Nanoparticles containing polymerized gamma globulin and methods of making and using same

Abstract

A diagnostic immunoassay reagent composition is disclosed, comprising nanoparticles formed of polymerized gamma globulin. The nanoparticles have at least one functional moiety on their surface, and at least one specific binding partner for a test substance is attached to the nanoparticle through the at least one functional moiety. Kits, devices, and systems containing the diagnostic immunoassay reagent composition are also disclosed, as well as methods of making and using the diagnostic immunoassay reagent composition.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS Not applicable.

[0002] Federally sponsored research or development Not applicable. [Background technology]

[0003] In the fields of pharmaceuticals and clinical chemistry, many tests and determinations regarding physiologically reactive species or analytes are performed by utilizing interactions between members of specific binding pairs. For example, a target analyte in a patient sample may be one member of a specific binding pair, which can be detected by using the corresponding member of the specific binding pair immobilized on a solid support. For example (but not limited to), the immobilized binding pair member can be an antigen for detection of a target antibody in the sample, or vice versa (i.e., the immobilized binding pair member can be an antibody for detection of a target analyte in the sample), or the immobilized binding pair member can be a ligand for detection of a target receptor in the sample, or vice versa (i.e., the immobilized binding pair member can be a receptor for detection of a target ligand in the sample). A variety of support or surface materials have been developed for these applications, but these materials require various coupling or "functionalization" techniques to immobilize the specific binding pair member to the support / surface.

[0004] Surfaces functionalized with binders are commonly used as a basic test architecture for the detection of substances. Key problems encountered in the process of functionalizing surfaces with binders include the need for a large number of materials and resources as well as a considerable amount of time, especially for "sensitive" systems. Furthermore, the coupling process and the resulting waste materials can be harmful, even for certain types of surfaces to which it is applied. The respective processes and waste management are usually associated with considerable safety measures and significant costs, and may even have to be stopped for regulatory reasons, with substantial impacts on the completion of projects.

[0005] For example, nonmagnetic latex particles (NMLPs) are widely used as surfaces (also called "solid phases") in homogeneous immunoassays. Binders, such as antigens or antibodies, are attached to such latex beads, which can be accomplished by chemical reactions. One non-limiting example of such an attachment reaction involves the use of an azomethine reaction, followed by a reduction process that may involve the application of sodium cyanoborohydride (NaBHCN), a hazardous chemical compound that requires proper management. Functionalized NMLPs and the analyte of interest combine in a multivalent manner to form aggregates that can be visualized or quantified spectrophotometrically, particularly (but not exclusively) turbidimetrically or nephelometrically. Quantifying this change in the turbidity (or opacity) or scattered light of the reaction mixture as a function of analyte concentration is the basis of homogeneous immunoagglutination assays. Summary of the Invention [Problem to be solved by the invention]

[0006] In addition to the use of hazardous materials in the manufacture of latex beads, such latex beads utilized in immunoassays also ultimately become waste. The waste disposal costs associated with immunoassay reagents containing such latex beads continue to be a challenge, and costs may rise significantly in the future. [Means for solving the problem]

[0007] Thus, there is a need in the art for new and improved diagnostic immunoassay reagents that overcome the difficulties and shortcomings of the prior art. It is to such reagents, as well as kits and microfluidic devices containing them, and methods of making and using them, that the present disclosure is directed. [Brief explanation of the drawings]

[0008] [Figure 1] 1 includes a photograph of one non-limiting embodiment of a protein nanoparticle constructed according to the present disclosure, where the protein nanoparticle is formed from polymerized goat gamma globulin (pGGG). The protein nanoparticle shown in this figure is present in solution at a concentration of 10 mg / ml. [Figure 2] FIG. 1 includes photographs of solutions (1 mg / ml) of pGGG protein nanoparticles that have been functionalized on their surfaces with biotin (left) or fluorescein (right). [Figure 3] Figure 1 graphically illustrates UV-VIS chromatograms of pGGG nanoparticles at 280, 340, and 600 nm. When pGGG nanoparticles were injected onto a Sepharose CL-2B column (1.0 x 30 cm), a high molecular weight peak (main peak (approximately 100 nm) excluded at approximately 7 ml) could be simultaneously detected at 280, 340, and 600 nm. The absorbance at 340 nm is commonly used for conventional solution agglutination immunoassays. This chromatographic behavior mimics that of prior art environmentally unfriendly microplastic polystyrene beads. [Figure 4]Figure 11 graphically illustrates the aggregation of pGGG nanoparticles as A340nm versus time. When biotinylated protein nanoparticles (1 mg) and avidin (1 mg) are mixed together, aggregation occurs over time. The buffer was 25 mM sodium phosphate, 75 mM NaCl, and 0.05% Tween 20, pH 7.4. After mixing avidin with protein nanoparticles that do not have biotin on their surface, no aggregation occurred. This demonstrates that protein nanoparticles can behave similarly to latex beads and liposomes to aggregate turbidimetrically. [Figure 5] Figure 10 graphically depicts the aggregation binding curve as A340nm versus μg-avidin. Binding curves were generated by gradually adding 20 μg of avidin to 1 mg of biotinylated protein nanoparticles. [Figure 6] FIG. 1 includes photographs illustrating biotinylated pGGG nanoparticles (1 mg / ml) in a test cuvette before (left) and after (right) avidin addition, illustrating visual detection of aggregation turbidimetrically. [Figure 7] 1 graphically illustrates the dynamic light scattering profile of biotinylated protein nanoparticles (approximately 100 nm on average) and after an aggregation event in the presence of avidin. The aggregated nanoparticles are now approximately 1000 nm in size. [Figure 8] Figure 1 graphically illustrates the dynamic light scattering profile of non-biotinylated protein nanoparticles and after the addition of avidin. The size remains unchanged, indicating that aggregation events can only occur if the nanoparticles are appropriately functionalized. [Figure 9] FIG. 1 graphically illustrates the dynamic light scattering profile of polymerized bovine gamma globulin (pBGG). The size of the pBGG nanoparticles shown here ranges from 100 to 500 nm. DETAILED DESCRIPTION OF THE INVENTION

[0009] Before describing at least one embodiment of the present disclosure in detail with illustrative language and results, it should be understood that the disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. Accordingly, the language used herein is intended to be accorded the broadest possible scope and meaning; the embodiments are meant to be illustrative and not exhaustive. It should also be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0010] Regardless of grammatical usage of the term, the term includes men, women, or people of other gender identities.

