the luminescent substrate and the electron transfer agent are provided to the container and incubated for 4-5 minutes.

[0161] In some embodiments, the luminescent substrate and electron transfer agent are dispensed into the vessels and incubated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes. In certain embodiments, the luminescent substrate and electron transfer agent are dispensed into the vessels and incubated for 3, 4, 5, or 6 minutes. In other embodiments, the luminescent substrate and electron transfer agent are incubated in each vessel for 4 minutes. In certain embodiments, the luminescent substrate and electron transfer agent are incubated in each vessel for 5 minutes.

[0162] The temperature of incubation can be readily determined by one of skill in the art and can vary based on a variety of factors. In one non-limiting example, the luminescent substrate and the electron transfer agent are dispensed into a container and incubated at a temperature between 32°C and 37°C, between 33°C and 37°C, between 34°C and 37°C, between 35°C and 37°C, or between 36°C and 37°C inclusive. In certain embodiments, the luminescent substrate and the electron transfer agent are dispensed into a container and incubated at a temperature of about 37°C. In one embodiment, the luminescent substrate and the electron transfer agent are dispensed into a container and incubated at a temperature of 37°C.

[0163] In one embodiment of this method, the luminescent substrate and the electron transfer agent are dispensed into a container and incubated for about 5 minutes at a temperature of 37° C. In another embodiment of this method, the luminescent substrate and the electron transfer agent are dispensed into a container and incubated for 4-5 minutes at 37° C.

[0164] The method can be expanded to the simultaneous or sequential detection of two or more different analytes using a single solid phase with high sensitivity and minimal interference from other analytes. In general, after applying the sample to the solid phase, the signal generated directly or indirectly from the detection antibody can be detected by any means known to those skilled in the art. Detection methods, including automated methods, are well known to those skilled in the art. For example, the signal can be detected visually or obtained by a device (analytical instrument) such as a reflectometer, a fluorometer, a transmittance photometer, etc.

[0165] In one non-limiting embodiment, a robotic instrument (i.e., an automated immunodiagnostic device), including but not limited to the Ortho Clinical Diagnostics VITROS® 3600, Ortho Clinical Diagnostics VITROS® ECiQ, Ortho Clinical Diagnostics VITROS® 5600, Beckman ACCESS®, Abbott AXSYM®, Roche ELECSYS®, or Dade Behring STRATUS® system, is used in conjunction with the method to measure the detectable signal. However, any automated immunodiagnostic device capable of detecting the particular signal used in the method can be used. By way of example only, any fluorometer can be used to detect fluorescent labels, any fluorometer can detect labels that emit wavelengths of light, and a reflectometer can be used to detect labels that absorb light.

[0166] In some embodiments, multiple analytes are detected sequentially, where a detectable signal generated in a first container is measured, followed by a detectable signal generated by another container until each container analyzed has been measured. Sequential detection of the presence of signals from different containers increases efficiency and eliminates the need for multiple fluorometers, luminometers, or reflectometers that would be required to simultaneously detect signals generated from multiple wells. Furthermore, sequential detection of analytes allows the use of detectable labels that emit detectable signals with overlapping wavelengths (spectrum) or absorption (color).

[0167] For example, after each vessel is removed from the solid phase, the first vessel is incubated for a predetermined time and passed to a reading station, which may include, for example, a reflectometer, luminometer, or electrometer, and read. Once the measurement is recorded, the vessel can be discarded. Then, the second vessel is passed to the reading station and read. The second measurement is recorded, and the second vessel is discarded from the solid phase.

[0168] In some embodiments, once a signal is generated and detected (indicating the presence of an analyte in the sample), the signal is measured and quantified. In certain embodiments, the measurement of the amount of signal in each vessel is quantified and provided as a single value. Any software or method for combining multiple measurements to provide a single quantified value known to those skilled in the art, such as those described in WO 2011 / 073741, can be used in the quantification step of the method.

