Systems and methods for analyte assessment, regardless of competing binders

Abstract

The present disclosure generally relates to systems and methods for determining or assessing analytes in a sample, e.g., in the presence of competitive binders able to bind to the analyte. In some cases, a sample suspected of containing an analyte and a competitive binder of the analyte may be partitioned within an aqueous multiphase partitioning system, which may cause both the analyte and the competitive binder to become partitioned within the various phases of the partitioning system. Within each of the phases, the competitive binder may be able to bind to the analyte, although the exact amounts of binding of the competitive binder with the analyte may vary depending on the physicochemical properties of each phase, i.e., there may be both free analyte and analyte bound to the competitive binder present in a given phase. Surprisingly, despite the different amounts of partitioning of the analyte and the competitive binder within the phases of the partitioning system, as well as the different concentrations of free analyte, analyte bound to the competitive binder, and free competitive binder in each of the phases of the partitioning system, it has been found that such partitioning systems may be effective for determining analytes in a sample in the presence of varying amounts of competitive binders.

Description

[0001] SYSTEMS AND METHODS FOR ANALYTE ASSESSMENT, REGARDLESS OF COMPETING BINDERS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 850,425, filed July 24, 2025, entitled “Systems and Methods for Analyte Assessment, Regardless of Competing Binders”; and U.S. Provisional Patent Application Serial No. 63 / 728,087, filed December 4, 2024, entitled “Systems and Methods for Analyte Assessment, Regardless of Competing Binders.” Each of these is incorporated herein by reference in its entirety.

[0004] FIELD

[0005] The present disclosure generally relates to systems and methods for determining analytes in a sample, e.g., in the presence of competitive binders.

[0006] BACKGROUND

[0007] The Enzyme-Linked ImmunoSorbent Assay (“ELISA”) is a well-known analytical technique that has been used for several decades. The ELISA assay typically uses a solidphase enzyme immunoassay (EIA) or similar techniques to detect the presence of an analyte (for example, a protein) in a sample, using antibodies directed against the analyte. ELISA has been widely used for a variety of applications, e.g., in diagnostics, medicine, pathology, biotechnology, or other uses. For example, ELISA has been used to detect and quantify a particular analyte in a complex mixture such as blood containing other analytes of the same type, e.g., other proteins. However, because of its reliance on antibodies that bind the analyte specifically, the presence of competing binders to that analyte (for example, other antibodies) can interfere with ELISA, rendering ELISA useless for detecting analytes in certain types of scenarios in which such competitive binders are expected to be present.

[0008] SUMMARY

[0009] The present disclosure generally relates to systems and methods for determining analytes in a sample, e.g., in the presence of competitive binders. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0010] Certain aspects are generally directed to a method. In one set of embodiments, the method comprises exposing a sample comprising an analyte and a binder of the analyte to an aqueous multiphase partitioning system, and determining partitioning of the analyte in the aqueous multiphase partitioning system.

[0011] #14460968vl The method, in another set of embodiments, comprises exposing a sample comprising an analyte and a binder of the analyte to an aqueous multiphase partitioning system, and assaying the aqueous multiphase partitioning system for the analyte using an immunological assay.

[0012] According to another set of embodiments, the method comprises determining partitioning of Thyroglobulin in an aqueous multiphase partitioning system.

[0013] According to yet another set of embodiments, the method comprises determining partitioning of Folate Receptor alpha in an aqueous multiphase partitioning system.

[0014] The method, in still another set of embodiments, comprises exposing a sample taken from a subject to an aqueous multiphase partitioning system, wherein the sample comprises Thyroglobulin, and determining partitioning of the Thyroglobulin in the aqueous multiphase partitioning system.

[0015] The method, in still another set of embodiments, comprises exposing a sample taken from a subject to an aqueous multiphase partitioning system, wherein the sample comprises Folate Receptor alpha, and determining partitioning of the Folate Receptor alpha in the aqueous multiphase partitioning system.

[0016] In one set of embodiments, the method includes exposing a sample comprising an analyte and a binder of the analyte to an aqueous multiphase partitioning system, and determining partitioning of the analyte in the aqueous multiphase partitioning system.

[0017] In another set of embodiments, the method comprises exposing a sample comprising an analyte and a binder of the analyte to an aqueous multiphase partitioning system, and assaying the aqueous multiphase partitioning system for the analyte using an immunological assay.

[0018] In yet another set of embodiments, the method comprises determining partitioning of Thyroglobulin in an aqueous multiphase partitioning system. The method, in still another set of embodiments, includes exposing a sample taken from a subject to an aqueous multiphase partitioning system, where the sample comprises Thyroglobulin, and determining partitioning of the Thyroglobulin in the aqueous multiphase partitioning system.

[0019] In one set of embodiments, the method is a method of diagnosing a subject. In certain cases, the method comprises exposing a sample taken from a subject to an aqueous multiphase partitioning system, where the sample contains Thyroglobulin and Thyroglobulin- binding autoantibodies, determining partitioning of the Thyroglobulin in the aqueous multiphase partitioning system, and diagnosing the subject based on partitioning of the Thyroglobulin.

[0020] #14460968vl In another set of embodiments, the method is a method of treating a subject. In certain instances, the method includes exposing a sample taken from a subject to an aqueous multiphase partitioning system, where the sample contains Thyroglobulin and Thyroglobulin- binding autoantibodies, determining partitioning of the Thyroglobulin in the aqueous multiphase partitioning system, and treating the subject for thyroid cancer based on partitioning of the Thyroglobulin.

[0021] In yet another set of embodiments, the method is a method of treating a subject. In one set of embodiments, the method comprises exposing a sample taken from a subject to an aqueous multiphase partitioning system, wherein the sample contains Thyroglobulin and Thyroglobulin-binding autoantibodies; determining partitioning of the Thyroglobulin in the aqueous multiphase partitioning system; and treating the subject for thyroid cancer based on partitioning of the Thyroglobulin.

[0022] In yet another set of embodiments, the method comprises exposing a sample taken from a subject to an aqueous multiphase partitioning system, where the sample contains Thyroglobulin and Thyroglobulin-binding autoantibodies; determining partitioning of the Thyroglobulin in the aqueous multiphase partitioning system; and diagnosing the subject based on partitioning of the Thyroglobulin.

[0023] In yet another set of embodiments, the method comprises determining partitioning of Folate Receptor alpha in an aqueous multiphase partitioning system. The method, in still another set of embodiments, includes exposing a sample taken from a subject to an aqueous multiphase partitioning system, where the sample comprises Folate Receptor alpha, and determining partitioning of the Folate Receptor alpha in the aqueous multiphase partitioning system.

[0024] In one set of embodiments, the method is a method of diagnosing a subject. In certain cases, the method comprises exposing a sample taken from a subject to an aqueous multiphase partitioning system, where the sample contains Folate Receptor alpha and Folate Receptor alpha-binding autoantibodies, determining partitioning of the Folate Receptor alpha in the aqueous multiphase partitioning system, and diagnosing the subject based on partitioning of the Folate Receptor alpha.

[0025] In another set of embodiments, the method is a method of treating a subject. In certain instances, the method includes exposing a sample taken from a subject to an aqueous multiphase partitioning system, where the sample contains Folate Receptor alpha and Folate Receptor alpha-binding autoantibodies, determining partitioning of the Folate Receptor alpha

[0026] #14460968vl in the aqueous multiphase partitioning system, and treating the subject for Ovarian cancer based on partitioning of the Folate Receptor alpha.

[0027] In yet another set of embodiments, the method is a method of treating a subject. In one set of embodiments, the method comprises exposing a sample taken from a subject to an aqueous multiphase partitioning system, wherein the sample contains Folate Receptor alpha and Folate Receptor alpha-binding autoantibodies; determining partitioning of the Folate Receptor alpha in the aqueous multiphase partitioning system; and treating the subject for Ovarian cancer based on partitioning of the Folate Receptor alpha.

[0028] In yet another set of embodiments, the method comprises exposing a sample taken from a subject to an aqueous multiphase partitioning system, where the sample contains Folate Receptor alpha and Folate Receptor alpha-binding autoantibodies; determining partitioning of the Folate Receptor alpha in the aqueous multiphase partitioning system; and diagnosing the subject based on partitioning of the Folate Receptor alpha.

[0029] In another aspect, the present disclosure encompasses methods of making one or more of the embodiments described herein, for example, an aqueous partitioning system. In still another aspect, the present disclosure encompasses methods of using one or more of the embodiments described herein, for example, an aqueous partitioning system.

[0030] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0033] Fig 1 illustrates that selective segregation of a competitive binder in an interface layer allows accurate measurement of analyte partitioning, in accordance with certain embodiments; and

[0034] Figs. 2A and 2B demonstrate that the analyte partition (“K”) is not dependent on concentration of autoantibodies, in some embodiments; and

[0035] #14460968vl Figs. 3 A-3B illustrates Receiver Operating Characteristic (ROC) curves for thyroid and ovarian cancer, respectively, in accordance with certain embodiments.

[0036] DETAILED DESCRIPTION

[0037] The present disclosure generally relates to systems and methods for determining or assessing analytes in a sample, e.g., in the presence of competitive binders able to bind to the analyte. In some cases, a sample suspected of containing an analyte and a competitive binder of the analyte may be partitioned within an aqueous multiphase partitioning system, which may cause both the analyte and the competitive binder to become partitioned within the various phases of the partitioning system. Within each of the phases, the competitive binder may be able to bind to the analyte, although the exact amounts of binding of the competitive binder with the analyte may vary depending on the physicochemical properties of each phase, i.e., there may be both free analyte and analyte bound to the competitive binder present in a given phase. Surprisingly, despite the different amounts of partitioning of the analyte and the competitive binder within the phases of the partitioning system, as well as the different concentrations of free analyte, analyte bound to the competitive binder, and free competitive binder in each of the phases of the partitioning system, it has been found that such partitioning systems may be effective for determining analytes in a sample in the presence of varying amounts of competitive binders.