[0011] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular. The foregoing techniques and procedures are generally performed in accordance with conventional methods well known in the art and described in various general and more specific references cited and discussed throughout this specification. The nomenclature utilized in connection with, and the laboratory procedures and techniques for, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0012] All patents, published patent applications, and non-patent publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any part of this application are expressly incorporated by reference in their entirety herein to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0013] All of the articles, compositions, kits, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles, compositions, kits, and / or methods have been described in terms of specific embodiments, it will be apparent to those skilled in the art that variations may be applied to the articles, compositions, kits, and / or methods, and in the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined by the appended claims.

[0014] As utilized in accordance with the present disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0015] When used in conjunction with the word "comprising" in the claims and / or specification, the use of the words "a" or "an" can mean "one," but it is also consistent with the meanings of "one or more," "at least one," and "one or more." Thus, the terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "plurality" refers to "two or more."

[0016] Use of the term "at least one" is understood to include one, and any quantity greater than one, including, but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend to 100 or 1000 or more, depending on the term to which it is attached; additionally, a 100 / 1000 quantity should not be considered limiting, as higher limits may yield satisfactory results. Additionally, use of the term "at least one of X, Y, and Z" is understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. Use of ordinal terminology (i.e., "first," "second," "third," "fourth," etc.) is for purposes of distinguishing two or more items only and is not intended to imply, for example, any permutation or order or importance of one item over another, or any order of addition.

[0017] The use of the word "or" in the claims is used to mean an inclusive "and / or" unless expressly stated to refer only to alternatives or unless the alternatives are mutually exclusive. For example, the condition "A or B" is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and A and B are both true (or exist).

[0018] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "an example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. For example, appearances of the phrases "in some embodiments" or "an example" in various places throughout this specification are not necessarily all referring to the same embodiment. Moreover, all references to one or more embodiments or examples should not be construed as limiting the scope of the claims.

[0019] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method used to determine the value, or the variation that exists among study subjects. For example, but not as a limitation, when the term "about" is used, the specified value can vary from the particular value by plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent, with such variations being appropriate for practicing the disclosed methods and as would be understood by one of ordinary skill in the art.

[0020] As used in this specification and claims, the words "comprising" (and any form of compris- ing such as "comprise" and "comprises"), "having" (and any form of having such as "have" and "has"), "including" (and any form of including such as "includes" and "include"), or "containing" (and any form of containing such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0021] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and, where order is important in the particular context, also intended to include BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing this example, combinations containing repeats of one or more items or terms are expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. One of ordinary skill in the art will understand that there is typically no limit to the number of items or terms in any combination, unless the context makes clear otherwise.

[0022] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs completely, or that the subsequently described event or circumstance occurs to a significant extent or degree. For example, when referring to a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs with at least an 80% probability, or at least an 85% probability, or at least a 90% probability, or at least a 95% probability. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are very close to each other but not 100% adjacent to each other, or that a portion of one of two items is not 100% adjacent to the other item but is very close to the other item.

[0023] As used herein, the phrases "associated with" and "coupled to" include both direct association / binding of two moieties to one another and indirect association / binding of two moieties to one another. Non-limiting examples of association / coupling include, for example, covalent binding of one moiety to another, either by a direct bond or through a spacer group, non-covalent binding of one moiety to another, either directly or with a member of a specific binding pair attached to both moieties, incorporation of one moiety into the other, such as by dissolving one moiety in the other or by synthesis, and coating one moiety onto the other.

[0024] The terms "analog" and "derivative" are used interchangeably herein and refer to a substance that contains in its structure the same basic carbon skeleton and carbon functionality as a given compound, but may also contain one or more substitutions therefor. The term "substituted" as used herein is understood to refer to the replacement of at least one substituent on a compound with a residue R. In certain non-limiting embodiments, R can include: H, hydroxyl, thiol, a halogenid selected from fluoride, chloride, bromide, or iodide, C1-C4 compounds selected from the following: optionally substituted linear, branched, or cyclic alkyl, and linear, branched, or cyclic alkenyl, where the optional substituents are one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocyclalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and aryl. and arylheterocycloalkyl, each of which is optionally substituted, wherein the optional substituents are selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cyclalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocyclalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocyclalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, —NH(alkyl), —NH(cycloalkyl), carboxy, and —C(O))-alkyl.

[0025] The term "sample," as used herein, is understood to include any type of biological sample that may be utilized in accordance with the present disclosure. Examples of biological samples that may be utilized include, but are not limited to, whole blood or any fraction thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, stool, pleural effusion, nasopharyngeal fluid, combinations thereof, and the like.

[0026] The term "specific binding partner," particularly (but not limited to) as used herein in the term "specific binding partner of a target analyte," is understood to refer to any molecule that can specifically associate with a target analyte. For example, but not limited to, a binding partner can be an antibody, a receptor, a ligand, an aptamer, a molecularly imprinted polymer (i.e., an inorganic or organic matrix), combinations or derivatives thereof, and any other molecule capable of specific binding to a target analyte.