[0169] In one non-limiting example, for quantitative measurements, a calibration curve is fitted using, for example, a modified 4-parameter or 5-parameter logarithmic software program on an Ortho Clinical Diagnostics Assay Data Disk (installed on the Vitros® 3600 Immunodiagnostic System, Vitros® 5600 Integrated System) or magnetic card (on the Vitros® ECiQ instrument), where the signal level from a calibrator present on the instrument used to measure the detectable signal from each vessel adjusts a master curve provided by the program, and the software determines the concentration of analyte in each well by applying the signal obtained from each well to the calibration curve.

[0170] In some embodiments, a single value can be provided that quantifies the total amount of all analytes present in a sample.For example, in the example where the method is deployed to detect the presence of two different analytes of interest, the detected amount of the first analyte in the first container is multiplied by the detected amount of the second analyte in the second container, and the total amount is divided by 1000.Therefore, the following formula can be used to obtain a single numerical value for the amount of the first and second analytes present in a sample: Numeric value = ([Analyte 1] × [Analyte 2]) / 1000 Units = (ng / mL) 2 / 1000

[0171] In certain embodiments, the amount of signal measured in each vessel correlates to the amount of analyte present in the sample.

[0172] In some embodiments, the signal or signals detected can be compared to the signal generated after the use of a control sample in the method. Such a comparison can facilitate quantification of the amount of analyte detected in the sample. In one embodiment, the amount of signal generated by each vessel is directly proportional to the concentration of the particular analyte present in the sample. This would be the case in a "sandwich assay".

[0173] In another embodiment, the amount of signal generated by each reservoir is inversely proportional to the concentration of a particular analyte of interest in the sample, as would be the case in a competitive immunoassay.

[0174] In certain embodiments, the method is carried out in an immunoassay. A variety of specific assay formats are useful in carrying out the method, including immunochemical assays such as enzyme immunoassays, sandwich assays, competitive binding assays, direct binding assays, and others known to those of skill in the art.

[0175] In certain embodiments, as shown in Figures 1-3, the method comprises a "sandwich assay", whereby one or more analytes of interest (e.g., antigens present in a sample) are simultaneously or sequentially complexed with at least a first capture antibody and a second detection antibody. For example, in certain embodiments, the capture antibody is immobilized on the surface of a solid phase. The sample is then provided to the solid phase, such that a second (detection) antibody specific for the same analyte of interest binds to the corresponding antigen in the sample, forming a capture antibody-analyte-detection antibody complex, i.e., a sandwich. If one antibody of the sandwich contains a detectable label (e.g., conjugated to a peroxidase, a fluorescent dye, or a radioactive label) or can be so labeled through an additional specific binding reaction (e.g., through an avidin-biotin complex), the amount of antigen (analyte) present in the sample can be determined.

[0176] Other embodiments include competitive binding assays, in which a particular analyte of interest competes with a detection antibody for the analyte and another ligand for the analyte. For example, in a competitive immunoassay format, an antigen in a sample may compete with the analyte or antigen provided to a solid phase for binding to a detection antibody with a labeled antigen provided as an assay reagent.

[0177] (kit) In some aspects of the present disclosure, kits are provided for carrying out the methods described herein. Suitable kits include sufficient reagents to carry out the methods of the present disclosure together with instructions for carrying out the described methods. Additional optional elements that may be provided as part of the assay are described herein.

[0178] In certain embodiments, reagents for carrying out the subject methods are provided in a kit, including one or more of the following: a solid phase, an antibody, a buffer solution, a luminescent substrate, an electron transfer agent, and instructions for carrying out the disclosed method.

[0179] In some embodiments, the kit comprises a solid phase having a plurality of containers coated with at least two capture antibodies, in certain embodiments, each container of the solid phase provided in the kit has at least two different capture antibodies immobilized thereon, whereby each of the at least two different capture antibodies recognizes a different antigen or epitope (analyte).

