EP4655591A1

EP4655591A1 - Nanoparticles comprising polymerized gamma globulin and methods of production and use thereof

Info

Publication number: EP4655591A1
Application number: EP24747588.2A
Authority: EP
Inventors: Huey Lee; William Bedzyk; Michelle SON; Michael Sommer; Frank Vitzthum
Original assignee: Siemens Healthcare Diagnostics Inc
Current assignee: Siemens Healthcare Diagnostics Inc
Priority date: 2023-01-26
Filing date: 2024-01-19
Publication date: 2025-12-03
Also published as: CN120677383A; WO2024158650A9; WO2024158650A1; AU2024211287A1; JP2026504163A

Abstract

Diagnostic immunoassay reagent compositions are disclosed that include nanoparticles formed of polymerized gamma globulin. The nanoparticles have at least one functional moiety on a surface thereof, and at least one analyte-specific binding partner is attached to the nanoparticles through the at least one functional moiety. Also disclosed are kits, devices, and systems that contain the diagnostic immunoassay reagent compositions, as well as methods of producing and using the diagnostic immunoassay reagent compositions.

Description

TITLE OF INVENTION NANOPARTICLES COMPRISING POLYMERIZED GAMMA GLOBULIN AND METHODS OF PRODUCTION AND USE THEREOF

REFERENCE TO RELATED APPLICATIONS

[0001] Not Applicable.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not Applicable.

BACKGROUND

[0003] In the fields of medicine and clinical chemistry, many studies and determinations of physiologically reactive species or analytes are carried out by taking advantage of the interaction between specific binding pair members. For example, the target analyte in a patient sample may be one member of a specific binding pair, and the target analyte is detectable by employing a corresponding member of the specific binding pair immobilized on a solid support. For example (but not by way of limitation), the immobilized binding pair member may be an antigen for the detection of a target antibody in a sample or vice versa (i.e., the immobilized binding pair member may be an antibody for detection of a target analyte in a sample), or the immobilized binding pair member may be a ligand for the detection of a target receptor in a sample or vice versa (i.e., the immobilized binding pair member may be a receptor for detection of a target ligand in a sample). Various support or surface materials have been developed for these applications and require various bonding or "functionalization" techniques to immobilize the specific binding pair member on 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 requirements of a large number of materials and resources as well as a substantial amount of time, in particular for "delicate" systems. Also, the coupling processes and waste material resulting therefrom may be hazardous, and the same is even true for certain surfaces that are applied. Management of respective processes and waste are typically associated with substantial safety measures as well as significant costs, too, and may even have to be discontinued for regulatory reasons with substantial consequences for a complete business.

[0005] For example, non-magnetic latex particles (NMLP) are widely used in homogeneous immunoassays as surfaces (also referred to as a "solid phase"). A binder, e.g., an antigen or antibody, is attached to these latex beads, and the attachment can be performed by a chemical reaction. One non-limiting example of such attachment reaction involves the use of an azomethine reaction followed by a reduction process that can apply sodium cyanoborohydride (NaBHsCN), a hazardous chemical compound that must be managed appropriately. The functionalized NMLPs and analytes of interest bind in a multivalent fashion, forming aggregates that can be visualized or quantified spectrophotometrically, in particular (but not by way of limitation) turbidimetrically or nephelometrically. Quantifying this change in the turbidity (or cloudiness) or scatter light of the reaction mixture as a function of an analyte concentration is the basis of a homogeneous immuno-agglutination assay.

[0006] In addition to the use of hazardous materials in the production thereof, these latex beads utilized in immunoassays also eventually represent waste. The waste handling costs associated with these latex bead-containing immunoassay reagents will continue to be a challenge, and costs are likely to rise significantly in the future.

[0007] Therefore, there is a need in the art for new and improved diagnostic immunoassay reagents that overcome the defects and disadvantages of the prior art. It is to such reagents and kits and microfluidics devices containing same, as well as methods of producing and using same, that the present disclosure is directed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 contains a photograph of one non-limiting embodiment of protein nanoparticles constructed in accordance with the present disclosure, wherein the protein nanoparticles are formed of polymerized goat gamma globulin (pGGG). The protein nanoparticles shown in this Figure are present in solution at a concentration of 10 mg/ml.

[0009] FIG. 2 contains photographs of solutions of pGGG protein nanoparticles (1 mg/ml) that have been functionalized with biotin (left) or fluorescein (right) on the surface thereof.

[0010] FIG. 3 graphically depicts UV-VIS chromatograms of pGGG nanoparticles at 280, 340, and 600 nm. When pGGG nanoparticles were injected into a Sepharose CL-2B column (1.0 x 30 cm), the high molecular weight peak [main peak (~100 nm) excluded at ~7 ml] can be detected simultaneously at 280, 340, and 600 nm. Absorbance at 340 nm is normally used for a typical solution agglutination immunoassay. This chromatographic behavior mimics that of the environmentally unfriendly microplastic polystyrene beads of the prior art.