[0027] The term "antibody" is used herein in the broadest sense and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof (e.g., but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, single-chain antibodies, and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody), antibody replacement proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof that exhibit the desired biological activity of binding a test substance. Antibodies can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0028] An "analyte" is a macromolecule that can be recognized by a specific binding partner of the analyte, such as (but not limited to) an antibody. Both the analyte and the hapten contain at least one antigenic determinant or "epitope," which is the region of the antigen or hapten that binds to the specific binding partner of the analyte (i.e., an antibody). Typically, the epitope on a hapten is the entire molecule.

[0029] Turning now to the inventive concept, certain non-limiting embodiments of the present disclosure are directed to diagnostic reagent compositions (such as, but not limited to, diagnostic immunoassay reagent compositions) for the detection of target analytes in biological samples. The diagnostic reagent composition includes protein nanoparticles comprising polymerized gamma globulin and associated therewith at least one specific binding partner for the analyte.

[0030] Any gamma globulin known in the art or otherwise contemplated herein can be utilized in accordance with the present disclosure, provided that such gamma globulin is capable of polymerization to form protein nanoparticles that naturally have at least one functional group disposed thereon. Non-limiting examples of gamma globulins that can be utilized include mammalian gamma globulins (such as, but not limited to, goat gamma globulin (GGG) and bovine gamma globulin (BGG)), and non-mammalian gamma globulins.

[0031] The protein nanoparticles can have disposed thereon any functional group naturally present in gamma globulin, non-limiting examples of which include carboxyl groups, amine groups, and / or sulfhydryl groups.

[0032] In certain specific (but non-limiting) embodiments, the protein nanoparticles may contain one or more proteins in addition to gamma globulin. A non-limiting example of an additional protein that may be included is serum albumin. However, it will be understood that any additional protein may be included as long as the protein nanoparticles can function as described in this disclosure.

[0033] Any specific binding partner of an analyte known in the art or otherwise contemplated herein can be utilized in accordance with the present disclosure, provided that such binding partner is capable of specifically binding to the analyte to be detected and thereby can be utilized for diagnostic detection of the analyte. Non-limiting examples of types of specific binding partners of an analyte that are utilized for diagnostic applications in accordance with the present disclosure include receptors, ligands, antigens, antibodies, aptamers, molecularly imprinted polymers, and the like, as well as derivatives and variants thereof, and any combination thereof.

[0034] A target analyte can be any molecule present in a biological sample whose detection and / or quantification is desired. For example (but not limited to), if the target analyte is an antigen, the specific binding partner of the target analyte can be an antibody or fragment thereof that specifically binds to the antigen; if the target analyte is an antibody, the specific binding partner of the target analyte can be the antigen to which the antibody specifically binds; if the target analyte is a ligand, the specific binding partner of the target analyte can be a receptor (or a part or derivative thereof) that specifically binds to the ligand; and if the target analyte is a receptor, the specific binding partner of the target analyte can be the ligand that specifically binds to the receptor.

[0035] Nanoparticles made from polymerized gamma globulin can have any size, shape, and dimensions, so long as the nanoparticles are capable of functioning in accordance with the present disclosure. Non-limiting examples of nanoparticle sizes that can be utilized in accordance with the present disclosure include about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 75 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 125 nm, about 130 nm, about 140 nm, and about 150 nm. , about 160nm, about 170nm, about 175nm, about 180nm, about 190nm, about 200nm, about 210nm, about 220nm, about 225nm, about 230nm, about 240nm, Approximately 250nm, approximately 260nm, approximately 270nm, approximately 275nm, approximately 280nm, approximately 290nm, approximately 300nm, approximately 325nm, approximately 350nm, approximately 375nm, approximately 400nm, approximately 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, about 1000 nm, and greater, as well as ranges formed by any of the above values ​​(i.e., from about 20 nm to about 1000 nm, from about 50 nm to about 1000 nm, from about 50 nm to about 500 nm, from about 100 nm to about 500 nm, from about 100 nm to about 200 nm, etc.).

[0036] In certain specific (but non-limiting) embodiments, the diagnostic reagent composition can comprise any element or property that allows for detection of a complex of the nanoparticle with the bound analyte. In certain specific (but non-limiting) embodiments, the diagnostic reagent composition can have at least one dye associated therewith to facilitate detection of the bound analyte. For example (but not limited to), the nanoparticles can have at least one dye incorporated or otherwise associated therewith. Dyes can be used for (for example, but not limited to) spectrophotometric, luminescent (i.e., chemiluminescent, or fluorescent) detection. Non-limiting examples include fluorescein, rhodamine, nitrobenzofurazan (NBD), and the like.

[0037] In certain specific, but non-limiting, embodiments, the nanoparticles can be biotinylated, thereby allowing for the indirect attachment of another biotinylated protein or molecule of interest to the particle via tetrameric streptavidin.

[0038] Certain non-limiting embodiments of the present disclosure are directed to kits containing one or more of any of the diagnostic reagent compositions disclosed or otherwise contemplated herein. In certain specific (but non-limiting) embodiments, the kit further comprises at least one additional assay reagent that interacts with the diagnostic reagent composition to detect the presence and / or concentration of a target analyte in a biological sample.

[0039] In a specific, but non-limiting, embodiment, the kit includes two or more diagnostic reagent compositions. The two or more diagnostic reagent compositions may be disposed in separate compositions, or the compositions may be disposed together in a single composition to perform a multiplex assay for two different target analytes. When disposed together in a single composition, the first and second diagnostic reagent compositions have different dyes (or other types of detection mechanisms that differ from each other) associated therewith that allow for the detection of both target analytes in a single reaction.