[0180] As described herein, the solid phase can include more than one capture antibody immobilized to each reservoir of the solid phase, depending on the number of analytes to be detected. In some embodiments, each reservoir of the solid phase has at least two, at least three, at least four, at least five, at least six or more different capture antibodies immobilized thereto. In some embodiments, each reservoir of the solid phase has 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different capture antibodies immobilized thereto.

[0181] In one embodiment, the capture antibodies immobilized on each solid phase container are mouse monoclonal antibodies specific for TIMP2 and IGFBP7. In a particular embodiment, the capture antibody specific for TIMP2 is mouse monoclonal antibody 6E2.1, and the capture antibody specific for IGFBP7 is mouse monoclonal antibody 1D6.

[0182] In other embodiments, the kit comprises a solid phase having at least two capture antibodies immobilized on the surface of a container, the capture antibodies being any of the following antibodies: any monoclonal or polyclonal antibody that specifically recognizes tau (e.g., HT7 and AT270 monoclonal antibodies, antibodies that recognize normally and abnormally phosphorylated tau (e.g., Alz50 (Ghanbari et al., 1990), HT7 (Mercken et al., 1992), and AT120 (Vandermeeren et al., 1993), any antibody that recognizes PSA or its binding partner (e.g., 2E9, 2H11 and 5A10, which are described in U.S. Pat. No. 5,501,983, the entire contents of which are incorporated herein by reference), any antibody that binds to Aβ or a portion thereof, which are described in U.S. Pat. No. 7,700,309, the entire contents of which are incorporated herein by reference, and other antibodies known to those skilled in the art, for example, which are disclosed in U.S. Pat. No. 9,174,097, the entire contents of which are incorporated herein by reference.

[0183] In certain embodiments of the method, the kit includes a solid phase comprised of multiple containers as part of a straw, sleeve, strip, plate, slide, etc. Specifically, the solid phase includes straws or strips of cups or wells, multi-well or microwell plates, etc. that can be used in automated immunodiagnostic devices. In one embodiment, the solid phase provided in the kit includes multiple tapered containers, such as Vitros® microwells.

[0184] In some embodiments, the solid phase provided in the kit comprises at least 2 containers, at least 10 containers, at least 15 containers, at least 20 containers, at least 25 or more containers. In embodiments, a solid phase is provided that comprises at least 2 VITROS® microwell straws or sleeves. In another embodiment, a solid phase is provided that comprises at least 20 VITROS® microwell straws or sleeves. In another embodiment, a solid phase is provided that comprises at least 25 VITROS® microwell straws or sleeves.

[0185] In certain embodiments, the solid phase provided in the kit is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 102, 104, 105, 106, 107, 108, 109, 110, 111, 112, The solid phase may comprise 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 receptacles. In some embodiments, the solid phase comprises 2-100, 2-75, 2-50, 2-25, 2-15, 2-10, or 2-5 receptacles. In one embodiment, the solid phase comprises 25 single receptacles. In a particular embodiment, the solid phase comprises 25 receptacles within a straw or sleeve. In a particular embodiment, the solid phase comprises 25 VITROS® microwells within a sealed straw.

[0186] In certain embodiments, the kits of the present disclosure include more than one solid phase. For example, the kits may include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 solid phases, each of which includes at least two containers. In certain embodiments, the kits include 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3 solid phases, each of which includes at least two containers. In one embodiment, the kits include 3-6, 3-5, or 3-4 solid phases, each of which includes at least two containers. In a particular embodiment, the kits include 4 solid phases, each of which includes at least two containers.

[0187] In one embodiment, the kit of the present disclosure includes four solid phases, each of which comprises at least 10 tapered vessel straws or sleeves. In another embodiment, the kit of the present disclosure includes four solid phases, each of which comprises at least 20 tapered vessel straws or sleeves. In yet another embodiment, the kit of the present disclosure includes four solid phases, each of which comprises 25 tapered vessel straws or sleeves. In one embodiment, the kit of the present disclosure includes four solid phases, each of which comprises 25 Vitros® microwell straws or sleeves.