[0011] FIG. 4 graphically depicts agglutination of pGGG nanoparticles at A340 nm vs. time. Agglutination occurs over time when biotinylated protein nanoparticles (1 mg) and avidin (1 mg) are mixed together. Buffer was 25 mM sodium phosphate, 75 mM NaCI, and 0.05%Tween 20, pH 7.4. Agglutination did not occur after mixing avidin with the protein nanoparticles without biotin on their surface. This demonstrates that the protein nanoparticles can work similarly to latex beads and liposomes to agglutinate turbidimetrically.

[0012] FIG. 5 graphically depicts a binding curve of agglutination at A340 nm vs. pg-avidin. When avidin 20 pg was added incrementally to 1 mg of the biotinylated protein nanoparticles, a binding curve was created.

[0013] FIG. 6 contains photographs illustrating the biotinylated pGGG nanoparticles (1 mg/ml) in a test cuvette before (left) and after avidin addition (right), illustrating the visual detection of agglutination turbidimetrically.

[0014] FIG. 7 graphically depicts dynamic light scattering profiles of biotinylated protein nanoparticles (~100 nm on average) and after the agglutination event in the presence of avidin. The agglutinated nanoparticles are now ~1000 nm in size.

[0015] FIG. 8 graphically depicts dynamic light scattering profiles of the unbiotinylated protein nanoparticles and after the addition of avidin. The sizes remain unchanged. This shows that the agglutination event can only occur when the nanoparticles are appropriately functionalized.

[0016] FIG. 9 graphically depicts a dynamic light scattering profile of polymerized bovine gamma globulin (pBGG). The sizes of pBGG nanoparticles shown here are 100-500 nm.

DETAILED DESCRIPTION

[0017] Before explaining at least one embodiment of the present disclosure in detail by way of exemplary language and results, it is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of the 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. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary - not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0018] Independent of the grammatical term usage, individuals with male, female, or other gender identities are included within the term.

[0019] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, 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 syntheses and chemical analyses.

[0020] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0021] 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 particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the articles, compositions, kits, and/or methods and in the steps or in the 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. [0022] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0023] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0024] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0025] The use of the term "or" in the claims is used to mean an inclusive "and/or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0026] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims. [0027] 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 being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0028] As used in this specification and claim(s), the words "comprising" (and any form of comprising, 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.

[0029] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items 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 if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0030] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item. [0031] As used herein, the phrases "associated with" and "coupled to" include both direct association/binding of two moieties to one another as well as indirect association/binding of two moieties to one another. Non-limiting examples of associations/couplings include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety, for example.

[0032] The terms "analog" and "derivative" are used herein interchangeably and refer to a substance which comprises the same basic carbon skeleton and carbon functionality in its structure as a given compound, but can also contain one or more substitutions thereto. The term "substitution" as used herein will be understood to refer to the replacement of at least one substituent on a compound with a residue R. In certain non-limiting embodiments, R may include H, hydroxyl, thiol, a halogenid selected from fluoride, chloride, bromide, or iodide, a C1-C4 compound selected one of the following: linear, branched or cyclic alkyl, optionally substituted, and linear branched or cyclic alkenyl, wherein the optional substitutents are selected from one or more alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocyclealkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, each of which is optionally substituted wherein the optional substitutents are selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cyclalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocyclealkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocyclalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH (alkyl), -NH(cycloalkyl)2, carboxy, and - C(O))-alkyl.

[0033] The term "sample" as used herein will be 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 portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like. [0034] The term "specific binding partner," as used in particular (but not by way of limitation) herein in the term "target analyte-specific binding partner," will be understood to refer to any molecule capable of specifically associating with the target analyte. For example, but not by way of limitation, the binding partner may be an antibody, a receptor, a ligand, aptamers, molecular imprinted polymers (i.e., inorganic or organic matrices), combinations or derivatives thereof, as well as any other molecules capable of specific binding to the target analyte.

[0035] The term "antibody" is used herein in the broadest sense and refers to, for example, intact monoclonal antibodies and polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof that exhibit the desired biological activity of analyte binding (such as, 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 substitute proteins or peptides (i.e., engineered binding proteins/peptides), and combinations or derivatives thereof. The antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl, and lgA2).

[0036] An "analyte" is a macromolecule that is capable of being recognized by an analytespecific binding partner, such as (but not limited to) an antibody. Both analytes and haptens comprise at least one antigenic determinant or "epitope," which is the region of the antigen or hapten which binds to the analyte-specific binding partner (i.e., antibody). Typically, the epitope on a hapten is the entire molecule.

[0037] Turning now to the inventive concepts, certain non-limiting embodiments of the present disclosure are directed to a diagnostic reagent composition (such as, but not limited to, a diagnostic immunoassay reagent composition) for detection of a target analyte in a biological sample. The diagnostic reagent composition comprises a protein nanoparticle comprising polymerized gamma globulin and at least one analyte-specific binding partner associated therewith.