[0040] The compositions / reagents of the kit can be supplied in any form that enables them to function in accordance with the present disclosure. For example, but not by way of limitation, each reagent can be supplied in liquid form and disposed within the kit in bulk and / or single-portion form. Alternatively, in a specific (but non-limiting) embodiment, one or more of the reagents can be disposed within the kit in the form of a single-portion lyophilized reagent. The use of dried reagents in kits / microfluidic devices is described in detail in U.S. Pat. No. 9,244,085 (Samproni), the entire contents of which are expressly incorporated herein by reference.

[0041] In addition to the compositions / reagents described in detail herein above, the kit may further contain other reagents for carrying out any of the specific assays described herein or otherwise contemplated. The nature of these additional reagents depends on the specific assay format, and their identification is well within the skill of those skilled in the art; therefore, further description thereof is not deemed necessary. In addition, the compositions / reagents present in the kit may each be present in separate containers / compartments, or various compositions / reagents may be combined in one or more containers / compartments depending on the cross-reactivity and stability of the compositions / reagents. In addition, the kit may also include a microfluidic device in which the compositions / reagents are arranged.

[0042] The relative amounts of the various compositions / reagents in the kit may vary widely to provide concentrations of the compositions / reagents that substantially optimize the reactions required to occur during the assay method and further optimize the sensitivity and selectivity of the assay. Under appropriate circumstances, one or more of the compositions / reagents in the kit may be supplied as a dry powder, such as a lyophilized powder, and the kit may further include an excipient for dissolving the dried reagent; in this way, a reagent solution having an appropriate concentration for performing a method or assay according to the present disclosure can be obtained from such compositions. Positive and / or negative controls may also be included in the kit. In addition, the kit may further include a set of written instructions describing how to use the kit. Kits of this type may be used in any of the methods described or otherwise contemplated herein.

[0043] Certain further non-limiting embodiments of the present disclosure are directed to microfluidic devices containing one or more of any of the diagnostic reagent compositions described herein above or otherwise contemplated herein. In particular, certain non-limiting embodiments include microfluidic devices for determining the concentration of at least one target analyte in a sample. The microfluidic device includes: (i) an inlet channel through which a sample is applied; and (ii) at least a first compartment in fluid communication with the inlet channel and capable of containing at least one of any of the diagnostic reagent compositions disclosed herein or otherwise contemplated. Compartment (ii) can further contain any additional reagents required to perform an assay for the detection of the target analyte. Any of the assay reagents disclosed or contemplated herein or otherwise known in the art can be utilized in the microfluidic devices of the present disclosure.

[0044] The microfluidic device can have any compartment arrangement and distribution of various compositions / reagents among the compartments that enables the device to function in accordance with the present disclosure. That is, when a diagnostic reagent composition is utilized in combination with a second assay reagent, the two reagents can be located in the same compartment or in different compartments. When the two reagents are separated between two compartments, the diagnostic reagent composition can be located in a first compartment in fluid communication with the inlet channel, and at least one additional assay reagent can be located in a second compartment in fluid communication with the first compartment.

[0045] In a specific, but non-limiting, embodiment, a microfluidic device includes two or more of any of the diagnostic reagent compositions disclosed herein or otherwise contemplated, where the two or more diagnostic reagent compositions are arranged together in the microfluidic device such that multiplex assays for two different target analytes can be performed within the microfluidic device. In this manner, the first and second diagnostic reagent compositions (and any additional diagnostic reagent compositions, if present) have different dyes (or other types of detection mechanisms that are different from each other) associated therewith that allow for detection of both target analytes in a single reaction. The two or more diagnostic reagent compositions may be arranged in the same compartment of the microfluidic device or in separate compartments.

[0046] Alternatively, a microfluidic device can include two or more of any of the diagnostic reagent compositions disclosed herein or otherwise contemplated, where the two or more diagnostic reagent compositions are disposed in separate compartments in the microfluidic device and have separate read chambers for their detection, thereby allowing for the detection of two different target analytes within the microfluidic device.

[0047] Microfluidic devices of the present disclosure can possess any design or configuration known in the art or otherwise contemplated herein for use in diagnostic analyte assays (e.g., but not limited to, diagnostic immunoassays). In certain specific (but non-limiting) embodiments, the microfluidic device may be in the form of a cassette configured for insertion into an automated diagnostic test instrument system that performs the diagnostic assay. Alternatively, the microfluidic device may be a stand-alone product that can be read without a diagnostic test instrument system. For example (but not limited to), the microfluidic device may be in the form of a lateral flow device that can be implemented at a point-of-care (POC) location.

[0048] Any of the compartments of a microfluidic device can be sealed to maintain a reagent placed therein in a substantially airtight environment until it is used; for example, a compartment containing a lyophilized reagent can be sealed to prevent any unintended reconstitution of the reagent. An inlet channel and a compartment, and two compartments can be described as "capable of fluid communication" with each other; this phrase indicates that each compartment may remain sealed, but that two compartments can allow fluid flow between them upon puncturing a seal formed within or between the two compartments.

[0049] The microfluidic devices of the present disclosure may include any other desired characteristics known in the art or otherwise contemplated herein. For example, but not limited to, the microfluidic devices of the present disclosure may further include a read chamber; the read chamber may be any of the reagent-containing compartments described herein above, or the read chamber may be in fluid communication with said compartments. The microfluidic devices may further include one or more additional compartments containing other solutions, such as (but not limited to) wash solutions, diluents, excipients, interference solutions, positive controls, negative controls, quality controls, etc. These additional compartments may be in fluid communication with one or more of the other compartments. For example, the microfluidic devices may further include one or more compartments containing wash solutions, which may be in fluid communication with any other compartment of the device. In another example, the microfluidic devices may further include one or more compartments containing excipients for dissolving one or more dried reagents, which may be in fluid communication with any other compartment of the device. In yet a further example, a microfluidic device may include one or more compartments containing a diluent, and the compartments may be in fluid communication with any other compartment of the device.