[0188] In some embodiments, the kit comprises at least two different detection antibodies, whereby each of the at least two different detection antibodies recognizes a different analyte of interest. In some embodiments, each of the at least two different detection antibodies comprises a detectable label, where each label is the same or produces the same detectable signal.

[0189] In some embodiments, the detection antibodies are monoclonal antibodies, polyclonal antibodies, fragments thereof, or any combination thereof. In certain embodiments, the detection antibodies are all monoclonal antibodies, each recognizing a different analyte of interest. In other embodiments, the detection antibodies are all polyclonal antibodies, each recognizing a different analyte of interest. In yet other embodiments, the detection antibodies provided include a combination of monoclonal and polyclonal antibodies, each specific for a different analyte of interest.

[0190] In some embodiments, the kit comprises antibodies specific for the TIMP2 and IGFP7 analytes. In certain embodiments, the detection antibodies of the present disclosure are monoclonal antibodies specific for TIMP2 and IGFBP7, respectively. In a particular embodiment, the detection antibody specific for TIMP2 is rabbit monoclonal antibody 40H2-40K3, and the detection antibody specific for IGFBP7 is mouse monoclonal antibody 6E2.1.

[0191] In certain embodiments, the kit comprises at least two different detection antibodies, each recognizing a different analyte of interest and conjugated to an enzyme such as horseradish peroxidase (HRP). In other embodiments, the detection antibodies provided are the following detection antibodies: any monoclonal or polyclonal antibody that specifically recognizes tau (e.g., HT7 and AT270 monoclonal antibodies, antibodies that recognize normally and abnormally phosphorylated tau (e.g., Alz50 (Ghanbari et al., 1990), HT7 (Mercken et al., 1992), and AT120 (Vandermeeren et al., 1993), any antibody that recognizes PSA or its binding partner (e.g., 2E9, 2H11 and 5A10, which are described in U.S. Pat. No. 5,501,983, the entire contents of which are incorporated herein by reference), any antibody that binds to Aβ or a portion thereof, which are described in U.S. Pat. No. 7,700,309, the entire contents of which are incorporated herein by reference, and other antibodies known to those skilled in the art, for example, which are disclosed in U.S. Pat. No. 9,174,097, the entire contents of which are incorporated herein by reference.

[0192] In some embodiments of the present disclosure, the kit includes a luminescent substrate. The luminescent substrate used in the present method and kit is known to those skilled in the art as its enhancer. Therefore, the specific combination or combinations of enzyme, substrate and enhancer are not intended to be limiting. In some embodiments, the luminescent substrate provided in the kit is luminol, or its derivatives and persalt salts. In some embodiments, the kit includes an electron transfer agent (enhancer), such as a substituted acetanilide, that can amplify the signal generated from the detectable label.

[0193] In some embodiments, the kit of the present disclosure includes a reference solution (calibration solution) containing a known amount of a particular analyte of interest. In one embodiment, the reference solution can contain one known amount or multiple known amounts of at least two analytes of interest. In another embodiment, a reference solution can be provided for each corresponding analyte detected by such a kit. As described herein, the reference solution can be used to form a calibration curve to further quantify the measured value of an analyte in a sample.

[0194] In some embodiments, the kits of the present disclosure include one or more solutions or buffers. For example, the kits can contain one or more of the following: phosphate buffer solution, detection antibody solution (e.g., TIMP2 detection antibody solution, IGFBP7 detection antibody solution), and water (e.g., deionized or sterile).