[0038] Any gamma globulins known in the art or otherwise contemplated herein that are capable of polymerization to form protein nanoparticles that naturally have at least one functional group disposed thereon may be utilized in accordance with the present disclosure. Non-limiting examples of gamma globulins that may be utilized include mammalian gamma globulins (such as, but not limited to, goat gamma globulin (GGG) and bovine gamma globulin (BGG)), as well as non-mammalian gamma globulins.

[0039] The protein nanoparticles may have any functional groups disposed thereon that naturally occur in gamma globulins. Non-limiting examples thereof include carboxyl, amine, and/or sulfhydryl groups.

[0040] In certain particular (but non-limiting) embodiments, the protein nanoparticles may include one or more proteins in addition to the gamma globulin. One non-limiting example of an additional protein that may be included is serum albumin. However it will be understood that any additional proteins may be included, so long as the protein nanoparticles are capable of functioning as described herein.

[0041] Any analyte-specific binding partners known in the art or otherwise contemplated herein that are capable of specifically binding to the analyte to be detected and that can thereby be utilized for the diagnostic detection of the analyte can be utilized in accordance with the present disclosure. Non-limiting examples of types of analyte-specific binding partners utilized for diagnostic use 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 combinations thereof.

[0042] The target analyte may be any molecule present in a biological sample for which detection and/or quantitation thereof are desired. For example (but not by way of limitation), when the target analyte is an antigen, the target analyte-specific binding partner may be an antibody or fragment thereof that specifically binds thereto; when the target analyte is an antibody, the target analyte-specific binding partner may be an antigen to which the antibody specifically binds; when the target analyte is a ligand, the target analyte-specific binding partner may be a receptor (or portion or derivative thereof) that specifically binds thereto; and when the target analyte is a receptor, the target analyte-specific binding partner may be a ligand that specifically binds thereto.

[0043] The nanoparticles produced from polymerized gamma globulin may be provided with any size, shape, and dimension, so long as the nanoparticles are capable of functioning in accordance with the present disclosure. Non-limiting examples of nanoparticle sizes that may 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, about 150 nm, about 160 nm, about 170 nm, about 175 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 225 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 275 nm, about 280 nm, about 290 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 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 larger, as well as a range formed of any of the above values (i.e., a range of from about 20 nm to about 1000 nm, a range of from about 50 nm to about 1000 nm, a range of from about 50 nm to about 500 nm, a range of from about 100 nm to about 500 nm, a range of from about 100 nm to about 200 nm, etc.).

[0044] In certain particular (but non-limiting) embodiments, the diagnostic reagent composition may be provided with any element(s) or feature(s) that allow for detection of complexes of nanoparticle(s) with analyte bound thereto. In certain particular (but nonlimiting) embodiments, the diagnostic reagent composition may have at least one dye associated therewith to facilitate detection of bound analyte. For example (but not by way of limitation), the nanoparticle may have at least one dye incorporated or otherwise associated therewith. Dyes could be used (for example, but not by way of limitation) for spectrophotometric, luminescence detection (i.e., chemiluminescence or fluorescence detection). Non-limiting examples thereof include fluorescein, rhodamine, nitrobenzofurazan (NBD), and the like.

[0045] In certain particular (but non-limiting) embodiments, the nanoparticles may be biotinylated. Biotinylation of the nanoparticles allows for an indirect attachment of another biotinylated protein or molecule of interest to the particles via a tetrameric streptavidin.

[0046] Certain non-limiting embodiments of the present disclosure are directed to kits that contain one or more of any of the diagnostic reagent compositions disclosed or otherwise contemplated herein. In certain particular (but non-limiting) embodiments, the kit further includes at least one additional assay reagent that interacts with the diagnostic reagent composition for detecting the presence and/or concentration of the target analyte in the biological sample. [0047] In a particular (but non-limiting) embodiment, the kit comprises 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 for performing a multiplex assay for two different target analytes. When disposed together in a single composition, the first and second diagnostic reagent compositions are provided with different dyes associated therewith (or other types of detection mechanisms that differ from one another) that allow for detection of both target analytes in a single reaction.

[0048] The compositions/reagents of the kits may be provided in any form that allows them to function in accordance with the present disclosure. For example, but not by way of limitation, each of the reagents may be provided in liquid form and disposed in bulk and/or single aliquot form within the kit. Alternatively, in a particular (but non-limiting) embodiment, one or more of the reagents may be disposed in the kit in the form of a single aliquot lyophilized reagent. The use of dried reagents in kits/microfluidics devices is described in detail in US Patent No. 9,244,085 (Samproni), the entire contents of which are hereby expressly incorporated herein by reference.

[0049] In addition to the compositions/reagents described in detail herein above, the kits may further contain other reagent(s) for conducting any of the particular assays described or otherwise contemplated herein. The nature of these additional reagent(s) will depend upon the particular assay format, and identification thereof is well within the skill of one of ordinary skill in the art; therefore, no further description thereof is deemed necessary. Also, the compositions/reagents present in the kits may each be in separate containers/compartments, or various compositions/reagents can be combined in one or more containers/compartments, depending on the cross-reactivity and stability of the compositions/reagents. In addition, the kit may include a microfluidics device in which the compositions/reagents are disposed.