[0038] As discussed in more detail herein, aqueous partitioning is an analytical technique in which an analyte, such as small or large molecule, is allowed to migrate and partition into the separate aqueous phases that can arise when water is mixed with polymer, salts and other additives. For example, for biological molecules, aqueous partitioning can provide an environment in which the molecule can be separated in two or more aqueous phases, e.g., depending on intermolecular interactions between the distinct aqueous solvents and the analyte, etc., as discussed herein. Aqueous partitioning can be used to determine molecules, or changes in the structure of the molecules, e.g., in proteins or other biological molecules. The partitioning of an analyte between the aqueous phases can be quantified, for example, via a ratio of the analyte concentrations in the phases, which may be determined as an index, e.g., the partition coefficient. This coefficient may be specific to an aqueous system composition and the analyte itself. The concentration of the analyte in each of the phases can be determined by various techniques, e.g., known to those skilled in the art.

[0039] If an analyte is introduced into an aqueous partitioning system as a part of a complex mixture of analytes, an analyte-specific analytical technique can be used to determine the concentrations within various phases to determine the partition coefficient. Accordingly,

[0040] #14460968vl certain embodiments as discussed herein are generally directed to accurately determining the partition coefficients of analytes in the presence of competing binders. In some cases, this may be achieved where individual concentrations of the analytes are determined without requiring first isolation of the analyte from competing binders or otherwise negating their potential interactions. In some cases, techniques such as ELISA, or other techniques, may be used to determine individual concentrations in various phases of the aqueous partitioning system.

[0041] Accordingly, certain embodiments are generally directed to systems and methods for detecting an analyte in the presence of a competitive binder. Prior art techniques such as ELISA depend on determining a chemical interaction between an analyte and other components of the assay to determine the analyte. For example, ELISA typically functions by using antibodies directed at an analyte (e.g., specifically). The analyte and the antibodies interact with each other to form a complex, which can be detected, for example, by detecting a change in color (e.g., using a secondary antibody or an enzyme covalently linked to the antibody). However, because of their reliance on such interactions (for example, specific antibody binding), techniques such as ELISA cannot be performed when an analyte is in the presence of a competitive binder (for example, an endogenous enzyme or antibody also able to recognize the analyte) that interferes with such interactions between the analyte and the components of the assay. Accordingly, techniques such as ELISA cannot be used to provide reliable or accurate results for samples containing an analyte and a competitive binder to that analyte.

[0042] In contrast, certain aspects of the present disclosure are generally drawn to systems and methods for determining an analyte in the presence of a competitive binder. In some cases, the analyte and the binder are partitioned in a system, such as an aqueous multiphase partitioning system. It should be understood that typically, two or more phases of the system will contain some concentration of analyte and some concentration of bound analyte. Furthermore, due to the presence of the binder, the amount of partitioning of the analyte between the phases of the system may change (i.e., as compared to a substantially identical system in which the analyte is still present, but the binder is completely absent). This change in partitioning behavior of the analyte due to the presence of the binder cannot be easily predictable, as compared to a substantially identical system in which the analyte is still present but the binder is completely absent.

[0043] Without wishing to be bound by any theory, it is believed that hydrophobic interactions may stabilize some antigen-antibody complexes. However, while the Fv regions

[0044] #14460968vl of autoantibodies harness hydrophobic interactions for less specific, multi-target binding (e.g., “polyreactive”), highly specific ELISA antibodies are often engineered to minimize non-specific surface hydrophobicity, thereby increasing their affinity and specificity. However, in partition systems such as those discussed herein, hydrophobic interactions may play an important in forming the interface between the two aqueous phases, directly stacking hydrophobic compounds between two liquid phases, often through aggregation of precipitation processes. In some embodiments, differential hydrophobic driving forces of autoantibodies versus ELISA antibodies may be used to create a hydrophobic pump that can selectively capture autoantibodies preferentially over ELISA antibodies. Thus, the equilibrium of analyte-bound and free autoantibodies may be shifted (Le Chatelier's principle) towards the reactants in certain embodiments, which may help to remove autoantibodies to the interface between the two aqueous phases.

[0045] Fig. 1 illustrates the partitioning behavior of an analyte and its endogenous or exogenous binders within an aqueous two-phase system (ATPS), as an illustrative nonlimiting example. This system in this non-limiting example may facilitate the selective exclusion of these binders from the solution phases into the interface. Within this interface, these binders (that might compete with assay binders) may undergo a phase transition in some cases, e.g., precipitation, which may render them unavailable or less able for competitive binding interactions with the analyte. Thus, this approach may allow an assessment of the analyte’s concentration, e.g., by minimizing or eliminating interference from competitive binders.

[0046] The phases can be analyzed, for example, by taking an aliquot from each. In some cases, an immunoassay binder may be added to some or all of the aliquots, e.g., to determine the analyte in the phases. The phases may be analyzed to determine the analyte in each phase, e.g., in the presence of binder. In some cases, concentrations and / or amounts of analyte in each phase may be used to define a partition coefficient K, which is the ratio of the concentrations of the analyte between the phases.

[0047] Given the differences and interactions between the analyte, the competitive binder, the first phase, and the second phase, and the independence of their partitioning behavior, it is unexpected that such a partitioning system can be used to determine an analyte in a sample despite the presence of competitive binders. Many other techniques that rely on competitive binders, such as ELISA, cannot function effectively while in the presence of additional competitive binders. Surprisingly, however, aqueous multiphase partitioning systems can be

[0048] #14460968vl used in certain embodiments to determine analytes while in the presence of competitive binders.

[0049] The above discussion is a non-limiting example of one embodiment of the present disclosure generally directed to systems and methods for determining analytes in a sample, e.g., in the presence of competitive binders. However, other embodiments are also possible, as discussed herein.

[0050] For example, certain aspects are generally directed to the determination of one or more analytes, typically in the presence of one or more binders of those analytes. As noted above, the presence of binders (for example, competitive binders, and / or other types of binders) can significantly interfere with immunological assays (e.g., ELISA) that quantify the analyte that use binding of the analyte to an antibody. However, certain embodiments as discussed herein are generally directed to the use of aqueous multiphase partitioning systems, comprising two phases, or more than two phases, that can be used to partition a mixture of bound and free analytes between two or more of the phases, along with determination of these in the phases. Such determinations may be qualitative and / or quantitative in various embodiments, for example determining the presence of, determining a concentration or an amount, etc. The partitioning systems may include a combination of polymers, buffers, and / or salts, e.g., as discussed herein. The analyte and / or the binder may, for example, be quantified in the phases, which may allow for a calculation of a ratio of the total quantities in each phase (e.g., a partition coefficient). Surprisingly, such systems may reduce or eliminate the interference of the binder to the analyte, even though such interference continues and is measurable in the individual phases. This is different from certain types of competitive ELISA assays (for example, cELISA or inhibition ELISA), which are used for detecting small antigens with only a single antibody epitope. Such ELISA assays cannot accommodate the presence of two or more different antibodies due to steric hindrance or small-molecule antigens in complex sample mixtures.

[0051] In some aspects, an analyte within a composition may be determined, where the composition comprises an analyte and a binder of the analyte (for example, Thyroglobulin and a Thyroglobulin-binding antibody, or folate receptor alpha and its binding antibody, such as discussed herein). The analyte may be a molecule, such as a biomolecule, to which a determination of the analyte, e.g., qualitatively and / or quantitatively, is desired. For example, the analyte may be a protein, a peptide, a nucleic acid, a carbohydrate, a sugar, a lipid, or the like.

[0052] #14460968vl The composition may arise from a sample taken from a subject. Certain systems and methods as discussed herein may be able to determine the analyte, despite the presence of the binder. The analyte may be any molecular species, whether biological, biochemical, chemical, or other species, and those of ordinary skill in the art will understand how the disclosure can be used in the context of other molecules. The composition may originate from a biological fluid (or other sample), such as a human clinical sample or other biological fluid, tissue, cells, a subject, etc., or the mixture may be a synthetic mixture. For example, the sample of biological fluid may comprise fluids such as whole blood, blood serum, blood plasma, saliva, nasal fluid, sputum, urine, CNS fluid, breast nipple aspirate fluid, cerebral spinal fluid, semen, or the like. The mixture can come from a biological system (e.g., a subject) which includes, but is not limited to, a human or non-human mammal. Non-human mammals include, but are not limited to, a dog, cat, horse, cow, pig, sheep, goat, chicken, primate, rat, and mouse.

[0053] A binder is usually able to bind to an analyte, e.g., in a specific association, such as with a biomolecule and its binding partner. Specific binding generally describes a binder that does not cross react substantially with any biomolecule other than the analyte or analytes specified. Generally, molecules which preferentially bind to each other, such as an analyte and a binder, are referred to as specific binding pairs. Such pairs include, but are not limited to, an antibody and its antigen, a lectin and a carbohydrate which it binds, an enzyme and its substrate, and a hormone and its cellular receptor.

[0054] In one set of embodiments, the binder is a competitive binder. For example, the competitive binder may bind to an analyte in such a way that another binder (e.g., an antibody, such as an ELISA antibody) is unable to also bind to the analyte while the competitive binder is bound to the analyte. In contrast, competitive binders cannot be used with prior art techniques such as ELISA; when the ELISA antibody is blocked from being able to interact with the analyte due to the presence of the competitive binder, techniques such as ELISA can produce erroneous measurements. However, as discussed herein, the use of aqueous multiphase partitioning systems may allow for accurate determination of analytes, even in the presence of a competitive binder to the analyte. In addition, it should be understood that the binder need not be a competitive binder. In other embodiments, the binder may be an uncompetitive binder, a noncompetitive binder, an allosteric binder, or the like.

[0055] In some cases, the competitive binder may be present in a sample; for example, the competitive binder may be one that is naturally-occurring. As a non-limiting example, as

[0056] #14460968vl discussed herein, blood or other samples taken from subjects suspected of having or being at risk of thyroid cancer are often tested to determine Thyroglobulin, which is often used as a tumor marker. However, samples frum such subjects also often contain Thyroglobulin antibodies, which can bind to Thyroglobulin.