[0195] Generally, the instructions provided in the kits of the present disclosure include the methods of the present disclosure. The methods described herein generally include contacting a sample containing or suspected to contain an analyte of interest with a first capture antibody immobilized on a solid phase and specifically binding to the analyte. The sample is also contacted with a detection antibody that includes a detection agent to form a capture antibody-analyte-detection antibody complex immobilized on a solid phase. A signal is then generated from the detection antibody, which indicates the presence or amount of a complex formed by the binding of the analyte in the sample with the capture and detection antibodies. The signal is then measured. In certain non-limiting examples, the methods of the present disclosure include chromatography, mass spectrometry, and protein detection assays. In one such example, robotic instruments (i.e., automated immunodiagnostic devices) including, but not limited to, Ortho Clinical Diagnostics VITROS® ECiQ, Ortho Clinical Diagnostics VITROS® 3600, Ortho Clinical Diagnostics VITROS® 5600, Beckman ACCESS®, Abbott AXSYM®, Roche ELECSYS®, and Dade Behring STRATUS® systems are among the automated immunodiagnostic devices that can be used in conjunction with the present methods. However, any suitable automated immunodiagnostic device may be used to measure the detectable signal.

[0196] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein should be understood to encompass any and all subranges subsumed therein. For example, a range stated as "1-10" should be interpreted to include any and all subranges between the minimum value of 1 and the maximum value of 10 (inclusive), i.e., all subranges beginning with a minimum value of 1 or more, e.g., 1-6.1, and ending with a maximum value of 10 or less, e.g., 5.5-10.

[0197] Additionally, any reference to "incorporated herein" should be understood to be incorporated in its entirety.

[0198] (Example) Example 1: Materials and Methods (Container formation) The vessels (e.g., wells) of the present disclosure are produced by the following two-day direct antibody coating process. Prior to coating, the vessels (e.g., wells coated with white polystyrene) are irradiated to 1 MRad to minimize well-to-well differences. A capture antibody coating solution is prepared containing 30 mM phosphate buffer composed of 2.04 mM KH2PO4 in 150 mM NaCl buffer, plus 0.96 mM K2HPO4, pH 6.3±0.2, and sunset yellow dye (7.5 mg / Kg solution). TIMP2 capture MAb 6E2.1 and IGFBP7 capture MAb 1D6 mouse antibodies are each added to a final concentration of 3 μg / mL. 200 μL of 3 μg / mL capture antibody coating solution is added to each well. The vessels are then incubated overnight (e.g., 16-32 hours) at 18-22°C. After overnight incubation with the capture antibody coating solution, the vessels are washed twice with TRIS wash buffer containing 0.1 M Tris / HCl at pH 8.5. After aspirating the wash buffer, the vessels are coated with TSBB (0.1 M Tris / HCl at pH 8.5 containing 5% sucrose, 0.45% sodium chloride and 0.1% BSA). All solutions are aspirated from each vessel and after the final aspiration, the vessels are allowed to dry. The washing, coating and final aspiration steps are an in-line process with no additional incubation steps. The plate containing the coated vessels is stored at 18-22 °C in a storage container (e.g., a plastic box) containing desiccant until use. The vessels are provided as 100 coated wells (25 wells per straw) sealed in four straws. Each vessel is coated with both TIMP2 capture antibody and IGFBP7 capture antibody.

[0199] (Preparation of detection conjugate reagent) TIMP2-HRP detection conjugate and IGFBP7-HRP detection conjugate are prepared from TIMP2 detection MAb 40H2-40K3, IGFBP7 detection MAb 6D2.1, and horseradish peroxidase (HRP) using standard conjugation methods known in the art. The TIMP2-HRP conjugate reagent contains potassium ferricyanide (0.001%), Tween® 20 (0.5%), ProClin TM The IGFBP7-HRP conjugate reagent was prepared using 110 mM phosphate buffer solution at pH 6.5, which also contained 950 (0.5%), anilinonaphthalene-1-sulfonate (ANS) (0.04%), and bovine serum albumin (BSA) (3.0%), and 0.0005 g / Kg of TIMP2 antibody conjugate. The IGFBP7-HRP conjugate reagent was prepared using potassium ferricyanide (0.001%), Tween® 20 (0.5%), ProClin TM The buffer was 110 mM phosphate buffer containing 100 mM NaCl solution at pH 6.5, which also contained 950 (0.5%), anilinonaphthalene-1-sulfonate (ANS) (0.04%) and bovine serum albumin (BSA) (3.0%), and 0.002 g / Kg of IGFBP7-HRP conjugate.