[0050] The relative amounts of the various compositions/reagents in the kits can vary widely to provide for concentrations of the compositions/reagents that substantially optimize the reactions that need to occur during the assay methods and further to optimize substantially the sensitivity and selectivity of an assay. Under appropriate circumstances, one or more of the compositions/reagents in the kit can be provided as a dry powder, such as a lyophilized powder, and the kit may further include excipient(s) for dissolution of the dried reagents; in this manner, a reagent solution having the appropriate concentrations for performing a method or assay in accordance with the present disclosure can be obtained from these compositions. Positive and/or negative controls may also be included with the kit. In addition, the kit can further include a set of written instructions explaining how to use the kit. A kit of this nature can be used in any of the methods described or otherwise contemplated herein.

[0051] Certain additional non-limiting embodiments of the present disclosure are directed to a microfluidics device that includes one or more of any of the diagnostic reagent compositions described herein above or otherwise contemplated herein. In particular, certain non-limiting embodiments include a microfluidics device for determining the concentration of at least one target analyte in a sample. The microfluidics device comprises: (i) an inlet channel through which a sample is applied; and (ii) at least a first compartment capable of being in fluidic communication with the inlet channel and containing at least one of any of the diagnostic reagent compositions disclosed or otherwise contemplated herein. The compartment(s) of (ii) may further contain any additional reagents required to perform the assay for detection of the target analyte. Any of the assay reagents disclosed or contemplated herein or otherwise known in the art may be utilized in the microfluidics devices of the present disclosure.

[0052] The microfluidics device may be provided with any arrangement of compartments and distribution of the various compositions/reagents therebetween that allows the device to function in accordance with the present disclosure. That is, when the diagnostic reagent composition is utilized in combination with a second assay reagent, the two reagents may be disposed in the same compartment or in different compartments. When the two reagents are separated between two compartments, the diagnostic reagent composition may be disposed in a first compartment that is in fluidic communication with the inlet channel, and the at least one additional assay reagent may be disposed in a second compartment that is in fluidic communication with the first compartment.

[0053] In a particular (but non-limiting) embodiment, the microfluidics device comprises two or more of any of the diagnostic reagent compositions disclosed or otherwise contemplated herein, wherein the two or more diagnostic reagent compositions are disposed together in the microfluidics device such that a multiplex assay for two different target analytes can be performed within the microfluidics device. In this manner, the first and second diagnostic reagent compositions (and any additional diagnostic reagent compositions, if present) are provided with different dyes associated therewith (or other types of detection mechanisms that differ from one another) that allow for detection of both target analytes in a single reaction. The two or more diagnostic reagent compositions may be disposed in the same or separate compartments of the microfluidics device.

[0054] Alternatively, the microfluidics device may comprise two or more of any of the diagnostic reagent compositions disclosed or otherwise contemplated herein, wherein the two or more diagnostic reagent compositions are disposed in separate compartments in the microfluidics device and have separate read chambers for detection thereof, thereby allowing for detection of the two different target analytes within the microfluidics device.

[0055] The microfluidics devices of the present disclosure may possess any design or configuration known in the art or otherwise contemplated herein for use in a diagnostic analyte assay (such as, but not limited to, a diagnostic immunoassay). In certain particular (but non-limiting) embodiments, the microfluidics device may be in the form of a cassette that is configured for insertion into an automated diagnostic test instrument system that performs the diagnostic assay. Alternatively, the microfluidics device may be a standalone product that can be read without a diagnostic test instrument system. For example (but not by way of limitation), the microfluidics device may be in the form of a lateral flow device that can be conducted at a point of care (POC) location.

[0056] Any of the compartments of the microfluidics device may be sealed to maintain reagent(s) disposed therein in a substantially air tight environment until use thereof; for example, compartments containing lyophilized reagent(s) may be sealed to prevent any unintentional reconstitution of the reagent. The inlet channel and a compartment, as well as two compartments, may be described as being "capable of being in fluidic communication" with one another; this phrase indicates that each of the compartment(s) may still be sealed, but that the two compartments are capable of having fluid flow therebetween upon puncture of a seal formed therein or therebetween.

[0057] The microfluidics devices of the present disclosure may be provided with any other desired features known in the art or otherwise contemplated herein. For example, but not by way of limitation, the microfluidics devices of the present disclosure may further include a read chamber; the read chamber may be any of the compartments containing the reagent(s) described herein above, or the read chamber may be in fluidic communication with said compartment. The microfluidics device may further include one or more additional compartments containing othersolutions, such as (but not limited to) wash solutions, dilution solutions, excipients, interference solutions, positive controls, negative controls, quality controls, and the like. These additional compartment(s) may be in fluidic communication with one or more of the other compartments. For example, the microfluidics device may further include one or more compartments containing a wash solution, and these compartment(s) may be capable of being in fluidic communication with any other compartment(s) of the device. In another example, the microfluidics device may further include one or more compartments containing an excipient for dissolution of one or more dried reagents, and the compartment(s) may be capable of being in fluidic communication with any other compartment(s) of the device. In yet a further example, the microfluidics device may include one or more compartments containing a dilution solution, and the compartment(s) may be capable of being in fluidic communication with any other compartment(s) of the device.