[0050] Certain non-limiting embodiments of the present disclosure are directed to methods of making any of the diagnostic reagent compositions disclosed or otherwise contemplated herein. In specific (but non-limiting) embodiments, the method comprises the steps of: polymerizing gamma globulin (GG) to form polymerized GG (pGG) nanoparticles; and attaching a specific binding partner of at least one test substance to the pGG nanoparticles via at least one naturally occurring functional moiety disposed on the surface of the nanoparticles.

[0051] The polymerization and attachment reactions can be carried out by any method known in the art or otherwise contemplated herein. For example (but not limited to), pGG nanoparticles can be formed through a technique selected from the group consisting of solvent evaporation, nanoprecipitation, salting out, and emulsification. In one non-limiting example, pGG nanoparticles are formed by heating GG to a temperature ranging from about 65°C to about 67°C for a certain period of time.

[0052] Additionally, in certain non-limiting embodiments, at least one additional element (such as, but not limited to, a dye) can be incorporated into the diagnostic reagent composition during the manufacturing process.

[0053] Certain non-limiting embodiments of the present disclosure are directed to a method for determining the presence and / or concentration of at least one target analyte in a biological sample. The method comprises combining the biological sample with at least one of the diagnostic reagent compositions disclosed herein or otherwise contemplated under conditions that allow the target analyte present in the biological sample to substantially bind to the specific binding partner of the analyte extending from the outer surface of the nanoparticles of the diagnostic reagent composition, thereby forming a complex; then determining the presence and / or concentration of the target analyte based on any complex formed. The determining step can be carried out using any assay method known in the art.

[0054] In certain specific (but non-limiting) embodiments, the method utilizes a homogeneous assay format. For example (but not limited to), binding of the target analyte to the diagnostic reagent composition results in agglutination, and therefore no additional reagents are required to detect the target analyte. The results of the agglutination assay can be detected manually or automatically (i.e., visually or spectrophotometrically), and can be detected turbidimetrically or nephelometrically.

[0055] Alternatively, the method can utilize a heterogeneous assay format, in which case additional reagents must be used in combination with the diagnostic reagent composition for detecting the target analyte in the biological sample. For example (but not limited to), the method may utilize a heterogeneous format such as a sandwich assay. If a second reagent is required, the biological sample can be contacted with the diagnostic reagent composition and the second reagent simultaneously or fully or partially sequentially. In addition, the detection step of the method involves detecting a complex comprising the diagnostic reagent composition / target analyte / second assay reagent.

[0056] Certain non-limiting embodiments of the present disclosure are directed to a method for determining the presence and / or concentration of at least two target analytes in a biological sample. In the method, the biological sample is combined with at least two of any of the diagnostic reagent compositions disclosed herein or otherwise contemplated (i.e., a first diagnostic reagent composition and a second diagnostic reagent composition) under the following conditions: (1) conditions that allow the first target analyte present in the biological sample to substantially bind to the specific binding partner of the analyte extending from the outer surface of the nanoparticles of the first diagnostic reagent composition, thereby forming a first complex, and (2) conditions that allow the second target analyte present in the biological sample to substantially bind to the specific binding partner of the analyte extending from the outer surface of the nanoparticles of the second diagnostic reagent composition, thereby forming a second complex. The presence and / or concentration of each of the first and second target analytes is then determined based on any first and second complexes formed, respectively. The determining step can be performed using any assay method known in the art.

[0057] Non-limiting examples of biological samples that can be utilized in accordance with various methods of the present disclosure include whole blood or any fraction thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, stool, pleural effusion, nasopharyngeal fluid, and combinations thereof. Specific non-limiting examples include lysed whole blood cells and lysed red blood cells.

[0058] As described above, when a diagnostic reagent composition is used in combination with a second reagent, the two compositions can be added simultaneously or sequentially. In addition, when two or more diagnostic reagent compositions are used in the same reaction, the two diagnostic reagent compositions can be added simultaneously or sequentially. When the various compositions used in the method are added sequentially, the order of addition of the compositions can be varied; those skilled in the art can determine the specific desired order of adding different compositions to the assay. The simplest addition order, of course, is to add all the materials simultaneously and determine the signal generated therefrom. Alternatively, each composition or group of compositions can be combined sequentially. In certain embodiments, an incubation step can follow one or more additions. [Example]

[0059] Examples are provided below. However, it should be understood that the present disclosure is not limited in its application to the specific experiments, results, and experimental procedures disclosed herein. Rather, the examples are provided merely as one of various embodiments and are intended to be illustrative and not exhaustive. [Example]

[0060] Nonmagnetic latex particles (NMLPs) have been widely used in the diagnostic industry, where antigens or antibodies are immobilized on the particles for use in competitive or sandwich assay formats. The analyte of interest brings the particles into close contact, resulting in a cloudy or turbid solution that can be quantified spectrophotometrically. However, these plastic beads are toxic and environmentally unfriendly, and they have significant associated waste disposal costs that will increase significantly in the future. In addition, certain surface functionalization procedures can require several materials, considerable time, and resources. Furthermore, the coupling process and waste materials can be hazardous. The respective processes and waste management typically involve significant safety measures and significant costs, and may even be discontinued for regulatory reasons.

[0061] The present disclosure solves these challenges by using biodegradable protein nanoparticles that naturally contain functional moieties (e.g., but not limited to, carboxyl, amine, or sulfhydryl moieties) to attach antibodies or antigens (or specific binding partners of other test substances) to the surface of the nanoparticles via conventional chemical reactions. Gamma globulins (GG, such as, but not limited to, goat gamma globulin (GGG) and bovine gamma globulin (BGG)) are polymerized (e.g., but not limited to, via heat treatment) to form polymerized gamma globulin (pGG) nanoparticles. This approach to producing pGG as biodegradable protein nanoparticles reduces the number of materials required and is environmentally neutral and therefore sustainable.