[0057] As another non-limiting example, blood or other samples taken from subjects suspected of having or being at risk of certain cancers, such as ovarian cancer, are often tested to determine folate receptor alpha, which can be used as a cancer market. However, samples from such subjects also often contain folate receptor alpha (FRa) autoantibodies (FRAAs), which can bind to folate receptor alpha.

[0058] Accordingly, a variety of compounds may interact with an analyte, e.g., as a competitive binder. For example, in one embodiment, the binder may be an antibody or an antibody fragment, e.g., able to specifically recognize the analyte. An antibody may include a protein or glycoprotein having one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below (i.e. toward the Fc domain) the disulfide linkages in the hinge region to produce F(ab)’2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)’2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab’)2 dimer into an Fab’ monomer. The Fab’ monomer is essentially a Fab with part of the hinge region. While various antibody fragments are defined in terms of the digestion of an intact antibody, such fragments may also be synthesized de novo, for example, chemically by utilizing recombinant DNA methodology, by “phage display” methods, or the like. Examples of antibodies include single chain antibodies, e.g., single chain Fv (scFv)

[0059] #14460968vl antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. Other examples include monoclonal antibodies and / or polyclonal antibodies, or fragments thereof, nanobodies, bispecific antibodies, or the like.

[0060] However, it should be understood that in other embodiments, other types of binders may be present, e.g., instead of or in addition to antibodies or antibody fragments. Nonlimiting examples include nucleic acid aptamers, affimers (peptide aptamers), DARPins, or the like. For example, an apatamer is an oligomer of a nucleic acid that is able to bind to a specific target molecule. An aptamer may be formed from nucleic acids such as DNA, RNA, XNA, or the like, and / or a peptide. One or more than one type of nucleic acid and / or peptide may be present. An affimer is a small protein or peptide that is able to bind to a target protein.

[0061] As previously discussed, aqueous multiphase partitioning systems may be used in various aspects to determine one or more analytes (or other species). The partition system may have two aqueous phases, or more than two aqueous phases in some embodiments. For instance, one or more analytes may be determined within an aqueous multiphase partitioning system, e.g., by determining the amount and / or concentration of the analytes in each of the phases using techniques such as those described herein. In addition, in some cases, one or more of the phases of the partitioning systems may be a non-aqueous phase.

[0062] Aqueous multiphase partitioning systems are known to those of ordinary skill in the art, and can arise in aqueous mixtures of different water-soluble polymers or a single polymer and a specific salt. When two or more certain polymers, e.g., dextran (“Dex”) and polyethylene glycol (“PEG”), or one or more certain polymers and one or more inorganic salts, e.g. polyvinylpyrrolidone (“PVP”) and sodium sulfate, are mixed in water above certain concentrations, the mixture can separate into two or more immiscible aqueous phases under certain conditions. There may be, in certain instances, a discrete interfacial boundary separating any two phases, for example, such that one is rich in one polymer and the other phase is rich in the other polymer or the inorganic salt. The interface or interfacial boundary between the aqueous phases may possess properties not available in the adjacent aqueousbased phases in certain embodiments. For example, the interface may provide a location in the aqueous partitioning system suitable for hosting of proteins or other molecules with hydrophobic moieties, such as antibodies. In some cases, the interface may be a region that is less hydrophilic, relative to the bulk of the aqueous phases, which may promote hydrophobic moieties to localize to the interface, e.g., to minimize interactions with the aqueous phases.

[0063] #14460968vl The aqueous solvent in one or more phases may provide a medium suitable for biological products. Two-phase systems can also be generalized to multiple phase system by using different chemical components, and aqueous systems with a dozen or more phases are known in the art.

[0064] When a species, such as an analyte, is introduced into such a two-phase system, it may distribute between two phases, and this understanding can be extended to three or more phases. In this and other systems, the analyte can be found at different concentrations within each phase, or can be at the same concentration within each phase. Partitioning of an analyte can be characterized by the partition coefficient “K,” defined as the ratio between the concentrations of the solute the two immiscible phases at equilibrium. It has previously been shown that phase separation in aqueous polymer systems may result from different effects of two polymers (or a single polymer and a salt) on the water structure (B. Zavlavsky, Aqueous Two-Phase Partitioning: Physical Chemistry and Bioanalytical Applications, Marcel Dekker, New York, 1995). As a result of the different effects on water structure, the solvent features of aqueous media in the coexisting phases can differ from one another. The difference between phases may be demonstrated by techniques such as dielectric, solvatochromic, potentiometric, and / or partition measurements. In some cases, the interface between two phases may provide distinct properties, for example, that are suitable for hosting of proteins or other entities having significant content of hydrophobic moi eties.

[0065] Without being bound by theory, Fig. 1 illustrates a mixture containing a free analyte, a competitive binder such as antibody, and a complex of such analyte and a binder being partitioned in an aqueous partitioning system, in accordance with one embodiment. In this example system, the binder may preferentially segregate to the interface, e.g., if the binder contains hydrophobic moieties. The interfacial area may thus serve as a “sink” to reduce or remove the binder from the aqueous phases. If the complex of the analyte and binder is in equilibrium with free binder in solution, then reducing or removing the binder may result in reducing the presence of the complex in the aqueous phases, e.g., under certain conditions, which may yield more purified free analyte that may partition between the phases. In some embodiments, the partition coefficient of the analyte may be determined based on the concentration of the analyte using, for example, ELISA, even when the original mixture contains both analyte and a binder to the analyte.

[0066] The basic rules of solute partitioning in aqueous two-phase systems have been shown to be similar to those in water-organic solvent systems (which can also be used as systems in the present disclosure), and can be extended to three or more phases in some embodiments.

[0067] #14460968vl However, the differences that do exist in the properties of the two phases in aqueous polymer systems are often very small, relative to those observed in water-organic solvent systems, as would be expected for a pair of solvents of the same (aqueous) nature. The small differences between the solvent features of the phases in aqueous two-phase or multiphase systems can be modified in some cases so as to amplify the observed partitioning that results when certain structural features are present.

[0068] It is known that the polymer and salt compositions of each of the phases usually depend upon the total polymer and / or salt composition of an aqueous system. The polymer and / or salt composition of a given phase, in turn, usually governs the solvent features of the aqueous media in this phase. These features include, but are not limited to, dielectric properties, solvent polarity, ability of the solvent to participate in hydrophobic hydration interactions with a solute, ability of the solvent to participate in electrostatic interactions with a solute, and hydrogen bond acidity and basicity of the solvent. All these and other solvent features of aqueous media in the coexisting phases may be manipulated in certain embodiments by selection of polymer and salt composition of an aqueous partitioning system. These solvent features of the media may govern the sensitivity of a given aqueous partitioning system toward a particular type of solvent accessible chemical groups in the receptor. This sensitivity, type, and topography of the solvent accessible groups in two different proteins, for example, can determine the possibility of separating proteins in a given aqueous two partitioning system.

[0069] In some cases, a particularly sensitive system may be required, i.e., a system that is very sensitive to, and able to determine a partition coefficient or a relative measure of interaction with respect to, two very similar species. This sensitivity may be of importance when, for example, subtle differences are being detected between the conformational changes in a receptor induced by binding of closely related chemical compounds. The present disclosure provides, in one set of embodiments, efficient and successful systems and methods for screening aqueous phase compositions to identify and / or amplify differences between the compositions of two mixtures. By utilizing a wide variety of different conditions to screen each analyte, e.g., as described herein, different partitioning behavior may be obtained reliably without the need to fully understand the underlying theory of aqueous partitioning systems, or any of the other related or substitutable techniques.

[0070] Evaluation of data from partitioning of an analyte can involve use of the partition coefficient, in some embodiments of the disclosure. (However, it should be understood that partition coefficients are not always required.) For example, the partition coefficient of an

[0071] #14460968vl analyte can be taken as the ratio of the analyte in first phase to that in the second phase in an aqueous partitioning system. When multiple phase systems are formed, there can be multiple independent partition coefficients, each of which can be defined between any two phases. It will be recognized that the partition coefficient for a given analyte may be constant if the conditions and the composition of the aqueous partitioning system to which it is subjected remain constant. The partition coefficient K, as used herein, is a specifically mathematically defined quantity as further described herein, and the term may include coefficients representing the relative measure of interaction between a species, such as an analyte, and at least two interacting components or phases. It should also be recognized that differences between partition coefficients of different species in two or more mixtures could indicate, in addition to potential structural changes, also binding or lack of binding of such analytes to other species in the mixtures, e.g., to binders within the mixture.

[0072] In a non-limiting example of one partitioning system, aqueous multiphase partitioning systems are known to be formable from a variety of substances. For example, in order to determine the partition coefficient of an analyte to be analyzed, concentrated stock solutions of all the components (polymer 1, e.g., dextran; polymer 2, e.g., PEG, polyvinylpyrrolidone, salts, etc.) in water can be prepared separately. The stock solutions of phase polymers, salts, etc. can be mixed in the amounts and conditions (e.g., pH from about 3.0 to about 9.0, temperature from about 4 °C to 60 °C, salt concentration from 0.001 to 5 mol / kg) appropriate to bring the system to the desired composition and vigorously shaken. The system can then be allowed to equilibrate (resolve the phases). Equilibration can be accomplished by allowing the solution to remain undisturbed, or it can be accelerated by centrifugation, e.g., for 2-30 minutes at about 1000 g to 4000 g, or higher. Aliquots of each settled (resolved) phase can be withdrawn from the upper and / or lower phases (or from one or more phases, if multiple phases are present). The concentration of analytes within the phases can be determined for one or more of the phases.

[0073] As mentioned elsewhere herein, aqueous multiphase (e.g., two-phase) partitioning systems are well-suited for use in many or most embodiments of the disclosure, but other partitioning systems can be used. Where terms such as “aqueous two-phase partitioning” or “aqueous multiphase partitioning” is used, it is to be understood that other systems can be used in other embodiments, such as those described herein. Partitioning of a biopolymer in aqueous two-phase systems may depend on its three-dimensional structure, type and topography of chemical groups exposed to the solvent, etc. Changes in the 3-D structure of a receptor induced by some effect, e.g., by binding of a ligand binding or by structural

[0074] #14460968vl degradation, also can change the topography of solvent accessible chemical groups in the biomolecule, or both the topography and the type of the groups accessible to solvent. One result of these changes may be an alteration in the partition behavior of the biomolecule or other species.