[0058] Certain non-limiting embodiments of the present disclosure are directed to a method of producing any of the diagnostic reagent compositions disclosed or otherwise contemplated herein. In a particular (but non-limiting) embodiment, the method includes the following steps: polymerizing gamma globulin (GG) to form polymerized GG (pGG) nanoparticles; and attaching at least one analyte-specific binding partner to the pGG nanoparticles via at least one naturally occurring functional moiety disposed on a surface of the nanoparticles.

[0059] The polymerization and attachment reactions can occur by any methods known in the art or otherwise contemplated herein. For example (but not by way of limitation), the pGG nanoparticles may be formed via a technique selected from the group consisting of solvent evaporation, nanoprecipitation, salting out, and emulsification techniques. In one non-limiting example, the pGG nanoparticles are formed by heating the GG to a temperature in a range of from about 65°C to about 67°C for a period of time.

[0060] In addition, in certain non-limiting embodiments, at least one additional element (such as, but not limited to, a dye) may be incorporated into the diagnostic reagent composition during the method of production.

[0061] Certain non-limiting embodiments of the present disclosure are directed to a method of determining the presence and/or concentration of at least one target analyte in a biological sample. In the method, the biological sample is combined with at least one of any of the diagnostic reagent compositions disclosed or otherwise contemplated herein under conditions that allow target analyte present in the biological sample to substantially bind to the analyte-specific binding partner extending from the outer surface of the nanoparticle of the diagnostic reagent composition, thereby forming a complex; then, the presence and/or concentration of the target analyte is determined based on any complex formed. The determination step may be performed using any assay methods known in the art.

[0062] In certain particular (but non-limiting) embodiments, the method utilizes a homogeneous assay format. For example (but not by way of limitation), binding of target analyte to the diagnostic reagent composition results in agglutination, and therefore no additional reagents are required for detection of target analyte. The results of the agglutination assay may be detected manually or automatically (i.e., visually or spectrophotometrically), and may be detected turbidimetrically or nephelometrically.

[0063] Alternatively, the method may utilize a heterogeneous assay format, in which an additional reagent must be used in combination with the diagnostic reagent composition for detection of the target analyte in the biological sample. For example (but not by way of limitation), the method may utilize a heterogenous format such as a sandwich assay. When a second reagent must be utilized, the biological sample may be contacted with the diagnostic reagent composition and the second reagent either simultaneously or wholly or partially sequentially. In addition, the detection step of the method will involve detection of the complex comprising diagnostic reagent composition/target analyte/second assay reagent.

[0064] Certain non-limiting embodiments of the present disclosure are directed to a method of 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 or otherwise contemplated herein (i.e., a first diagnostic reagent composition and a second diagnostic reagent composition) under conditions that: (1) allow a first target analyte present in the biological sample to substantially bind to the analyte-specific binding partner extending from the outer surface of the nanoparticle of the first diagnostic reagent composition, thereby forming a first complex, and (2) allow a second target analyte present in the biological sample to substantially bind to the analyte-specific binding partner extending from the outer surface of the nanoparticle of the second diagnostic reagent composition, thereby forming a second complex. Then, the presence and/or concentration of each of the first and second target analytes is determined based on any first and second complexes, respectively, formed. The determination steps may be performed using any assay methods known in the art.

[0065] Non-limiting examples of biological samples that may be utilized in accordance with the various methods of the present disclosure include whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof. Particular non-limiting examples include lysed whole blood cells and lysed red blood cells.

[0066] As mentioned above, when the diagnostic reagent composition is utilized in combination with a second reagent, the two compositions may be added either simultaneously or sequentially. In addition, when two or more diagnostic reagent compositions are utilized in the same reaction, the two diagnostic reagent compositions may be added simultaneously or sequentially. When the various compositions utilized in the method are added sequentially, the order of addition of the compositions may be varied; a person having ordinary skill in the art can determine the particular desired order of addition of the different compositions to the assay. The simplest order of addition, of course, is to add all the materials simultaneously and determine the signals produced therefrom. Alternatively, each of the compositions, orgroups of compositions, can be combined sequentially. In certain embodiments, an incubation step may be involved subsequent to one or more additions.

EXAMPLES

[0067] Examples are provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.

Example 1

[0068] Non-magnetic latex particles (NMLP) have been widely used in the diagnostics industry, in which antigen or antibody is immobilized on the particles for use in a competitive or sandwich assay format. An analyte of interest brings the particles together, resulting in a cloudy or turbid solution that can be quantified spectrophotometrically. However, these plastic beads are toxic and environmentally unfriendly and have significant waste handling costs associated therewith, which will rise significantly in the future. In addition, certain procedures to functionalize surfaces can require a number of materials, substantial time, and resources. Also, the coupling processes and waste material may be hazardous. Management of respective processes and waste are typically associated with substantial safety measures as well as significant costs, too, and may even have to be discontinued for regulatory reasons. [0069] The present disclosure solves these challenges by using biodegradable protein nanoparticles that naturally contain functional moieties (such as (but not limited to) carboxyl, amine, or sulfhydryl moieties) to attach antibody or antigen (or other analyte-specific binding partner) to the surface of the nanoparticles via conventional chemistry. Gamma globulin (GG, such as, but not limited to, goat gamma globulin (GGG) and bovine gamma globulin (BGG)) is polymerized (such has, but not limited to, via heat treatment) to form polymerized gamma globulin (pGG) nanoparticles. This approach to produce pGG as biodegradable protein nanoparticles reduces the number of materials required and is environmentally neutral and consequently sustainable.