[0062] These functionalized beads and the analyte of interest bind in a multivalent manner, forming aggregates that can be visualized or quantified spectrophotometrically, particularly turbidimetrically or nephelometrically. Quantifying this change in turbidity (or cloudiness) of the reaction mixture as a function of analyte concentration is the basis of homogeneous immunoagglutination assays. In contrast, prior art latex beads ultimately result in waste, and waste disposal costs remain a challenge that may rise significantly in the future. The present disclosure replaces these currently used toxic beads with environmentally friendly and biodegradable protein nanoparticles.

[0063] The present disclosure is based on the use of biodegradable protein nanoparticles, which are between about 20 nm and about 1000 nm (typically <500 nm) and contain amino acid residues with reactive groups (e.g., COOH, NH, SH, etc.) that are ready for attachment of biotin, fluorescein, antibodies, antigens, or ligands to their surfaces via conventional chemical reactions. These protein nanoparticles are colloidal and fully suspendable in buffers and solutions commonly used for diagnostic reagents. They can be prepared by various methods known in the art (e.g., solvent evaporation, nanoprecipitation, salting out, emulsification, etc.). In this example, a 10 mg / ml solution of goat gamma golubin (GGG) was heated appropriately at 65–67°C until it reached approximately 30% transmittance at 340 nm. Similar to latex beads, the polymerized GGG (pGGG) product was white and milky in appearance (Figure 1).

[0064] These protein nanoparticles could be modified by attaching specific binding partners of target analytes to them. For example, the image on the left of Figure 2 demonstrates that protein nanoparticles were functionalized to contain biotin on their surfaces. In addition, protein nanoparticles could also be fabricated with (or post-fabrication modified to contain) dyes associated therewith; the image on the right of Figure 2 demonstrates that protein nanoparticles were functionalized with fluorescein on their surfaces.

[0065] pGGG nanoparticles behave similarly to NMLPs in chromatography. For example, as shown in Figure 3, pGGG nanoparticles are normally excluded from size-exclusion columns (e.g., CL-2B, with an exclusion limit of approximately 100 nm) and can be detected at 280, 340, and 600 nm. Based on these data, it was determined that these pGGG particles could be used for aggregation assays. As shown in Figure 4, when biotinylated nanoparticles (1 mg) were exposed to avidin (1 mg), aggregation occurred almost instantaneously, resulting in an increase in absorbance readings at 340 nm over time. In the absence of biotin on the particle surface, the absorbance readings remained unchanged. This data demonstrates that pGGG protein nanoparticles behave similarly to latex beads and liposomes to aggregate turbidimetrically.

[0066] Next, a calibration curve (A340 nm vs. μg avidin) was generated when increasing amounts of avidin were added to a solution containing biotinylated pGGG nanoparticles (Figure 5). Thus, quantitative analysis of avidin can be performed based on this homogeneous aggregation assay format using functionalized biotinylated pGGG nanoparticles. Figure 6 contains images of the test cuvette at the end of the reaction, before (left) and after (right) the addition of avidin. This aggregation event was also readily observed with a dynamic light scattering (DLS) detector, as shown in Figure 7. The aggregated particles were observed to be approximately 1 μm in size on the DLS. In contrast, in the absence of biotin on the surface of the pGGG nanoparticles, the particle size remained unchanged even in the presence of avidin, as shown in Figure 8.

[0067] The functionalized protein nanoparticles (pGG) of the present disclosure can replace the "toxic" plastic beads currently used in homogeneous immunoassays. This replacement avoids the need for expensive waste disposal units and provides an alternative to NMLPs, which is especially important if NMLPs are banned in the future. Key characteristics of the protein pGG nanoparticles of the present disclosure are their biodegradability, favorable size (e.g., 20-1000 nm), ease of attachment of functionalized molecules or proteins, and similar behavior to conventional NMLPs. The pGG nanoparticles of the present disclosure can also be used in immunoassays in various formats. Non-limiting examples of other uses include as nonspecific interference blockers, signal amplifiers and enhancers, colloidal stationary phases to help stabilize proteins, and functionalized dye carriers in multiplex formats (e.g., flow cytometry immunoassays). [Example]

[0068] Example 1 describes the use of goat gamma globulin to prepare polymeric protein nanoparticles (pGGG nanoparticles) for use in preparing diagnostic reagent compositions to which specific binding partners of target analytes can be attached. However, the present disclosure is not limited to the use of goat gamma globulin in preparing nanoparticles; it will be understood that other gamma globulins can be utilized in a similar manner, and thus the present disclosure encompasses the use of other gamma globulins in preparing diagnostic reagent compositions in accordance with the present disclosure.

[0069] For example, Figure 9 illustrates the use of bovine gamma globulin (BGG) to produce polymerized BGG (or pBGG) protein nanoparticles. pBGG protein nanoparticles were produced using the same heating technique as described above in Example 1.

[0070] These pBGG protein nanoparticles were attached to specific binding partners of target analytes in a manner similar to that described above in Example 1, and a diagnostic reagent composition containing the pBGG protein nanoparticles was incorporated into an agglutination immunoassay for detecting analytes bound by the specific binding partners of the target analytes.