[0200] (Detection of Analyte) To obtain the amount of an exemplary analyte present in a sample, two assays are performed in succession. A first coated container is provided from the solid phase, a patient sample is dispensed into the first container, and a detection antibody solution reagent containing a first detection antibody that binds to a first analyte conjugated with horseradish peroxidase (e.g., TIMP2 HRP conjugate) is then dispensed into the first container containing the patient sample. A second coated container is then provided from the solid phase, a patient sample is dispensed into the second container, and a detection antibody solution containing a second detection antibody that binds to a second analyte conjugated with horseradish peroxidase (e.g., IGFBP7 HRP conjugate) is then dispensed into the second container containing the patient sample. Both the first and second wells are incubated at 37° C. for 8 minutes to promote the formation of a capture antibody-analyte-detection antibody complex immobilized on the surface of each well. Each container is then washed and aspirated to remove excess detection antibody solution. A signal reagent containing a luminescent substrate (luminol derivative and persalt salt) and a solution containing an electron transfer agent as described in U.S. Pat. No. 5,846,756 is added to each well. A detection reagent (HRP), conjugated to each detection reagent, catalyzes the oxidation of the luminol derivative and generates light that is emitted from the well. An electron transfer agent (substituted acetanilide) present in the signal reagent increases the level of light generated from the luminol derivative and prolongs the emission of light from the well. The light emission is measured for each well. Each container is placed near a luminometer (Vitros® 3600 Immunodiagnostic System or Vitros® Integrated System) so that the luminometer can read the amount of light emitted from each container.

[0201] Due to the efficiency of the present invention, measured amounts of both exemplary analytes, TIMP2 and IGFBP7, can be obtained in 16 minutes using an automated immunodiagnostic device (i.e., the Vitros® 3600 Immunodiagnostic System or the Vitros® 5600 Integrated System) as shown in Table 1.

[0202] Table 1: Conditions of an exemplary method and times of first detection results obtained by multiple automated immunodiagnostic devices. [Table 1]

[0203] (quantification) The amount of light emitted from each well is directly proportional to the concentration of the analyte present in the sample in each container. It may be desirable to obtain a single value derived from the two measured signals by a certain method. In this example, the detected amount of the first analyte in the first well is multiplied by the detected amount of the second analyte in the second well, and the total amount is divided by 1000 to obtain a single numerical value. For example, the following formula can be used to obtain a single numerical value for the amount of the first and second analytes present in the sample: Numeric value = ([Analyte 1] × [Analyte 2]) / 1000 Units = (ng / mL) 2 / 1000

[0204] Example 2: Determining the amount of two separate analytes in a sample Using the methods of the present disclosure, immunoassay techniques are used to detect the presence of two separate analytes (TIMP-2 and IGFBP-7) present in a urine sample, as shown in Figures 1-3, where a solid phase is provided containing wells having immobilized on the surface of each well a mouse monoclonal antibody (capture antibody) specific for the analytes TIMP2 or IGFBP7.

[0205] 35 μL of urine (sample) obtained from a subject was dispensed into the first well of the solid phase together with an anti-TIMP2 rabbit monoclonal detection antibody conjugate labeled with horseradish peroxidase. During incubation at 37° C. for 8 minutes, analytes present in the sample bind to the TIMP2 detection antibody to form analyte-detection antibody complexes within the well. These complexes are captured by a mouse monoclonal anti-TIMP2 capture antibody immobilized on the surface of the well.

[0206] 20 μL of urine obtained from a subject is dispensed into another well of the solid phase along with a horseradish peroxidase-labeled anti-IGFBP7 mouse monoclonal detection antibody conjugate. The mixture is incubated at 37° C. for 8 minutes to form an analyte-detection antibody complex that is captured by the mouse monoclonal anti-IGFBP7 capture antibody immobilized on the well surface.

[0207] After incubation, unbound material is removed by washing and the remaining solution in each well is thoroughly aspirated.

[0208] The amount of horseradish peroxidase bound by the conjugate to each detection antibody is measured by luminescence detection. As shown in Figure 3, 100 μL of signal reagent containing luminescent substrates (luminol derivatives and persalt salts) and 100 μL of enhancer solution containing electron transfer agent (substituted acetanilide) are added to each well and the wells are incubated for 4-5 minutes. Horseradish peroxidase conjugated to each detection antibody catalyzes the oxidation of the substrate (luminol derivative) and generates light (luminescence) of a detectable wavelength in each well. In addition, the electron transfer agent present in the signal reagent mixture increases the level of light generated by the substrate and prolongs the emission of light. The first and second wells each emit light signals of the same wavelength and are measured consecutively by a luminometer.

[0209] Measurement of the luminescent signal from each well is performed by placing a first well near a luminometer as it would be in an automated immunodiagnostic device (e.g., the Vitros® 3600 Immunodiagnostic System). The luminometer then reads the luminescent signal of the first well. A second well can then be placed near the luminometer, which in turn can read the luminescent signal emitted from the second well. In a quantification assay, a positive or signal is provided by the luminometer or automated immunodiagnostic device to indicate whether or not an analyte is present in the sample being tested if present, and not if absent.

[0210] Example 3: Quantification of Analytes For quantification of the measurements, fit a calibration curve using a modified 4 or 5 parameter log-logistic program such as that provided by the manufacturer (Ortho Clinical Diagnostics, Assay Data Disk (included in the Vitros® 3600 Immunodiagnostic System, Vitros® 5600 Integrated System)) or magnetic card (Vitros® ECiQ instrument). Here, the signal levels form a calibrator to adjust the master curve provided by the program, and the software determines the concentration of the analyte in each well by applying the luminescence signal obtained from each well to the calibration curve.

[0211] The amount of light emitted is directly proportional to the amount of horseradish peroxidase-conjugated detection antibody that binds to the antigen (analyte) present in the sample, and therefore the amount of light measured from each well is proportional to the concentration of analyte present in each sample (i.e., nanograms of analyte per milliliter of urine sample).

[0212] The measured amount of each analyte can also be calculated as a single number, where the detected amount of a first analyte in a first well is multiplied by the detected amount of a second analyte in a second well, and then the total amount is divided by 1000. For example, the following formula can be used to arrive at a single number for the amount of first and second analytes present in a sample: Numeric value = ([TIMP-2] x [IGFBP-7]) / 1000 Units = (ng / mL) 2 / 1000

[0213] [Note] [Appendix 1] providing a solid phase comprising two containers, each of the two containers comprising a first capture antibody and a second capture antibody immobilized thereon, the first capture antibody binding to a first analyte and the second capture antibody binding to a second analyte, the first analyte and the second analyte being different; providing a sample, a portion of the sample being provided in each of the two containers; providing on the solid phase a first detection antibody specific for the first analyte and a second detection antibody specific for the second analyte, wherein a portion of the sample in the first container is contacted with the first detection antibody and a portion of the sample in the second container is contacted with the second detection antibody, and wherein the first detection antibody and the second detection antibody generate the same detectable signal; detecting the first analyte and the second analyte in the sample; 4. A method for detecting an analyte, comprising:

[0214] [Appendix 2] The two containers are tapered containers. The method according to claim 1,

[0215] [Appendix 3] Quantifying the amount of analyte detected in each of the two containers. The method according to claim 1,

[0216] [Appendix 4] the quantifying step includes obtaining a detected amount of analyte for each container and providing a single numerical value. The method according to claim 3,

[0217] [Appendix 5] and dispensing into each of the two containers a luminescent substrate and an electron transfer agent, wherein the luminescent substrate generates a detectable signal in each of the first container and the second container. The method according to claim 1,

[0218] [Appendix 6] the detectable signal in each of the first and second containers is light; The method according to claim 5, characterized in that

[0219] [Appendix 7] the detectable signal in each of the first and second containers is the same wavelength of light; 7. The method according to claim 6,

[0220] [Appendix 8] the amount of the first analyte in the sample is directly proportional to the amount of light detected in the first container. 8. The method according to claim 7,

[0221] [Appendix 9] the amount of the second analyte in the sample is directly proportional to the amount of light detected in the second container. 9. The method according to claim 8,

[0222] [Appendix 10] the luminescent substrate is oxidized by contacting the detection antibody in the first container or by contacting the detection antibody in the second container; The method according to claim 5, characterized in that

[0223] [Appendix 11] The luminescent substrate comprises a luminol derivative and a persalt. The method according to claim 5, characterized in that

[0224] [Appendix 12] The electron transfer agent is a substituted acetanilide. The method according to claim 5, characterized in that

[0225] [Appendix 13] the first detection antibody and the second detection antibody are each conjugated to horseradish peroxidase; The method according to claim 1,

[0226] [Appendix 14] prior to detecting the amount of each of the first analyte in the sample and the second analyte in the sample, further comprising the step of incubating a portion of the sample in the first container with the first detection antibody and a portion of the sample in the second container with the second detection antibody for at least 5 minutes. The method according to claim 1,

[0227] [Appendix 15] The incubation lasts for 8 minutes. 15. The method according to claim 14,

[0228] [Appendix 16] the amount of the first analyte in the sample and the amount of the second analyte in the sample are detected sequentially. The method according to claim 1,

[0229] [Appendix 17] the sample, the first detection antibody specific for the first analyte, and the second detection antibody specific for the second analyte are simultaneously provided to the solid phase; The method according to claim 1,

[0230] [Appendix 18] The sample is a body fluid sample. The method according to claim 1,

[0231] [Appendix 19] The body fluid sample is urine. 19. The method according to claim 18,

[0232] [Appendix 20] the first capture antibody, the second capture antibody, the first detection antibody, and the second detection antibody are monoclonal antibodies; The method according to claim 1,

[0233] [Appendix 21] The solid phase is configured for use in an automated immunodiagnostic device. The method according to claim 1,

[0234] [Appendix 22] the portion of the sample is provided into each of the two containers by an automated immunodiagnostic device; 22. The method according to claim 21,

[0235] [Appendix 23] the automated immunodiagnostic device providing the first detection antibody to a portion of the sample in the first container and providing the second detection antibody to a portion of the sample in the second container; 23. The method of claim 22,

[0236] [Appendix 24] the detecting includes determining, by the automated immunodiagnostic device, the amount of each of the first analytes in the sample and detecting the amount of the second analyte in the sample. 24. The method according to claim 23,

[0237] [Appendix 25] the step of detecting each analyte comprises providing the solid phase to an automated immunodiagnostic device, the automated immunodiagnostic device detecting the first analyte in the sample in the first container and detecting the second analyte in the sample in the second container. The method according to claim 1,

[0238] [Appendix 26] the automated immunodiagnostic device includes a fluorometer, the fluorometer sequentially detecting each of the first analyte and the second analyte; 26. The method according to claim 25,

Claims

[Claim 1] providing a solid phase in the form of a closed straw or sleeve comprising two tapered vessels, each of the two tapered vessels comprising a first capture antibody and a second capture antibody immobilized thereon, the first capture antibody binding to a first analyte and the second capture antibody binding to a second analyte, the first analyte and the second analyte being different; providing a sample, a portion of the sample being provided in each of the two tapered vessels; providing on said solid phase a first detection antibody specific for said first analyte and a second detection antibody specific for said second analyte, wherein a detectable label that produces a detectable signal is conjugated to said first detection antibody and said second detection antibody, wherein a portion of said sample in a first of said two tapered vessels is contacted with said first detection antibody and a portion of said sample in a second of said two tapered vessels is contacted with said second detection antibody, and wherein said first detection antibody and said second detection antibody produce the same detectable signal; detecting the first analyte and the second analyte in the sample; 1. A method for detecting an analyte in a body fluid sample, comprising:

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