[0070] These functionalized beads and analytes of interest bind in a multivalent fashion, forming aggregates that can be visualized or quantified spectrophotometrically, in particular tu r bid i met rica I ly or nephelometrically. Quantifying this change in the turbidity (or cloudiness) of the reaction mixture as a function of an analyte concentration is the basis of a homogeneous immuno-agglutination assay. In contrast, the latex beads of the prior art eventually represent waste, and their waste handling costs will continue to be a challenge that are likely to rise significantly in the future. The present disclosure replaces these currently used toxic beads with environmentally friendly and biodegradable protein nanoparticles.

[0071] The present disclosure is based on the use of biodegradable protein nanoparticles that are between about 20 nm to about 1000 nm (typically < 500 nm) and contain amino acid residues with reactive groups (e.g., COOH, NH3, SH, etc.) ready to attach biotin, fluorescein, antibody, antigen, or ligand, etc. to the surface via conventional chemistry. These protein nanoparticles are colloidal and suspend well in buffer and solution typically used in the diagnostic reagents. They can be manufactured by various methods known in the art (e.g., solvent evaporation method, nanoprecipitation, salting out methods, emulsification, etc.). For this Example, a goat gamma golubin (GGG) solution at 10 mg/ml was conveniently heated

Y1 at 65-67°C until it reached approximately 30% transmission at 340 nm. Like the latex beads, this polymerized GGG (pGGG) product was white and milky in appearance (FIG. 1).

[0072] These protein nanoparticles were modifiable by attaching a target analyte-specific binding partner thereto. For example, the left image of FIG. 2 demonstrates that the protein nanoparticles were functionalized to contain biotin on the surface thereof. In addition, the protein nanoparticles could be produced with (or modified after production to contain) a dye associated therewith; the right image of FIG. 2 demonstrates that the protein nanoparticles were functionalized with fluorescein on the surface thereof.

[0073] The pGGG nanoparticles behave similarly to the NMLPs chromatographically. For example, as shown in FIG. 3, the pGGG nanoparticles are normally excluded from a sizeexclusion column (e.g., CL-2B with exclusion limit ~100 nm) and can be detected at 280, 340, and 600 nm. Based on these data, it was determined that these pGGG particles can be used for an agglutination assay. As shown in FIG. 4, when the biotinylated nanoparticles (1 mg) were exposed to avidin (1 mg), agglutination occurred almost instantaneously, and this resulted in increased absorbance readings at 340 nm over time. In the absence of biotin on the surface of the particles, the absorbance readings remained unchanged. This data demonstrates that the pGGG protein nanoparticles perform similarly to latex beads and liposomes to agglutinate turbidimetrically.

[0074] Next, a calibration curve (A340 nm vs. pg-avidin) was formed when an incremental amount of avidin was added to a solution containing the biotinylated pGGG nanoparticles (FIG. 5). As such, quantitative analysis of avidin can be achieved based on this homogeneous agglutination assay format with the functionalized biotinylated pGGG nanoparticles. FIG. 6 contains images of the test cuvettes before (left) and after (right) the addition of avidin at the end of the reaction. This agglutination event was readily observed on a dynamic light scattering (DLS) detector as well, as shown in FIG. 7. The agglutinated particles were now seen as large as about 1 pm on the DLS. In contrast, in the absence of biotin on the surface of the pGGG nanoparticles, the particle sizes remained unchanged in the presence of avidin, as shown in FIG. 8.

[0075] The functionalized protein nanoparticles (pGG) of the present disclosure can replace the "toxic" plastic beads currently used for homogeneous immunoassays. This replacement circumvents the need for a costly waste processing unit and provides a substitute for NMLP's, which is especially important should NMLP's be banned in the future. The key features of the protein pGG nanoparticles of the present disclosure are their biodegradability, favorable sizes (e.g., 20-1000 nm), convenience for attaching functionalized molecules or proteins, and behavior similarity to conventional NMLP's. The pGG nanoparticles of the present disclosure may also be used in immunoassays in various ways. Non-limiting examples of other uses include as a non-specific interference blocker, a signal amplifier and enhancer, a colloidal stationary phase to help stabilize proteins, a functionalized dye-carrier in a multiplexing format (e.g., flow cytometry immunoassay), and the like.