[0071] Non-limiting exemplary embodiments The following is a list of non-limiting exemplary embodiments disclosed herein:

[0072] Exemplary embodiment 1. 1. A diagnostic immunoassay reagent composition for the detection of a target analyte in a biological sample, comprising: Biodegradable protein nanoparticles comprising polymerized gamma globulin (pGG) and having at least one functional moiety thereon; and a specific binding partner for at least one test substance attached to the biodegradable protein nanoparticle through at least one functional moiety; A composition comprising:

[0073] Exemplary Embodiment 2. The composition of Exemplary Embodiment 1, wherein the at least one functional moiety is selected from the group consisting of a carboxyl group, an amine group, or a sulfhydryl group.

[0074] Exemplary Embodiment 3. The composition of exemplary embodiment 1 or 2, wherein the nanoparticles have a diameter ranging from about 20 nm to about 1000 nm.

[0075] Exemplary Embodiment 4. The composition of any one of Exemplary Embodiments 1-3, wherein the specific binding partner of the test agent comprises an antibody or fragment thereof that specifically binds to the target analyte.

[0076] Exemplary Embodiment 5. The composition of any one of Exemplary Embodiments 1-4, wherein the specific binding partner of the analyte comprises an antigen to which the target analyte specifically binds.

[0077] Exemplary Embodiment 6. The composition of any one of Exemplary Embodiments 1-5, wherein the specific binding partner of the at least one test agent comprises at least one receptor or a portion or derivative thereof that specifically binds to the target test agent.

[0078] Exemplary Embodiment 7 The composition of any one of Exemplary Embodiments 1-6, wherein the specific binding partner of at least one analyte comprises a ligand for the target analyte.

[0079] Exemplary Embodiment 8 The composition of any one of Exemplary Embodiments 1-7, wherein the nanoparticles are biotinylated.

[0080] Exemplary Embodiment 9 The composition of any one of Exemplary Embodiments 1-8, wherein the nanoparticles have at least one dye attached thereto.

[0081] Exemplary Embodiment 10 The composition of Exemplary Embodiment 9, wherein the dye comprises fluorescein.

[0082] Exemplary Embodiment 11 The composition of any one of Exemplary Embodiments 1-10, wherein the gamma globulin is goat gamma globulin.

[0083] Exemplary Embodiment 12 The composition of any one of Exemplary Embodiments 1-11, wherein the gamma globulin is bovine gamma globulin.

[0084] Exemplary embodiment 13. A kit comprising at least one diagnostic reagent composition of any one of exemplary embodiments 1-12.

[0085] Exemplary Embodiment 14 The kit of exemplary embodiment 13, further comprising at least one additional reagent for use in a diagnostic immunoassay.

[0086] Exemplary Embodiment 15. The kit of exemplary embodiment 13 or 14, further comprising at least two diagnostic reagent compositions of any one of exemplary embodiments 1-12.

[0087] Exemplary Embodiment 15A. The kit of exemplary embodiment 15, wherein the kit is for use in a multiplexed assay.

[0088] Exemplary embodiment 16. 1. A microfluidic device comprising: (i) an inlet channel through which the sample is applied; and (ii) at least one compartment capable of fluid communication with the inlet channel; Including, At least one compartment contains at least one diagnostic reagent composition of any one of exemplary embodiments 1-12; Microfluidic devices.

[0089] Exemplary Embodiment 17. The microfluidic device of Exemplary Embodiment 16, further defined as a microfluidic device for performing multiplexed assays, and wherein (ii) comprises at least two diagnostic reagent compositions.

[0090] Exemplary embodiment 18. 1. A method for preparing a diagnostic reagent composition for the detection of a target analyte in a biological sample, comprising: polymerizing gamma globulin (GG) to form polymerized GG (pGG) nanoparticles; attaching a specific binding partner of at least one test substance to the pGG nanoparticles; A method comprising:

[0091] Exemplary Embodiment 19. The method of Exemplary Embodiment 18, wherein the pGG nanoparticles are formed by heating the GG to a temperature ranging from about 65°C to about 67°C for a period of time.

[0092] Exemplary Embodiment 20. The method of Exemplary Embodiment 18 or 19, wherein the pGG nanoparticles are formed via a technique selected from the group consisting of a solvent evaporation technique, a nanoprecipitation technique, a salting-out technique, and an emulsification technique.

[0093] Exemplary Embodiment 21 The method of any one of Exemplary Embodiments 18-20, wherein the at least one functional moiety is selected from the group consisting of a carboxyl group, an amine group, or a sulfhydryl group.

[0094] Exemplary Embodiment 22 The method of any one of Exemplary Embodiments 18-21, wherein the nanoparticles have a diameter ranging from about 20 nm to about 1000 nm.

[0095] Exemplary Embodiment 23 The method of any one of Exemplary Embodiments 18-22, wherein the specific binding partner of the analyte comprises an antibody or fragment thereof that specifically binds to the target analyte.

[0096] Exemplary Embodiment 24 The method of any one of Exemplary Embodiments 18-23, wherein the specific binding partner of the analyte comprises an antigen to which the target analyte specifically binds.

[0097] Exemplary Embodiment 25 The method of any one of Exemplary Embodiments 18-24, wherein the specific binding partner of the at least one analyte comprises at least one receptor or a portion or derivative thereof that specifically binds to the target analyte.

[0098] Exemplary Embodiment 26 The method of any one of Exemplary Embodiments 18-25, wherein the specific binding partner of at least one analyte comprises a ligand for the target analyte.

[0099] Exemplary Embodiment 27 The method of any one of Exemplary Embodiments 18-26, wherein the nanoparticles are biotinylated.

[0100] Exemplary Embodiment 28 The method of any one of Exemplary Embodiments 18-27, wherein the nanoparticles have at least one dye attached thereto.

[0101] Exemplary Embodiment 29 The method of Exemplary Embodiment 28, wherein the dye comprises fluorescein.

[0102] Exemplary Embodiment 30 The method of any one of Exemplary Embodiments 18-29, wherein the gamma globulin is goat gamma globulin.