[0075] Different assay methods may be used to determine partition coefficients between a species (such as an analyte) and interacting components, e.g. in the form of the concentration of the targets in each phase of a multiphase system. The assays will often depend upon the identity and type of analytes that may be present. Examples of suitable assay techniques include, but are not limited to, spectroscopic, immunochemical, chemical, fluorescent, radiological, enzymatic assays, electrochemiluminescence immunoassays, radioimmunoassay, or the like.

[0076] The concentration of a species, such as an analyte, in two or more phases can be used to determine the partition coefficient of the sample under the particular system conditions. Since the partition coefficient reflects the ratio of the two concentrations, the absolute values are not typically required. It will be recognized that this can allow certain analytical procedures to be simplified, e.g., calibration can be eliminated in some instances. It also may have a significant advantage for negating the effect of natural variability in the absolute concentration in samples obtained from, e.g., various subjects, when comparing two or more samples, thus focusing on those changes detected as differences in the partition coefficient relevant to changes to the structure of the individual species in the samples.

[0077] It should be recognized by those of ordinary skill in the art that the steps in above description of obtaining the partition coefficient could be substituted by other steps or measurements. Depending on the size, volumes, amount of the analyte, detection system, discrete or continuous operation using either liquid-liquid or liquid-solid portioning, other processes that effectively result in results described herein could be developed. Such modifications and different processes should not limit the scope of this complete disclosure.

[0078] The partition coefficient can be compared with other partition coefficients in some cases. For example, a partition coefficient for an analyte can be compared to the partition coefficients for the analyte under different conditions, a partition coefficient for an analyte can be compared to the partition coefficients for the target analyte combined with a competitive binder of the analyte, a set of partition coefficients for a species can be compared to other sets of partition coefficients, etc. This comparative information can be obtained at the same time or near the same time and in the same system or a similar system as is used to determine the interaction characteristics of the analyte, or can be provided as pre-prepared

[0079] #14460968vl data in the form of charts, tables, or electronically stored information (available on the Internet, disc, etc.)

[0080] In one embodiment, proteins or other biomolecular mixtures from an experimental sample and from a reference sample (determined simultaneously, previously, or subsequently, as described above) may be caused to partition in a variety of different aqueous two-phase systems, e.g. formed by different types of polymers, such as Dextran and PEG or Dextran and Ficoll, by the same types of polymers with different molecular weights, such as Dextran-70 and PEG-600 or Dextran-70 and PEG-8,000, by the same polymers but containing different in type and / or concentration salt additives, different buffers of different pH and concentration, etc. The overall partition coefficients for the mixtures determined using a particular assay procedure (e.g., same for both samples) can be determined in all of the systems. In one embodiment, the systems displaying different partition coefficients for the mixtures under comparison may be selected as a separation medium, for example, for further fractionation and / or characterization of the mixtures. In another embodiment, mixtures are partitioned using one or more standard systems with known properties, e.g., those providing enhanced sensitivity levels towards hydrophobic or ionic interactions. In such a case, the individual partition coefficients of the species comprising the mixtures may be determined following separation of the mixtures in the phases and / or compared between two or more mixtures.

[0081] The reasons for the observed differences in the partition behavior of the two samples do not have to be scientifically characterized for such differences to be useful for many applications, e.g., for diagnostics. Such differences, resulting in partitioning behavior, may arise due to multiple reasons, including relative compositional, structural, or conformational differences in the samples when exposed to aqueous media of different solvent structures.

[0082] In addition, one set of embodiments as discussed herein are generally drawn to systems and methods of determining Thyroglobulin (Tg) in subject samples, in accordance with certain aspects. In some cases, Thyroglobulin may be present in a sample at a concentration of at least 0.1 ng / ml, at least 0.2 ng / ml, at least 0.3 ng / ml, at least 0.5 ng / ml, at least 1 ng / ml, at least 2 ng / ml, at least 3 ng / ml, at least 5 ng / ml, at least 10 ng / ml, at least 20 ng / ml, at least 30 ng / ml, at least 50 ng / ml, at least 75 ng / ml, at least 100 ng / ml, etc. As mentioned, Thyroglobulin is often found in association with Thyroglobulin-binding antibodies (TgAbs), which may be endogenously produced, e.g., by the subject. Accordingly, in certain embodiments, samples such as blood samples suspected of containing Thyroglobulin often also contain thyroglobulin-binding antibodies. Such endogenously-

[0083] #14460968vl produced antibodies can interfere with ELISA and other assay techniques that rely on Thyroglobulin-binding antibodies.

[0084] Several functional disorders and diseases of the thyroid are known, which can be determined in various embodiments, e.g., as discussed herein. Non-limiting examples include thyroid cancer, hormonal imbalance, hypothyroidism (Reduced thyroid function), hyperthyroidism (Increased thyroid function), autoimmune diseases such as Hashimoto's disease (chronic lymphocytic thyroiditis), and Graves' disease (toxic diffuse goiter). Additionally, most common thyroid cancers are papillary (PTC), follicular (FTC), anaplastic (ATC), and medullary (MTC). In addition, in some embodiments, the subject may be one that has experienced a partial or complete thyroidectomy.

[0085] Thus, certain embodiments are generally directed to systems and methods for diagnosing a subject, e.g., based on Thyroglobulin levels or concentrations, having or at risk of a Thyroid disease or disorder, such as described herein. In addition, in some embodiments, the subject may also be treated, for example, for the thyroid disease or disorder.

[0086] In addition, one set of embodiments as discussed herein are generally drawn to systems and methods of determining Folate Receptor alpha (FRa or FRa) in subject samples, in accordance with certain aspects. In some cases, Folate Receptor alpha may be present in a sample at a concentration of at least 0.1 ng / ml, at least 0.2 ng / ml, at least 0.3 ng / ml, at least 0.5 ng / ml, at least 1 ng / ml, at least 2 ng / ml, at least 3 ng / ml, at least 5 ng / ml, at least 10 ng / ml, at least 20 ng / ml, at least 30 ng / ml, at least 50 ng / ml, at least 75 ng / ml, at least 100 ng / ml, etc. As mentioned, folate receptor alpha is often found in association with Folate Receptor alpha autoantibodies, which may be endogenously produced, e.g., by the subject. Accordingly, in certain embodiments, samples such as blood samples suspected of containing folate receptor alpha often also contain Folate Receptor alpha autoantibodies. Such endogenously-produced antibodies can interfere with ELISA and other assay techniques that rely on antibodies that bind to folate receptor alpha.

[0087] A variety of cancers can be determined based on Folate Receptor alpha. These include, but are not limited to, ovarian cancer, endometrial cancer, lung cancer, breast cancer, or the like. Accordingly, certain embodiments are generally directed to systems and methods for diagnosing a subject having or at risk for cancer, e.g., based on folate receptor alpha levels or concentrations, for a cancer, e.g., described herein. In addition, in some embodiments, the subject may also be treated, for example, for the cancer.

[0088] #14460968vl In addition, in one embodiment, the subject may have or be at risk of cerebral folate deficiency (CFD). FRAAs are the most common cause of CFD. They may bind to folate receptor alpha, impairing its ability to transport folate into the cerebrospinal fluid, leading to abnormally low folate levels in the brain despite normal folate levels in the blood. Treatments for CFD include, but are not limited to, folinic acid (leucovorin calcium). Folinic acid can bypass the dysfunctional folate receptor alpha by utilizing the Reduced Folate Carrier (RFC) to enter the brain. Early intervention with folinic acid may lead to significant clinical improvement in neurological and developmental symptoms.

[0089] In another embodiment, the subject may have or be at risk of autism spectrum disorder (ASD). FRAAs are found in a significant percentage (around 70%) of children diagnosed with ASD. FRAAs can contribute to the neurological symptoms of ASD by causing cerebral folate deficiency. Maternal FRAAs during pregnancy may be associated with an increased risk of ASD in offspring, as these antibodies can cross the placenta and affect fetal brain development and folate transport.

[0090] In yet another embodiment, the subject may have or be at risk of pregnancy complications and / or neural tube defects (NTDs). FRAAs can block folate transport to the fetus, even with adequate maternal folic acid supplementation. This may contribute to subfertility, recurrent miscarriages, preterm birth, and an increased risk of neural tube defects (NTDs) in the developing fetus. Early testing for FRAAs in women planning pregnancy or during pregnancy may identify those at risk, allowing for targeted intervention with folinic acid to ensure sufficient folate supply to the fetus.

[0091] The subject may have or be at risk of other neuropsychiatric disorders in other embodiments. FRAAs have been found in some patients with treatment-resistant depression, suggesting a potential link between folate transport impairment and mood disorders. Folinic acid treatment may be a possible therapeutic strategy in these cases. Folate receptor autoantibodies have also been identified in children with pediatric acute-onset neuropsychiatric syndrome (PANS) and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS), indicating a possible autoimmune and folate-related component to these conditions.

[0092] Some embodiments are related to developing and determining characteristics (quantitative and / or qualitative) of a mixture that are obtained, for example, via processing using multiphase partitioning, which can reflect certain structural and functional characteristics of analytes that may be present within the sample. These characteristics can be used, for example, for establishing relationships between the composition of the sample

[0093] #14460968vl and the physiological state of the biological source of the sample, e.g., the state of health or disease of a subject, such as a human subject. These characteristics can also be used to design experimental conditions for subsequent fractionation of the mixtures into subsets enriched in the molecule(s) of interest for the purpose of the analysis, while simultaneously reduced in the total number of different molecule(s) in some cases. Certain systems and methods can also be useful for detecting, classifying, and / or predicting changes in samples containing certain analytes, such as Thyroglobulin. For example, the sample may be one associated with a particular disease (e.g., cancer) or physiological state of a living organism, cells, tissues, or biological liquids. Certain systems and methods can also be used to detect changes to the analyte in a biological sample, and these changes could further be used to detect and classify a diagnostic that is related to such changes (for example, the development or spread of a cancer).