Example 2

[0076] Example 1 describes the use of goat gamma globulin to produce polymerized protein nanoparticles (pGGG nanoparticles) for use in preparing diagnostic reagent compositions to which target analyte-specific binding partners can be attached. However, the present disclosure is not limited to the use of goat gamma globulin in the production of the nanoparticles; it will be understood that other gamma globulins may be utilized in a similar manner, and thus the present disclosure encompasses the use of other gamma globulins in the production of diagnostic reagent compositions in accordance with the present disclosure. [0077] For example, FIG. 9 illustrates the use of bovine gamma globulin (BGG) to produce polymerized BGG (or pBGG) protein nanoparticles. The pBGG protein nanoparticles were produced using a similar heating technique as described above in Example 1.

[0078] These pBGG protein nanoparticles are attached to a target analyte-specific binding partner in a similar manner as described above in Example 1, and the diagnostic reagent composition containing the pBGG protein nanoparticles is incorporated into an agglutination immunoassay for detection of the analyte to which the target analyte-specific binding partner binds.

NON-LIMITING ILLUSTRATIVE EMBODIMENTS

[0079] The following is a list of non-limiting illustrative embodiments disclosed herein: [0080] Illustrative embodiment 1. A diagnostic immunoassay reagent composition for detection of a target analyte in a biological sample, comprising: a biodegradable protein nanoparticle comprising polymerized gamma globulin (pGG) and having at least one functional moiety thereon; and at least one analyte-specific binding partner attached to the biodegradable protein nanoparticle through the at least one functional moiety. [0081] Illustrative embodiment 2. The composition of illustrative embodiment 1, wherein the at least one functional moiety is selected from the group consisting of a carboxyb amine, or sulfhydryl group.

[0082] Illustrative embodiment 3. The composition of illustrative embodiment 1 or 2, wherein the nanoparticle has a diameter in a range of from about 20 nm to about 1000 nm.

[0083] Illustrative embodiment 4. The composition of any of illustrative embodiments 1- 3, wherein the analyte-specific binding partner comprises an antibody or fragment thereof that specifically binds to the target analyte.

[0084] Illustrative embodiment 5. The composition of any one of illustrative embodiments 1-4, wherein the analyte-specific binding partner comprises an antigen to which the target analyte specifically binds.

[0085] Illustrative embodiment 6. The composition of any one of illustrative embodiments 1-5, wherein the at least one analyte-specific binding partner comprises at least one receptor or portion or derivative thereof that specifically binds to the target analyte.

[0086] Illustrative embodiment 7. The composition of any one of illustrative embodiments 1-6, wherein the at least one analyte-specific binding partner comprises a ligand for the target analyte.

[0087] Illustrative embodiment 8. The composition of any one of illustrative embodiments 1-7, wherein the nanoparticle is biotinylated.

[0088] Illustrative embodiment 9. The composition of any one of illustrative embodiments 1-8, wherein the nanoparticle has at least one dye attached thereto.

[0089] Illustrative embodiment 10. The composition of illustrative embodiment 9, wherein the dye comprises fluorescein.

[0090] Illustrative embodiment 11. The composition of any one of illustrative embodiments 1-10, wherein the gamma globulin is goat gamma globulin.

[0091] Illustrative embodiment 12. The composition of any one of illustrative embodiments 1-11, wherein the gamma globulin is bovine gamma globulin.

[0092] Illustrative embodiment 13. A kit, comprising: at least one diagnostic reagent composition of any one of illustrative embodiments 1-12.

[0093] Illustrative embodiment 14. The kit of illustrative embodiment 13, further comprising at least one additional reagent for use in the diagnostic immunoassay. [0094] Illustrative embodiment 15. The kit of illustrative embodiment 13 or 14, further comprising at least two diagnostic reagent compositions of any one of illustrative embodiments 1-12.

[0095] Illustrative embodiment 15A. The kit of illustrative embodiment 15, wherein the kit is for use in a multiplexed assay.

[0096] Illustrative embodiment 16. A microfluidics device, comprising: (i) an inlet channel through which a sample is applied; and (ii) at least one compartment capable of being in fluidic communication with the inlet channel, wherein the at least one compartment comprises at least one diagnostic reagent composition of any one of illustrative embodiments 1-12.

[0097] Illustrative embodiment 17. The microfluidics device of illustrative embodiment 16, further defined as a microfluidics device for performing a multiplexed assay, and wherein (ii) comprises at least two diagnostic reagent compositions.

[0098] Illustrative embodiment 18. A method of producing a diagnostic reagent composition for detection of a target analyte in a biological sample, the method comprising the steps of: polymerizing gamma globulin (GG) to form polymerized GG (pGG) nanoparticles; and attaching at least one analyte-specific binding partner to the pGG nanoparticles.

[0099] Illustrative embodiment 19. The method of illustrative embodiment 18, wherein the pGG nanoparticles are formed by heating the GG to a temperature in a range of from about 65°C to about 67°C for a period of time.

[0100] Illustrative embodiment 20. The method of illustrative embodiment 18 or 19, wherein the pGG nanoparticles are formed via a technique selected from the group consisting of solvent evaporation, nanoprecipitation, salting out, and emulsification techniques.