[0103] Exemplary Embodiment 31 The method of any one of Exemplary Embodiments 18-29, wherein the gamma globulin is bovine gamma globulin.

[0104] Exemplary embodiment 32. 1. A method for determining the presence and / or concentration of a target analyte in a biological sample, comprising: combining a biological sample with at least one diagnostic reagent composition of any one of exemplary embodiments 1-12 under conditions that allow a specific binding partner of the at least one analyte to bind to a target analyte present in the sample to form a complex; determining the presence and / or concentration of the target analyte based on any complexes formed; A method comprising:

[0105] Exemplary Embodiment 33 The method of exemplary embodiment 32, comprising a homogeneous assay format.

[0106] Exemplary Embodiment 34 The method of Exemplary Embodiment 33, wherein the assay format comprises an agglutination assay.

[0107] Exemplary Embodiment 35 The method of Exemplary Embodiment 34, wherein aggregate formation is detected visually or spectrophotometrically.

[0108] Exemplary Embodiment 36 The method of Exemplary Embodiment 35, wherein the detection is performed turbidimetrically or nephelometrically.

[0109] Exemplary Embodiment 37 The method of exemplary embodiment 32, comprising a heterogeneous assay format.

[0110] Exemplary Embodiment 38 The method of Exemplary Embodiment 37, wherein the assay format comprises a sandwich assay.

[0111] Exemplary embodiment 39. A method for determining the presence and / or concentration of at least two target analytes in a biological sample, comprising: combining a biological sample with the first diagnostic reagent composition of any one of exemplary embodiments 1-12 and the second diagnostic reagent composition of any one of exemplary embodiments 1-12 under conditions that allow a specific binding partner of the analyte of the first diagnostic reagent composition to bind to a first target analyte present in the sample to form a first complex, and that allow a specific binding partner of the analyte of the second diagnostic reagent composition to bind to a second target analyte present in the sample to form a second complex; determining the presence and / or concentration of a first target analyte based on any first complexes formed; determining the presence and / or concentration of a second target test substance based on any second complexes formed; A method comprising:

[0112] Exemplary Embodiment 40. The method of any one of Exemplary Embodiments 32-39, wherein the biological sample is selected from the group consisting of whole blood or any portion thereof, urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, stool, pleural effusion, nasopharyngeal fluid, and combinations thereof.

[0113] Thus, in accordance with the present disclosure, there have been provided compositions, kits, and devices, as well as methods for making and using the same, that fully satisfy the objects and advantages set forth above. While this disclosure has been described in conjunction with the specific figures, experiments, results, and phraseology set forth above, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the description is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

1. 1. A diagnostic immunoassay reagent composition for the detection of a target analyte in a biological sample, comprising: Biodegradable protein nanoparticles comprising polymerized gamma globulin (pGG) and having at least one functional moiety thereon; and a specific binding partner for at least one test substance attached to the biodegradable protein nanoparticle through at least one functional moiety; A composition comprising:

2. at least one functional moiety is selected from the group consisting of a carboxyl group, an amine group, or a sulfhydryl group; The composition of claim 1.

3. The nanoparticles have a diameter ranging from about 20 nm to about 1000 nm. The composition of claim 1.

4. The specific binding partner of the test substance comprises an antibody or fragment thereof that specifically binds to the target test substance; The composition of claim 1.

5. The specific binding partner of the analyte comprises an antigen to which the target analyte specifically binds; The composition of claim 1.

6. the specific binding partner of at least one analyte comprises a receptor or a portion or derivative thereof that specifically binds to the target analyte; The composition of claim 1.

7. the specific binding partner of at least one analyte comprises a ligand for the target analyte; The composition of claim 1.

8. The nanoparticles have at least one dye attached thereto. The composition of claim 1.

9. The gamma globulin is goat gamma globulin, The composition of claim 1.

10. The gamma globulin is bovine gamma globulin, The composition of claim 1.

11. 1. A kit comprising: comprising at least one diagnostic reagent composition according to any one of claims 1 to 10, kit.

12. further comprising at least one additional reagent for use in a diagnostic immunoassay, The kit of claim 11.

13. Further comprising at least two diagnostic reagent compositions according to any one of claims 1 to 10. The kit of claim 11.

14. 1. A microfluidic device comprising: (i) an inlet channel through which the sample is applied; and (ii) comprises at least one compartment capable of fluid communication with the inlet channel; At least one compartment contains at least one diagnostic reagent composition according to any one of claims 1 to 10. Microfluidic devices.

15. 1. A method for preparing a diagnostic reagent composition for the detection of a target analyte in a biological sample, comprising: polymerizing gamma globulin (GG) to form polymerized GG (pGG) nanoparticles; attaching a specific binding partner of at least one test substance to pGG nanoparticles; A method comprising:

16. pGG nanoparticles are formed by heating GG to a temperature ranging from about 65°C to about 67°C for a period of time.

16. The method of claim 15.

17. The pGG nanoparticles are formed via a technique selected from the group consisting of a solvent evaporation technique, a nanoprecipitation technique, a salting-out technique, and an emulsification technique.

16. The method of claim 15.

18. 1. A method for determining the presence and / or concentration of a target analyte in a biological sample, comprising: combining a biological sample with at least one diagnostic reagent composition according to any one of claims 1 to 10 under conditions that allow a specific binding partner of the at least one analyte to bind to a target analyte present in the sample to form a complex; determining the presence and / or concentration of the target analyte based on any complexes formed; A method comprising:

19. including homogeneous assay formats, The assay format comprises an agglutination assay.

20. The method of claim 18.

20. The biological sample is selected from the group consisting of whole blood or any fraction thereof, urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, stool, pleural effusion, nasopharyngeal fluid, and combinations thereof; 20. The method of claim 18.

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