[0094] Examples of such changes in a mixture can be the differences in a property of an analyte in a composition, such as its conformation, structure and / or interaction tendency with respect to another molecule or molecules (e.g., its binding affinity or other interaction characteristic with respect to another molecule or molecules, for example, a competitive binder of the analyte). For example, if a composition includes an analyte, such changes may be induced through primary sequence modification, by degradation of the composition through chemical, thermal, biological, or other degradation mechanisms, by interaction with other molecules and / or biomolecules, by interaction with low molecular weight compounds (e.g., hormones, peptides, vitamins, cofactors, etc.), by changes in the relative content or concentration of the constituents of the composition, by reactions such as enzymatic reactions, etc. Some systems and methods can be used, in certain cases, to detect, analyze and / or characterize biological materials as they interact with an analyte, including but not limited to, polypeptides, proteins, carbohydrates, nucleic acids, polynucleotides, lipids, sterols, and mixtures or derivatives thereof, e.g., for the purpose of detection of, or onset of, a particular disease or physiological state, monitoring its progress, treatment, etc.

[0095] Comparison and classification steps involved in the disclosure can make use of additional information not necessarily related to (not directly derived from) the analytical methods of the disclosure, in certain embodiments. For example, blood pressure, temperature, blood glucose level, and / or essentially any other measurable physiological condition can be used in conjunction with various techniques of the disclosure to analyze one or more diseases or conditions.

[0096] #14460968vl It will be recognized by those of ordinary skill in the art that these biological materials can be found in any suitable form, for example, in the form of extracts from natural sources, biological liquids, collections of molecules generated by combinatorial chemical or biochemical techniques and combinations thereof, synthetically created, etc. In one set of embodiments, the biological materials arise from a biological fluid (e.g., withdrawn from a subject), and such biological materials may include one or more analytes suspected of being present within the subject.

[0097] In one embodiment, the present disclosure provides a method to determine certain conditions under which variations among samples representing different species (e.g., an analyte), or mixtures of species could be detected, i.e., determining a set of criteria and / or system components as a “tool,” or a part of a tool, to determine information, as well as the subsequent use of the tool. For example, the ability of a system to determine a partition coefficient or a relative measure of interaction between a species, such as an analyte, and one or more interacting components that can define one or more phases of the system can serve as an important tool or component of such a tool. Specifically, as one example, the partitioning of the constituents of a composition between two phases having different chemical or biochemical affinities or other characteristics, such as solvent structures, may separate the constituents by their relative affinity for media of different properties or composition. This separation technique thus can include or, alternatively, can be unlike those typically used in proteomics or similar techniques, e.g., 2-D gel electrophoresis, in which charge and size differences are the two dimensions used to separate the constituents of a sample. Some embodiments provide the ability for performing sequential or serial partitioning, with the same or different conditions, which may result in additional amplification of differences in the fractionated samples. These fractions may be further analyzed using standard proteomics techniques.

[0098] In some cases, an analyte can be determined to diagnose or determine an underlying physiological condition or disease. The concentrations of an analyte in two or more phases can be used to calculate the values of the partition coefficients. Changes to the individual values of the partition coefficients thus may indicate certain changes to the analyte. In some cases, the change to the partition coefficient of one or more analytes, can result in a diagnosis of a disease or condition. In yet other cases, partitioning of a composition in multiple systems and performing the steps above, then observing the pattern of values for one or more analytes, can provide a diagnostics method.

[0099] #14460968vl Thus, for example, a sample may be obtained from a subject and partitioned in one or more aqueous two-phase (or multiphase) partitioning systems. Partition coefficients for one or more analytes may be determined and used to determine a physiological condition of the subject, e.g., a progress of cancer. In some cases, the partition coefficients may be compared to reference partition coefficients, e.g., reference values previously determined for biomolecules taken from subjects with and without a disease or condition. For example, in connection with certain aspects, a variety of studies can take place. For example, the studies may include determining analysis procedures that involve taking samples from a single subject or multiple subjects.

[0100] Similarly, changes may be detected using other systems and methods which have an underlying dependence upon the topography and / or the types of solvent accessible groups. Examples of such other methods include, but are not limited to, column Liquid-Liquid Partition Chromatography (LLPC), a heterogeneous two-phase system, or a multiphase heterogeneous system. In some cases, an apparent partition coefficient may be generated that expresses the relative changes in the average partitioning between a first and a second phase, e.g., of an analyte. For example, in LLPC, the retention volume of a receptor may be used as the apparent partition coefficient.

[0101] In some embodiments, one or more of the fluid manipulations may occur within a microfluidics device. “Microfluidic,” as used herein, refers to a device, article, or system including at least one fluid channel having a cross-sectional dimension of less than about 1 mm. The “cross-sectional dimension” of the channel is measured perpendicular to the direction of net fluid flow within the channel. Thus, for example, some or all of the fluid channels in an article can have a maximum cross-sectional dimension less than about 2 mm, and in certain cases, less than about 1 mm. In one set of embodiments, all fluid channels in an article are microfluidic and / or have a largest cross sectional dimension of no more than about 2 mm or about 1 mm. In certain embodiments, the fluid channels may be formed in part by a single component (e.g. an etched substrate or molded unit). Of course, larger channels, tubes, chambers, reservoirs, etc. can be used to manipulate in other embodiments of the disclosure. In one set of embodiments, the maximum cross-sectional dimension of the channels in an article is less than about 1 mm, less than about 500 micrometers, less than about 300 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75 micrometers, less than about 50 micrometers, less than about 30 micrometers, less than about 25 micrometers, less than about 20 micrometers, less than about 15 micrometers, less than about 10 micrometers, less than about 5 micrometers, less than

[0102] #14460968vl about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 500 nm, less than about 300 nm, less than about 100 nm, or less than about 50 nm. In some cases, suitable microfluidics devices may be readily obtained commercially.

[0103] In addition, according to some aspects of the present disclosure, a computer and / or an automated system is provided able to automatically and / or repetitively perform any of the methods described herein. As used herein, “automated” devices refer to devices that are able to operate without human direction, i.e., an automated device can perform a function during a period of time after any human has finished taking any action to promote the function, e.g. by entering instructions into a computer. Typically, automated equipment can perform repetitive functions after this point in time. One specific example of a technique that can make use of a computer or other automated system is in a process in which a physiological condition of a system as determined by determining a relative measure of interaction between one or more species from a sample from the system and various interacting components of a partitioning system. In the clinical setting, this may be accomplished by drawing a sample of blood (milliliter-sized or a very small sample such as a drop or less) and subjecting the blood sample or a subset thereof (e.g., plasma) to a multiphase partitioning process. The results of this process can then be compared to similar behavior of markers in a similar system, which can take the form of data stored electronically.

[0104] Various embodiments of the present disclosure can also be implemented exclusively in hardware, or in a combination of software and hardware. For example, in one embodiment, rather than a conventional personal computer, a Programmable Logic Controller (PLC) is used. As known to those skilled in the art, PLCs are frequently used in a variety of process control applications where the expense of a general purpose computer is unnecessary. PLCs may be configured in a known manner to execute one or a variety of control programs, and are capable of receiving inputs from a user or another device and / or providing outputs to a user or another device, in a manner similar to that of a personal computer. Accordingly, although embodiments of the present disclosure are described in terms of a general purpose computer, it should be appreciated that the use of a general purpose computer is exemplary only, as other configurations may be used.

[0105] “Aqueous,” as used herein, refers to the characteristic properties of a solvent / solute system wherein the solvating substance has a predominantly hydrophilic character. Examples of aqueous solvent / solute systems include those where water, or compositions containing water, are the predominant solvent.

[0106] #14460968vl “Partitioning system,” as used herein, refers to any material having at least two phases, sections, areas, components, or the like, at least two of which can interact differently with at least one species to which they are exposed. For example, a partitioning system can include different areas of a solid surface, which can interact differently with a particular molecule exposed to the different sections, a multiphase system such as a multiphase liquid system, e.g., an aqueous / non-aqueous system or an aqueous multiphase system (as defined herein) to which one or more species can be exposed and optionally dissolved, at least some of which species can interact differently with different phases. For example, a particular species may have a greater affinity for one phase rather than another phase to the extent that a multiphase partitioning system can isolate a species from a mixture, or cause a species to partition at least in some way differently between the phases.

[0107] “Aqueous multiphase system,” as used herein, refers to an aqueous system which includes greater than one aqueous phase in which a species can reside, and which can be used to characterize the structural state of the species according to the methods described herein. For example, an aqueous multiphase system can separate at equilibrium into two, three, or more immiscible phases. Aqueous multiphase systems are known in the art and this phrase, as used herein, is not meant to be inconsistent with accepted meaning in the art. Examples of various aqueous multiphase systems, and their compositions, are discussed herein.

[0108] An “interacting component” means a component, such as a phase of multiphase system, that can interact with a species and provide information about that species (for example, an affinity for the species). Multiple interacting components, exposed to a species, can define a system that can provide a “relative measure of interaction” between each component and the species. An interacting component can be aqueous or non-aqueous, can be polymeric, organic (e.g. a protein, small molecule, etc.), inorganic (e.g. a salt), or the like, or any combination thereof. A set of interacting components can form a system useful in and in part defining any experimental method which is used to characterize the structural state of a species according to the methods described herein. Typically, a system of interacting components can measure the relative interaction between the species and at least two interacting components. An aqueous multiphase system is an example of a system of interacting components, and it is to be understood that where “aqueous system” or “aqueous multiphase system” is used herein, this is by way of example only, and any suitable system of interacting components can be used.

[0109] Where aqueous two-phase and aqueous multiphase systems are described herein, it is to be understood that other systems, as used herein, systems analogous to those comprising

[0110] #14460968vl only aqueous solutions or suspensions can be used. For example, an aqueous two-phase system can include non-aqueous components in one or more phases that are not liquid in character. In this aspect, multiphase systems also refer to related techniques that rely on differential affinity of the biomolecule to one media versus another, wherein the transport of the biomolecule between one medium and, optionally, another medium occurs in an aqueous environment. Examples of such multiphase systems include, but are not limited to, HPLC columns or systems for liquid-liquid partition chromatography, as are known to those of ordinary skill in the art.