[0101] Illustrative embodiment 21. The method of any one of illustrative embodiments 18-20, wherein the at least one functional moiety is selected from the group consisting of a carboxyl, amine, or sulfhydryl group.

[0102] Illustrative embodiment 22. The method of any one of illustrative embodiments 18-21, wherein the nanoparticle has a diameter in a range of from about 20 nm to about 1000 nm.

[0103] Illustrative embodiment 23. The method of any one of illustrative embodiments 18-22, wherein the analyte-specific binding partner comprises an antibody or fragment thereof that specifically binds to the target analyte. [0104] Illustrative embodiment 24. The method of any one of illustrative embodiments 18-23, wherein the analyte-specific binding partner comprises an antigen to which the target analyte specifically binds.

[0105] Illustrative embodiment 25. The method of any one of illustrative embodiments 18-24, wherein the at least one analyte-specific binding partner comprises at least one receptor or portion or derivative thereof that specifically binds to the target analyte.

[0106] Illustrative embodiment 26. The method of any one of illustrative embodiments 18-25, wherein the at least one analyte-specific binding partner comprises a ligand for the target analyte.

[0107] Illustrative embodiment 27. The method of any one of illustrative embodiments 18-26, wherein the nanoparticle is biotinylated.

[0108] Illustrative embodiment 28. The method of any one of illustrative embodiments 18-27, wherein the nanoparticle has at least one dye attached thereto.

[0109] Illustrative embodiment 29. The method of illustrative embodiment 28, wherein the dye comprises fluorescein.

[0110] Illustrative embodiment 30. The method of any one of illustrative embodiments 18-29, wherein the gamma globulin is goat gamma globulin.

[0111] Illustrative embodiment 31. The method of any one of illustrative embodiments 18-29, wherein the gamma globulin is bovine gamma globulin.

[0112] Illustrative embodiment 32. A method of determining the presence and/or concentration of a target analyte in a biological sample, the method comprising the steps of: combining the biological sample with at least one diagnostic reagent composition of any one of illustrative embodiments 1-12 under conditions that allow for binding of the at least one analyte-specific binding partner to target analyte present in the sample to form a complex; and determining the presence and/or concentration of the target analyte based on any complex formed.

[0113] Illustrative embodiment 33. The method of illustrative embodiment 32, wherein the method comprises a homogeneous assay format.

[0114] Illustrative embodiment 34. The method of illustrative embodiment 33, wherein the assay format comprises an agglutination assay.

[0115] Illustrative embodiment 35. The method of illustrative embodiment 34, wherein formation of aggregates is detected visually or spectrophotometrically. [0116] Illustrative embodiment 36. The method of illustrative embodiment 35, wherein the detection is performed tu rbidimetrica I ly or nephelometrically.

[0117] Illustrative embodiment 37. The method of illustrative embodiment 32, wherein the method comprises a heterogeneous assay format.

[0118] Illustrative embodiment 38. The method of illustrative embodiment 37, wherein the assay format comprises a sandwich assay.

[0119] Illustrative embodiment 39. A method of determining the presence and/or concentration of at least two target analytes in a biological sample, the method comprising the steps of: combining the biological sample with a first diagnostic reagent composition of any one of illustrative embodiments 1-12 and a second diagnostic reagent composition of any of illustrative embodiments 1-12 under conditions that allow for binding of the analytespecific binding partner of the first diagnostic reagent composition to first target analyte present in the sample to form a first complex and that allow for binding of the analyte-specific binding partner of the second diagnostic reagent composition to the second target analyte present in the sample to form a second complex; determining the presence and/or concentration of the first target analyte based on any first complex formed; and determining the presence and/orconcentration of the second target analyte based on any second complex formed.

[0120] Illustrative embodiment 40. The method of any one of illustrative 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, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof.

[0121] Thus, in accordance with the present disclosure, there have been provided compositions, kits, and devices, as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

What is claimed is:

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

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

3. The composition of claim 1, wherein the nanoparticle has a diameter in a range of from about 20 nm to about 1000 nm.

4. The composition of claim 1, wherein the analyte-specific binding partner comprises an antibody or fragment thereof that specifically binds to the target analyte.

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

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

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

8. The composition of claim 1, wherein the nanoparticle has at least one dye attached thereto.

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

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

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

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

13. The kit of claim 11, further comprising at least two diagnostic reagent compositions of any one of claims 1-10.

14. A microfluidics device, comprising:

(i) an inlet channel through which a sample is applied; and

(ii) at least one compartment capable of being in fluidic communication with the inlet channel, wherein the at least one compartment comprises at least one diagnostic reagent composition of any one of claims 1-10.

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

16. The method of claim 15, wherein the pGG nanoparticles are formed by heatingthe GG to a temperature in a range of from about 65°C to about 67°C for a period of time.

17. The method of claim 15, wherein the pGG nanoparticles are formed via a technique selected from the group consisting of solvent evaporation, nanoprecipitation, salting out, and emulsification techniques.

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

19. The method of claim 18, wherein the method comprises a homogeneous assay format, and wherein the assay format comprises an agglutination assay.

20. The method of claim 18, 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, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof.