Compositions, kits, and methods for dual immunoassays for anti-SARS-COV-2 antibodies

Abstract

Disclosed are kits, microfluidic devices and methods comprising a multiplexed chemiluminescent detection system for detecting the presence and / or concentration of anti-SARS-CoV-2 antibodies in a sample. The kits, microfluidic devices and methods utilize singlet oxygen activatable chemiluminescent compounds in combination with two or more fluorescent molecules that emit at different wavelengths. In certain non-limiting embodiments, the kits, microfluidic devices and methods are capable of distinguishing between anti-SARS-CoV-2 antibodies generated in response to vaccination and anti-SARS-CoV-2 antibodies generated in response to infection.

Description

[Technical field]

[0001] CROSS REFERENCE TO RELATED APPLICATIONS / INCORPORATION-BY-REFERENCE STATEMENT Not applicable.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable. [Background technology]

[0003] In the field of medical diagnostics, many different types of assay techniques are utilized: When a patient is suspected of being infected with a microorganism (such as, but not limited to, a bacteria or virus), an assay is performed on a biological sample from the patient to detect antibodies against the microorganism produced by the patient's immune system.

[0004] When detection of anti-viral or anti-bacterial antigen antibodies (such as, but not limited to, IgG, IgM, and / or IgA) in patient serum and plasma is desired, bridging serological assays have been utilized in which immobilized and labeled viral / bacterial antigens are often used to formulate the assay reagents. In another example, latex particle agglutination assays utilize latex particles coated with viral / bacterial antigens as the single reagent that agglutinate in the presence of anti-viral / bacterial antigen antibodies in patient samples.

[0005] One example of a commercially used assay is the luminescent oxygen channeling assay (LOCI®) technology. LOCI® advanced chemiluminescent assays are described, for example, in U.S. Pat. No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference. Currently available LOCI® technologies have high sensitivity and use several reagents. In particular, the LOCI® assay requires that two of these reagents (called "sensibeads" and "chemibeads") are held by other specific binding partner assay reagents in a manner that the sensibeads and chemibeads are in close proximity to each other to achieve a signal. When exposed to light of a certain wavelength, the sensibeads release singlet oxygen, which is transferred to the chemibeads when the two beads are in close proximity; this triggers a chemical reaction that results in the chemibeads emitting light that can be measured at a different wavelength.

[0006] In June 2020, the U.S. Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for a laboratory-based pan-antibody test developed by Siemens Healthineers (Tarrytown, NY) to detect the presence of all SARS-CoV-2 antibodies, including IgM and IgG, in blood. The immunoassay is based on the LOCI® platform and is referred to as the CV2T LOCI® Immunoassay.

[0007] The spike protein on the surface of the SARS-CoV-2 virus allows the virus to enter and infect human cells found in multiple organs and blood vessels. Siemens Healthineers' whole antibody COV2T CV2T LOCI® immunoassay was designed to detect antibodies against the receptor binding domain (RBD) of the spike protein. Some of these antibodies are believed to neutralize the SARS-CoV-2 virus, thus blocking infection.

[0008] Additionally, multiple vaccines against SARS-CoV-2 that contain the spike protein in their focus have been developed and are available, including the widely used Pfizer / BioNTech, Moderna, Johnson & Johnson (Janssen), and Oxford-AstraZeneca COVID-19 vaccines, and thus immunized patients are now producing antibodies against the SARS-CoV-2 spike protein. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is a need in the art for new and improved immunoassays for detecting the presence of SARS-CoV-2 antibodies that can distinguish between antibodies produced in response to vaccination and antibodies produced in response to infection, thereby overcoming the disadvantages and shortcomings of the prior art. Directed to this disclosure are such assays, as well as kits and microfluidic devices comprising same, and methods of using same. [Brief description of the drawings]

[0010] [Figure 1] Schematic representation of the SARS-CoV-2 Total (COV2T) assay (Siemens Healthineers, Tarrytown, NY). [Diagram 2] FIG. 2 shows a schematic of one non-limiting embodiment of a dual immunoassay format constructed in accordance with the present disclosure. FIG. [Diagram 3] Schematic diagram of one non-limiting embodiment of a dual immunoassay format constructed according to the present disclosure: Figure 3 shows the complex formed in the presence of anti-SARS-CoV-2 antibodies. [Figure 4] FIG. 4 shows a schematic diagram of another non-limiting embodiment of a duplex immunoassay format constructed in accordance with the present disclosure, with the reagents utilized. [Diagram 5]FIG. 5 shows a schematic of another non-limiting embodiment of a dual immunoassay format constructed according to the present disclosure. FIG. 5 shows the complex formed in the presence of anti-SARS-CoV-2 antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Before describing at least one embodiment of the present disclosure in detail by exemplary language and results, it should be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components described in the following description. The present disclosure is capable of other embodiments or can be practiced or carried out in various ways. Therefore, the language used in this specification is intended to give the broadest possible scope and meaning; the embodiments are meant to be illustrative, not comprehensive. It should also be understood that the phraseology and terminology used in this specification are for the purpose of description and should not be regarded as limiting.

[0012] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The techniques and procedures described above are generally performed according to conventional methods well known in the art and described in various general and more specific references cited and discussed throughout this specification. The nomenclatures utilized in connection with analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the experimental procedures and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0013] 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 herein 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.

[0014] All of the articles, compositions, kits, and / or methods disclosed herein can be made and carried out without undue experimentation in light of this disclosure.Although the articles, compositions, kits, and / or methods are described with reference to specific embodiments, it will be apparent to those skilled in the art that modifications can be made 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 this disclosure.All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope and concept of this disclosure, as defined by the appended claims.

[0015] As utilized in accordance with the present disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0016] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the term "a" or "an" may mean "one," but it is also consistent with the meanings of "one or more," "at least one," and "one or more than one." Thus, 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 a greater number of compounds. The term "plurality" refers to "two or more."

[0017] Use of the term "at least one" will be understood to include one and any amount 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" may extend to 100 or 1000 or more, depending on the term to which it is attached; furthermore, an amount of 100 / 1000 should not be considered limiting, since higher limits may also provide satisfactory results. Furthermore, 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. Use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is for the purpose of distinguishing between two or more items only, and is not intended to imply, for example, any permutation or order or any order of importance or addition of one item to another item.

[0018] Use of the term "or" in the claims is used to mean inclusive "and / or" unless expressly indicated to refer only to alternatives or the alternatives are mutually exclusive. For example, a 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).

[0019] 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, the appearances of the phrases "in some embodiments" or "one example" in various places in the specification are not necessarily all referring to the same embodiment. Moreover, any reference to one or more embodiments or examples should not be construed as limiting the scope of the claims.

[0020] 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, and without limitation, when the term "about" is used, the specified value may vary 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 from the specified value, such variations being appropriate to perform the disclosed methods and as would be understood by one of ordinary skill in the art.

[0021] As used in the specification and claims, 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.

[0022] 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 also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, if order is important in the particular context. Following this example, combinations containing one or more repeats of an item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are also expressly included. Those skilled in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0023] As used herein, the term "substantially" means that the event or circumstance described thereafter occurs completely, or that the event or circumstance described thereafter occurs to a large extent or to a large extent. For example, when referring to a particular event or circumstance, the term "substantially" means that the event or circumstance described thereafter 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" can mean that two items are 100% adjacent to each other, or that two items are within a range of proximity to each other but are not 100% adjacent to each other, or that a portion of one of the two items is not 100% adjacent to the other item but is within a range of proximity to the other item.

[0024] As used herein, the phrases "associated" and "linked" include both direct association / binding of two moieties to each other and indirect association / binding of two moieties to each other. Non-limiting examples of association / linkage include, for example, covalent binding of one moiety to another moiety by a direct bond or via a spacer group, non-covalent binding of one moiety to another moiety directly or by a specific binding pair member attached to both moieties, incorporation of one moiety into another moiety, such as by dissolving or synthesizing one moiety into another moiety, and coating one moiety onto another moiety.

[0025] 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 functional groups as a given compound, but may also contain one or more substitutions thereon. As used herein, the term "substitution" will be understood to refer to the replacement of at least one substituent on a compound with a residue R.

[0026] As used herein, the term "sample" will be understood to include any type of biological sample that may be utilized in accordance with the present disclosure. Examples of fluid 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, intraperitoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.

[0027] As used herein specifically (but not exclusively) in the terms "biotin-specific binding partner" or "target analyte-specific binding partner," the term "specific binding partner" will be understood to refer to any molecule that can specifically associate with biotin or a target analyte, respectively. For example, but not limited to, the binding partner may be an antibody, a receptor, a ligand, an aptamer, a molecularly imprinted polymer (i.e., an inorganic matrix), combinations or derivatives thereof, and any other molecule that can specifically bind to biotin or a target analyte, respectively.

[0028] The term "antibody" is used herein in the broadest sense to refer to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof that exhibit the desired biological activity of analyte binding (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. Antibodies may 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).

[0029] As used herein, the term "hapten" refers to a small proteinaceous or non-proteinaceous antigenic determinant (or "epitope") capable of being recognized by a target analyte-specific binding partner, such as, but not limited to, an antibody. As used herein, the term "polyhapten" will be understood to refer to a synthetic molecule that contains multiple epitopes / antigenic determinants attached to the synthetic molecule.

[0030] An "analyte" is a macromolecule that can be recognized by an analyte-specific binding partner, such as, but not limited to, an antibody. Both analytes and haptens contain at least one antigenic determinant or "epitope," which is the region of an antigen or hapten that binds to an analyte-specific binding partner (i.e., an antibody). Typically, an epitope on a hapten is the entire molecule.

[0031] Certain non-limiting embodiments of the present disclosure are directed to multiplex assays for detecting anti-SARS-CoV-2 antibodies in a sample, as well as kits comprising same and methods of using same. In some assay embodiments, the signal producing system (sps) members include a sensitizer, such as, for example, a photosensitizer, and two or more chemiluminescent fluorescent molecular compositions, where a first chemiluminescent composition generates a signal related to the presence of anti-SARS-CoV-2-spike protein antibodies, while at least a second chemiluminescent composition generates a signal related to the presence of anti-SARS-CoV-2 antibodies against another SARS-CoV-2 protein other than the spike protein, such as, but not limited to, SARS-CoV-2 nucleocapsid protein, envelope protein, or membrane protein; in these assay embodiments, activation of the sensitizer produces a product that activates the chemiluminescent composition, thereby producing a detectable signal related to the amount of bound anti-SARS-CoV-2 antibodies to be detected. An exemplary (but non-limiting) embodiment of an assay platform on which the present disclosure can be based is the Luminescence Oxygen Channeling Assay (LOCI®; Siemens Healthcare Diagnostics Inc., Tarrytown, NY). The LOCI® assay is described, for example, in U.S. Pat. No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference.

[0032] Certain non-limiting embodiments of the present disclosure are directed to kits for performing a multiplex assay that utilizes a chemiluminescence detection system for determining the presence and / or concentration of multiple anti-SARS-CoV-2 antibodies in a sample. The kit includes: (a) a composition comprising a singlet oxygen activatable chemiluminescent compound having a first target antigen bound directly or indirectly to the chemiluminescent compound and a fluorescent molecule excited by the activated chemiluminescent compound; (b) a composition comprising a singlet oxygen activatable chemiluminescent compound having a second target antigen bound directly or indirectly to the chemiluminescent compound and a fluorescent molecule excited by the activated chemiluminescent compound, the fluorescent molecule being different from the fluorescent molecule of (a) and emitting light at a different wavelength than the fluorescent molecule of (a); (c) a biotinylated first target antigen; (d) a biotinylated second target antigen; and (e) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, the composition having a biotin-specific binding partner bound directly or indirectly to the sensitizer; the first target antigen is at least a portion of a SARS-CoV-2 spike protein and the second target antigen is at least a portion of a SARS-CoV-2 protein other than the spike protein.