[0111] “Relative measure of interaction,” with reference to a particular species as used herein, means the degree to which the species interacts with another species or with a phase of a multiphase system in a relative sense. For example, a particular species may have a greater affinity for one phase of a multiphase system rather than another phase or phases, the degree to which it interacts with or resides in, that phase as opposed to other phases defines its relative measure of interaction. Relative measures of interaction, in the context of the present disclosure, are generally determined in a ratiometric manner, rather than an absolute manner. That is, where a species can interact with each phase of a two-phase system but resides more preferably in one than the other, the present disclosure typically makes use of information as to the ratio of concentration of the species in each of the two phases, but not necessarily of the absolute concentration of the species in either phase. In other cases, the interaction can be an interaction based not upon residence of a particular species within a particular solvent or fluid carrier, but interaction with a solid surface such as a solid phase of a chromatography column where the relative measure manifests itself in elution time, or can involve geometric or spatial interaction such as a particular species interaction with a porous substrate as opposed to that of a different species or a different substrate.

[0112] “Partition coefficient,” as used herein, refers to the coefficient which is defined by the ratio of chemical activity or the concentrations of a species in two or more phases of a multiphase system at equilibrium. For example, the partition coefficient (K) of a species in a two-phase system can be defined as the ratio of the concentration of species in the first phase to that in the second phase. For multiphase systems, there can be multiple partition coefficients, where each partition coefficient defines the ratio of species in first selected phase and a second selected phase. It will be recognized that the total number of partition coefficients in any multiphase system will be equal to the total number of phases minus one.

[0113] For heterogeneous phase systems, an “apparent partition coefficient,” as used herein, refers to a coefficient which describes information obtained from alternative techniques that

[0114] #14460968vl is correlated to the relative partitioning between phases. For example, if the heterogeneous two-phase system used is an HPLC column, this “apparent partition coefficient” can be the relative retention time for the species. It will be recognized by those of ordinary skill in the art that the retention time of a species, in such a case, reflects the average partitioning of the species between a first, mobile phase and a second, immobile phase. Also, it will be recognized that other, similarly determinable properties of species can also be used to quantify differences in physical properties of the species (e.g. in other techniques) and are, therefore, suitable for use as apparent partition coefficients.

[0115] “Molecule-molecule interaction,” such as biomolecule-biomolecule interaction, protein-protein interaction, and the like means an interaction that typically is weaker than “binding,” i.e., an interaction based upon hydrogen bonding, van der Waals binding, London forces, and / or other non-covalent interactions that contribute to an affinity of one molecule for another molecule, which affinity can be assisted by structural features such as the ability of one molecule to conform to another molecule or a section of another molecule. Moleculemolecule interactions can involve binding, but need not.

[0116] “Biomolecule,” as used herein, means a molecule typically derived from a subject, and which typically includes building blocks including nucleotides, and the like. Examples include, but are not limited to, peptides, polypeptides, proteins, protein complexes, nucleotides, oligonucleotides, polynucleotides, nucleic acid complexes, saccharides, oligosaccharides, carbohydrates, lipids, etc., as well as combinations, enantiomers, homologs, analogs, derivatives and / or mimetics thereof.

[0117] “Species,” as used herein, refers to a molecule or collection of molecules, for example, an inorganic chemical, an organic chemical, a biomolecule, or the like. In the present disclosure, species generally are biomolecules.

[0118] “Corresponding species,” as used herein, means at least two different species that are identical chemically or, if they differ chemically and / or by molecular weight, differ only slightly. Examples of corresponding species include structural isoforms of proteins, proteins or other molecules that are essentially identical but that differ in binding affinity with respect to another species or plural species, have different higher-order structure, e.g., differing in secondary or tertiary structure but not differing or not differing significantly in chemical sequence. In general, corresponding species are species that may be arranged differently (isoforms, isomers, etc.) but are composed of the same or essentially the same chemical building blocks.

[0119] #14460968vl “Detectable,” as used herein, refers the ability of a species and / or a property of the species to be discerned. One example method of rendering a species detectable is to provide further species that bind or interact with the first species, where the species comprise(s) a detectable label. Examples of detectable labels include, but are not limited to, nucleic acid labels, chemically reactive labels, fluorescence labels, enzymatic labels and radioactive labels.

[0120] “Mimetic,” as used herein, includes a chemical compound, an organic molecule, or any other mimetic, the structure of which is based on, or derived from, a binding region of an antibody or antigen. For example, one can model predicted chemical structures to mimic the structure of a binding region, such as a binding loop of a peptide. Such modeling can be performed using standard methods. The mimetics identified by methods such as this can be further characterized as having the same binding function as the originally identified molecule of interest, according to the binding assays described herein.

[0121] “Structure,” “structural state,” “configuration” or “conformation,” as used herein, all refer to the commonly understood meanings of the respective terms, for example, as they apply to biomolecules such as proteins and nucleic acids, as well as pharmacologically active small molecules. In different contexts, the meaning of these terms will vary, as is appreciated by those of skill in the art. The structure or structural state of a molecule refers generally not to the building blocks that define the molecule but the spatial arrangement of these building blocks. The configuration or confirmation typically defines this arrangement. For instance, the use of the terms primary, secondary, tertiary or quaternary, in reference to protein structure, have accepted meanings within the art, which differ in some respects from their meaning when used in reference to nucleic acid structure (see, e.g., Cantor and Schimmel, Biophysical Chemistry, Parts I-III). Unless otherwise specified, the meanings of these terms will be those generally accepted by those of skill in the art.

[0122] “Physiological conditions,” as used herein, means the physical, chemical, or biophysical state of a subject. As most typically used in the context of the present disclosure, physiological condition refers to a normal (e.g., healthy in the context of a human) or abnormal (e.g., in a diseased state in the context of a human) condition.

[0123] “Marker,” as used herein, is a species that can be a carrier of information regarding a physiological state of a biological environment within which it resides. A marker can exhibit at least two different properties or values of a specific property or properties (e.g., structural conformation, binding affinity for another species, etc. but not solely different amounts of the species) that correspond to and / or that represent information regarding the two or more

[0124] #14460968vl physiological states of environments within which they reside. For example, a marker may be a protein that is structurally modified between a first state representative of a healthy system within which it resides and a second structural state (different conformation) representative of a disease system within which it resides.

[0125] The following documents are each incorporated herein by reference in their entireties: U.S. Pat Nos. 7,968,350, 8,099,242, 8,211,714, 9,354,229, 10,613,087, 11,422,135, 11,796,544, and 11,971,408. In addition, U.S. Provisional Patent Application Serial No. 63 / 850,425, filed July 24, 2025, entitled “Systems and Methods for Analyte Assessment, Regardless of Competing Binders,” and U.S. Provisional Patent Application Serial No. 63 / 728,087, filed December 4, 2024, entitled “Systems and Methods for Analyte Assessment, Regardless of Competing Binders,” are each incorporated herein by reference in its entirety.

[0126] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0127] EXAMPLE 1

[0128] This example illustrates an immune-based method (“IsoTg”) to determine thyroglobulin (“Tg”) in the absence or presence of Thyroglobulin autoantibodies (“TgAbs”). Thyroid cancer (TC) management guidelines typically mandate that Thyroglobulin testing should always include measurement of Thyroglobulin autoantibodies, thus introducing an inherent inaccuracy for immunometric assays of Thyroglobulin. The method presented in this example improves the existing methodology by adding a step prior to the immunoassay, which allows for accurate determination of thyroglobulin levels in body fluids. This method in this example was also shown to improve specificity and sensitivity of current thyroglobulin test for TC.

[0129] Thyroid cancer (TC) management guidelines typically mandate that thyroglobulin testing should always include measurement of thyroglobulin autoantibodies, thus introducing an inherent inaccuracy for immunometric assays of thyroglobulin. The thyroid is a 2-lobes gland, butterfly-shaped located in front of the neck below the larynx (voice box) and Adam's apple. The thyroid is a spherical follicle, lined with follicular (thyrocytes) and parafollicular cells. The thyroid gland secretes three hormones: the two thyroid hormones — triiodothyronine (T3), thyroxine (T4), and a peptide hormone, calcitonin — to regulate metabolic rate, energy levels, body temperature, protein synthesis, growth and development in children, and stress responses.

[0130] Several functional disorders and diseases of the thyroid are known: hormonal imbalance, hypothyroidism (Reduced thyroid function), hyperthyroidism (Increased thyroid

[0131] #14460968vl function), autoimmune diseases such as Hashimoto's disease (chronic lymphocytic thyroiditis), and Graves' disease (toxic diffuse goiter). Additionally, most common thyroid cancers are papillary (PTC), follicular (FTC), anaplastic (ATC), and medullary (MTC).

[0132] Thyroglobulin (Tg) is used as a tumor marker for thyroid cancer post-thyroidectomy. However, TgAbs, detected in about 1 / 3 of thyroid cancers and in 10-30% of healthy adults, limit the value of Tg test, as TgAbs affect thyroglobulin immunometric assay, causing falsely low results. Conversely, heterophilic antibodies may cause falsely elevated results. Yet, thyroid cancer management guidelines mandate that Tg testing should always include the measurement of TgAb. The discrepancies in acceptance of patient samples for serum Tg evaluation in the presence of TgAb, thus illustrate a diagnostic dilemma.

[0133] Samples acquired from patients were added to an aqueous two-phase partition system (ATPS) that was able to partition protein isoforms between top and bottom phases. In this sample, the isoforms of the protein deviate from healthy or normal conformations as a result of the disease conditions. The concentrations of the protein in both phases was determined, and the ratio between the phases was then calculated. Larger differences between the ratios of healthy versus disease conditions allowed for more reliable tests (e.g., to distinguish between a given normal protein and a given diseased protein).

[0134] The aqueous two-phase partition system was able to detect conformational changes of Tg. It is believed that the malignant conditions cause altered processes within the thyroid, which casuses the Tg forms to deviate from healthy conformations (i.e., Tg isoforms). Differences in partitioning of the Tg, despite the presence of thyroglobulin autoantibodies, could be used for diagnostic interpretation. Concentrations of Tg in the phases of the aqueous two-phase partition system were measured using a cobas e411 Tg test available from Roche.

[0135] It was found that 95% of healthy serum samples had <77 ng / mL Tg, and 93% of them had <40 ng / mL Tg. In contrast, 61% of the thyroid cancer serum samples had <77 ng / ml Tg.

[0136] EXAMPLE 2

[0137] Thyroglobulin (Tg) determinations can be affected by the presence of Tg autoantibodies (TgAb) present in some patient samples. This example shows that analysis of partitioning of thyroglobulin (Tg) present in blood serum, which is being assayed in a single aqueous two-phase partition system (ATPS), may display differential behavior from subjects with thyroid malignant tumor (TCa) and healthy subjects, without being affected by the presence of TgAb.

[0138] #14460968vl The cobas e411 analyzer (Roche Diagnostics, Indianapolis, IN) that uses ElectroChemiLuminescence (ECL) technology for immunoassay analysis was used in this example to quantify Tg and TgAb present in the native (neat) patient samples and following partitioning.

[0139] Sample Preparation. Human serum samples corresponding to malignant phenotype (thyroid cancer, papillary carcinoma) were purchased from i Specimen (Lexington, MA) and BioIVT (Hicksville, NY). The diagnostic status of each sample was provided by the supplier and samples corresponded to stage I and II. The sample aliquots of 1.0-1.8 mL were frozen and stored at -80 °C until time of use.

[0140] Human sample samples corresponding to healthy clinical phenotypes were purchased from iSpecimen (Lexington, MA) and Precision for Medicine (Frederick, MD). The diagnostic status of each sample was provided by the suppliers. Sample aliquots of 0.5-1.0 mL were frozen and stored at -80 °C until time of use.

[0141] Sample aliquots were thawed, equilibrated to room temperature, before being tested.

[0142] Aqueous Two-Phase System (IsoClear™). An aqueous two-phase system (“ATPS 243”) containing 11.9 wt.% dextran-65 (average molecular weight of 65,000), 15.7 wt.% polyethylene glycol 600 (PEG-600) (average molecular weight of 600), 0.010 M sodium / potassium phosphate buffer, pH 7.4, and 0.320 M sodium perchlorate was prepared by mixing the appropriate amounts of stock polymers, buffer and salt solutions dispensed using a liquid handling workstation (Hamilton ML-4000, Reno, NV or CDx ALD, Cleveland, OH) into a 2.0 mL tube up to a total volume ~1.072 mL of the mixture.

[0143] Partitioning Experiments. Partitioning was conducted by adding 0.200 mL of each sample into to an individual prepared system tube of ATPS 243. The sample aliquot was thawed, equilibrated to room temperature, and mixed prior to addition to the ATPS. The final weight of each system after sample addition was 1.40 g (total volume of -1.272 mL) and the ratio between the volumes of the two phases of each system was approximately equal. Each system tube was vigorously shaken and then centrifuged at 10,000 RPM / 9500 RCF in a refrigerated centrifuge (Hettich, Universal 320R, with a fixed angle rotor (30 places)), at 22 + / - 1 °C, for 60 min to accelerate phase settling. The tubes were removed from the centrifuge, and aliquots (0.120 mL) were withdrawn from the top and the bottom phases for further analysis. Each aliquot was transferred to individual vials, diluted 2-fold using the appropriated diluent (Phosphate-buffered saline (PBS, IX)), vortexed, and centrifuged at 4000 RPM / 1620 RCF (Hettich, Universal 320R, 1460 rotor), at 22 + / - 1 °C, for 10 min, and

[0144] #14460968vl 0.200 mL aliquots transferred to cobas cups (catalogue # 11706802001) for protein concentration analysis.

[0145] An immunoassay analyzer capable of measuring Tg and TgAb concentrations (cobas e411 (Roche Diagnostics, Indianapolis, IN) was used to measure the concentration in native and both top and bottom diluted phases (aliquots of 0.200 mL). All Roche cobas e411 Tg and TgAb reagent packs were handled, stored, and calibrated according to their respective Method Sheets (Roche Diagnostics, REF 08906556190 (Tg) and REF 09004998190 (TgAb)). For quality control, each reagent pack was tested at the start of the day and following each calibration with the recommend control materials (PreciControl Universal (Roche Diagnostics, REF 11731416190), PreciControl Thyro Sensitive (Roche Diagnostics, Ref 06445918190) was used for Tg reagent assay and PreciControl ThyroAB (Roche Diagnostics, REF 05042666191) were used for TgAb reagent assay).

[0146] For each sample tested, the partition coefficient (K) for Tg was calculated as the ratio of the Tg concentration determined in the top phase to that in the bottom phase. The calculated K-values were plotted versus each native TgAb concentration measured in each native (neat) sample.

[0147] The results are shown in Fig. 2A, showing the samples Thyroglobulin (Tg) partition coefficients in ATPS 243 (labeled as K243), plotted versus native Tg autoantibodies (TgAb) concentration present in the corresponding tested patient sample, where TgAb was determined prior to partitioning. In particular, it can be seen that the partition coefficients for Tg were not affected by variable concentrations of TgAb.

[0148] EXAMPLE 3

[0149] In this example, an aqueous two-phase system (ATPS 80) containing 12.0 wt.% Dextran-70 (average molecular weight of 70,000), 8.0 wt.% Polyethylene glycol 2000 (PEG- 2000) (average molecular weight of 2000), 0.010 M sodium / potassium phosphate buffer (K / NaPB), pH 7.4, and 0.150 M sodium sulfate was prepared by mixing the appropriate amounts of stock polymers, buffer and salt solutions dispensed using liquid handling workstation(s) (CDx Automated Liquid Dispenser (ALD), Cleveland, OH) into a 2.0 mL tube up to a total volume of 1.09 mL of the mixture.

[0150] Partitioning was conducted by adding 0.200 mL of each serum sample into to an individual prepared system tube of ATPS 80. The sample aliquot was thawed, equilibrated to room temperature, and mixed prior to addition to the ATPS. The final weight of each system after sample addition was 1.40 g (total volume of 1.29 mL) and the ratio between the volumes of the two phases of each system was approximately equal. Each system tube was vigorously

[0151] #14460968vl shaken and then centrifuged at 10,000 RPM / 10845xg RCF in a refrigerated centrifuge (Hettich, Universal 320R, with a fixed angle rotor, 1689-A (30 places) rotor), at 22 + / - 1 °C, for 60 min to accelerate phase settling. The tubes were removed from the centrifuge, and aliquots (0.05 mL) from the top and the bottom phases were withdrawn using a liquid handler ML-4000 (Hamilton Company, Reno, NV, USA). Each aliquot was transferred to individual vials, diluted 2-fold using the appropriated diluent (Meso Scale Discovery (MSD), Diluent 57), vortexed, and centrifuged at 4000 RPM / 2218xg RCF (Hettich, Universal 320R, 1460 rotor), at 22 + / - 1 °C, for 5 min, before Folate Receptor Alpha (FRa) quantitation by MSD. The FRa concentration in each native sample, diluted 5-fold (Diluent 57), was also determined.

[0152] For each sample tested, the FRa partition coefficient (K) was calculated as the ratio of the FRa concentration determined in the top phase to that in the bottom phase. An alternative result output, Index 1, was calculated as the ratio of FRa concentration determined in the native serum sample to that in the top phase fraction. These results are shown in Fig. 2B, illustrating that KFR« is not affected by anti-FRa antibody concentration.

[0153] EXAMPLE 4

[0154] This example illustrates the leveraging of protein modifications for differentiated thyroid cancer risk stratification, including diagnosis and reduced over-treatment. This example also illustrates the evaluation of the diagnostic utility of certain embodiments for identifying protein modifications associated with thyroid malignancy using serum Thyroglobulin, aiming to differentiate thyroid cancers from benign nodules and reduce unnecessary interventions

[0155] In this example, 101 archived serum samples from subjects with Tg of less than or equal to 40 ng / mL with and without confirmed thyroid cancer (prevalence = 0.297) were partitioned with an Aqueous Two-Phase System (ATPS) such as discussed herein and assessed using Receiver Operating Characteristic (ROC) analysis. Sensitivity and specificity were calculated at a predefined clinical cutoff selected for balance and potential clinical utility.

[0156] The ATPS system demonstrated statistically significant diagnostic performance. The area under the ROC curve (AUC) for the assay in Fig. 3A was 0.805 (95% CI: 0.710-0.900), versus 0.533 (95% CI: 0.399-0.667) for the concentration-based Tg assay. At the selected cutoff, the ATPS system achieved a sensitivity of 0.933 (95% CI: 0.787-0.982) and a

[0157] #14460968vl specificity of 0.380 (95% CI: 0.276-0.497). Importantly, the presence of TgAbs had no measurable effect on assay performance, supporting broader clinical applicability.

[0158] The rising incidence of thyroid cancer, driven by sensitive imaging, highlights a critical challenge: overdiagnosis and overtreatment of indolent lesions. These subclinical cancers often lead to unnecessary surgeries and radioactive iodine therapy, imposing a significant patient burden. An unmet need is for robust, preoperative tools to distinguish truly aggressive malignancies from indolent forms suitable for active surveillance. While serum Tg monitors recurrence, its use in initial diagnosis is limited by its presence in benign conditions and normal thyroid tissue. Traditional protein quantification falls short. This example overcomes this by sensitively detecting specific protein modifications linked to thyroid malignancy. This example thus illustrates enhanced preoperative risk stratification, minimizing diagnostic surgeries for low-risk nodules and optimizing management for clinically significant diseases. The diagnosis may also be coupled in some embodiments with certain therapies (e.g., “companion diagnostics”), thus leading to better treatment of the disease.

[0159] EXAMPLE 5

[0160] In this example, an electrochemiluminescent (ECL) sandwich immunoassay was employed for the quantitative detection of Folate Receptor a (FRa) in human samples. A 96- well streptavidin-coated plate (Cat. No. K151AASR, Meso Scale Diagnostics, Rockville, MD, USA) was utilized as the solid phase for the capture of biotinylated monoclonal anti- FRa antibodies. Following immobilization of the capture antibody, calibrators, diluted human samples, and diluted Aqueous Two-Phase System (ATPS) phases were added to the wells and incubated. A total of 84 human samples were analyzed, 42 ovarian cancer samples and 42 normal controls. Detection of captured FRa was achieved using a SULFO-TAG-labeled polyclonal anti- FRa detection antibody, followed by the addition of MSD Read Buffer. Plates were subsequently read on a MESO QuickPlex SQ120MM instrument (Meso Scale Diagnostics, Rockville, MD, USA). Signal intensities were quantified against a standard curve generated from known concentrations of calibrators. The diagnostic performance of the assay was evaluated by Receiver Operating Characteristic (ROC) curve analysis using Analyze-it software (Analyze-it Software Ltd., Leeds, UK), and is shown in Fig. 3B. The Area Under the Curve (AUC) was calculated to assess the assay's sensitivity and specificity.

[0161] While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other

[0162] #14460968vl means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0163] In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0164] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0165] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one

[0166] #14460968vl embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0167] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0168] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0169] When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

[0170] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts

[0171] #14460968vl of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0172] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0173] What is claimed is:

[0174] #14460968vl

Claims

CLAIMS1. A method, compri sing : exposing a sample comprising an analyte and a binder of the analyte to an aqueous multiphase partitioning system; and determining partitioning of the analyte in the aqueous multiphase partitioning system.

2. The method of claim 1, wherein the binder of the analyte is competitive.

3. The method of any one of claims 1 or 2, wherein the aqueous multiphase partitioning system is an aqueous two-phase partitioning system.

4. The method of any one of claims 1-3, wherein the sample is taken from a subject.

5. The method of claim 4, wherein the subject is human.

6. The method of any one of claims 1-5, wherein the sample comprises blood.

7. The method of any one of claims 1-6, wherein the sample comprises saliva.

8. The method of any one of claims 1-7, wherein the sample comprises nasal fluid.

9. The method of any one of claims 1-8, wherein the sample comprises urine.

10. The method of any one of claims 1-9, wherein the sample comprises semen.

11. The method of any one of claims 1-10, comprising determining the analyte in a phase of the aqueous multiphase partitioning system using an electrochemiluminescence immunoassay.12807703.1#14460968vl12. The method of any one of claims 1-11 , comprising determining the analyte in a phase of the aqueous multiphase partitioning system using a radioimmunoassay.

13. The method of any one of claims 1-12, further comprising determining a relative measure of interaction of the analyte in the aqueous multiphase partitioning system.

14. The method of any one of claims 1-13, further comprising determining a partition coefficient of the analyte in the aqueous multiphase partitioning system.

15. The method of any one of claims 1-14, further comprising determining a relative measure of interaction of the binder in the aqueous multiphase partitioning system.

16. The method of any one of claims 1-15, further comprising determining a partition coefficient of the binder in the aqueous multiphase partitioning system.

17. The method of any one of claims 1-16, wherein the analyte comprises a protein.

18. The method of any one of claims 1-17, wherein the analyte comprises a nucleic acid.

19. The method of any one of claims 1-18, wherein the binder comprises an antibody or an antibody fragment.

20. The method of claim 19, wherein the binder comprises a monoclonal antibody.

21. The method of any one of claims 19 or 20, wherein the binder comprises a polyclonal antibody.

22. The method of any one of claims 19-21, wherein the binder comprises a bispecific antibody.12807703.1#14460968vl23. The method of any one of claims 19-22, wherein the binder comprises an immunoglobulin light chain.

24. The method of any one of claims 19-23, wherein the binder comprises a single-chain variable fragment.

25. The method of any one of claims 1-24, wherein the binder comprises a nucleic acid.

26. The method of any one of claims 1-25, wherein the binder comprises an aptamer.

27. The method of any one of claims 1-26, wherein the binder comprises an affimer.

28. The method of any one of claims 1-27, wherein the binder comprises a nanobody.

29. The method of any one of claims 1-28, wherein the binder comprises a DARPin.

30. A method, compri sin : exposing a sample comprising an analyte and a binder of the analyte to an aqueous multiphase partitioning system; and assaying the aqueous multiphase partitioning system for the analyte using an immunological assay.

31. The method of claim 30, wherein the binder is a competitive binder of the analyte.

32. The method of any one of claims 30 or 31, wherein the immunological assay comprises a second binder of the analyte.

33. The method of claim 32, wherein the binder and the second binder are competitive binders of the analyte.12807703.1#14460968vl34. A method, comprising: determining partitioning of Thyroglobulin in an aqueous multiphase partitioning system.

35. The method of claim 34, wherein the aqueous multiphase partitioning system further comprises Thyroglobulin-binding antibodies.

36. The method of claim 35, wherein the Thyroglobulin-binding antibodies are present in the sample at a concentration of at least 1 ng / ml.

37. The method of any one of claims 34-36, wherein the Thyroglobulin arises from a sample taken from a subject.

38. The method of claim 37, wherein the sample comprises blood.

39. The method of any one of claims 37 or 38, wherein the sample comprises saliva.

40. The method of any one of claims 37-39, wherein the sample comprises nasal fluid.

41. The method of any one of claims 37-40, wherein the subject is human.

42. The method of any one of claims 37-41, wherein the subject is suspected of having thyroid cancer.

43. The method of any one of claims 37-42, wherein the subject has undergone a thyroidectomy.

44. A method, comprising: exposing a sample taken from a subject to an aqueous multiphase partitioning system, wherein the sample comprises thyroglobulin; and12807703.1#14460968vldetermining partitioning of the thyroglobulin in the aqueous multiphase partitioning system.

45. The method of claim 44, wherein the sample further comprises thyroglobulin-binding antibodies.

46. The method of any one of claims 44 or 45, wherein the thyroglobulin-binding antibodies are present in the sample at a concentration of at least 1 ng / ml.

47. The method of any one of claims 44-46, wherein the sample comprises blood.

48. The method of any one of claims 44-47, wherein the sample comprises saliva.

49. The method of any one of claims 44-48, wherein the sample comprises nasal fluid.

50. The method of any one of claims 44-49, wherein the subject is human.

51. The method of any one of claims 44-50, wherein the subject is suspected of having thyroid cancer.

52. The method of any one of claims 44-51, wherein the subject has undergone a thyroidectomy.

53. A method of treating a subject, comprising: exposing a sample taken from a subject to an aqueous multiphase partitioning system, wherein the sample contains thyroglobulin and thyroglobulin-binding autoantibodies; determining partitioning of the thyroglobulin in the aqueous multiphase partitioning system; and treating the subject for thyroid cancer based on partitioning of the Thyroglobulin.12807703.1#14460968vl54. The method of claim 53, wherein the subject is human.

55. A method, comprising: determining partitioning of Folate Receptor alpha in an aqueous multiphase partitioning system.

56. The method of claim 55, wherein the aqueous multiphase partitioning system further comprises folate Receptor Alpha autoantibodies.

57. The method of claim 56, wherein the Folate Receptor alpha autoantibodies are present in the sample at a concentration of at least 1 ng / ml.

58. The method of any one of claims 55-57, wherein the Folate Receptor alpha arises from a sample taken from a subject.

59. The method of claim 58, wherein the sample comprises blood.

60. The method of any one of claims 58 or 59, wherein the subject is human.

61. The method of any one of claims 58-60, wherein the subject is suspected of having ovarian cancer.

62. The method of any one of claims 58-60, wherein the subject is suspected of having cerebral folate deficiency.

63. The method of any one of claims 58-60, wherein the subject is suspected of having autism spectrum disorder.

64. The method of any one of claims 58-60, wherein the subject is suspected of having neural tube defect.12807703.1#14460968vl65. The method of any one of claims 58-60, wherein the subject is suspected of having pediatric acute-onset neuropsychiatric syndrome.

66. The method of any one of claims 58-60, wherein the subject is suspected of having pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections.

67. A method, comprising: exposing a sample taken from a subject to an aqueous multiphase partitioning system, wherein the sample comprises Folate Receptor alpha; and determining partitioning of the folate receptor alpha in the aqueous multiphase partitioning system.

68. The method of claim 67, wherein the sample further comprises Folate Receptor alpha autoantibodies.

69. The method of any one of claims 67 or 68, wherein the Folate Receptor alpha autoantibodies are present in the sample at a concentration of at least 1 ng / ml.

70. The method of any one of claims 67-69, wherein the sample comprises blood.

71. The method of any one of claims 67-70, wherein the subject is human.

72. The method of any one of claims 67-71, wherein the subject is suspected of having ovarian cancer.

73. The method of any one of claims 67-71, wherein the subject is suspected of having cerebral folate deficiency.

74. The method of any one of claims 67-71, wherein the subject is suspected of having autism spectrum disorder.12807703.1#14460968vl75. The method of any one of claims 67-71, wherein the subject is suspected of having neural tube defect.

76. The method of any one of claims 67-71, wherein the subject is suspected of having pediatric acute-onset neuropsychiatric syndrome.

77. The method of any one of claims 67-71, wherein the subject is suspected of having pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections.

78. A method of treating a subject, comprising: exposing a sample taken from a subject to an aqueous multiphase partitioning system, wherein the sample contains folate receptor alpha and folate receptor alpha autoantibodies; determining partitioning of the folate receptor alpha in the aqueous multiphase partitioning system; and treating the subject for ovarian cancer based on partitioning of the folate receptor alpha.

79. The method of claim 78, wherein the subject is human.

80. A method of treating a subject, comprising: exposing a sample taken from a subject to an aqueous multiphase partitioning system, wherein the sample contains an analyte specific to a disease and binding antibodies of said analyte; determining partitioning of the analyte in the aqueous multiphase partitioning system; and treating the subject for said disease based on partitioning of the analyte.

81. The method of claim 79, whereas the subject is human.12807703.1#14460968vl

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