[0033] All or any portion of the SARS-CoV-2 spike protein can be utilized as the first target antigen in accordance with the present disclosure. In certain non-limiting embodiments, the first target antigen comprises at least a portion of the SARS-CoV-2 S1 protein, such as, but not limited to, at least a portion of the receptor binding domain (RBD) of the S1 protein.

[0034] In one particular (but non-limiting) embodiment, the first target antigen is the receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike protein. The RBD S1 antigen can be obtained from any source known in the art. For example (but not by way of limitation), certain antigens are commercially available from GenScript (Piscataway, NJ); Meridian Life Sciences, Inc. (Memphis, TN); Sino Biological US Inc. (Wayne, PA); ACRO Biosystems (Newark, DE); Biorbyt, LLC (St. Louis, MO); Icosagen, AS (San Francisco, CA); and Bios Pacific Inc. (Emeryville, CA).

[0035] All or any portion of another SARS-CoV-2 protein can be utilized as the second target antigen. In certain non-limiting embodiments, the second target antigen comprises at least a portion of a SARS-CoV-2 nucleocapsid protein, at least a portion of a SARS-CoV-2 membrane protein, or at least a portion of a SARS-CoV-2 envelope protein.

[0036] A chemiluminescent compound (chemiluminescer) is a compound that can be chemically activated and, as a result of such activation, emits light at a certain wavelength. For illustrative purposes and without limitation, examples of chemiluminescent materials include: olefins that can react with singlet oxygen or peroxides to form hydroperoxides or dioxetanes, which can decompose to ketones or carboxylic acid derivatives; stable dioxetanes that can decompose under the action of light; acetylenes that can react with singlet oxygen to form diketones; hydrazones or hydrazides that can form azo compounds or azocarbonyls, such as (but are not limited to) luminol; and aromatic compounds that can form endoperoxides, for example. As a result of the activation reaction, chemiluminescent materials directly or indirectly cause light emission.

[0037] In certain embodiments, a singlet oxygen activatable chemiluminescent compound can be a substance that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose with simultaneous or subsequent emission of light. A composition comprising a chemiluminescent compound can be directly excited by the activated chemiluminescent compound; alternatively, the composition can further comprise at least one fluorescent molecule that is excited by the activated chemiluminescent compound.

[0038] A sensitizer is a molecule, usually a compound, that generates a reactive intermediate, such as, for example, singlet oxygen, for activation of a chemiluminescent compound. In some non-limiting embodiments, the sensitizer is a photosensitizer. Other sensitizers that are chemically activated (e.g., by enzymes and metal salts) include, by way of example and not limitation, other substances and compositions that can generate singlet oxygen with or without activation by an external light source. For example, certain compounds have been shown to catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Non-limiting examples of other sensitizer substances and compositions include: oxides of the alkaline earth metals Ca, Sr, and Ba; d 0 Derivatives of elements of groups 3A, 4A, SA, and 6A of the configuration; oxides of actinides and lanthanides; and the oxidizing agent ClO - ,BrO - , Au 3+ ,IO 3 - and I.O. 4 -and in particular, molybdate, peroxomolybdate, tungstate and peroxotungstate ions, and acetonitrile. The following references, expressly incorporated herein by reference in their entireties, provide further disclosure regarding sensitizer materials and compositions that are also within the scope of this disclosure: Aubry, J. Am. Chem. Soc., 107:5844-5849 (1985); Aubry, J. Org. Chem., 54:726-728 (1989); Bohme and Brauer, Inorg. Chem., 31:3468-3471 (1992); Niu and Foo ... Bohme and Brauer, Inorg. Chem., 31:3468-3471 (1992); Niu and Foo, J. Am. Chem. Soc., 107:5844-5849 (1985); Bohme and Brauer, Inorg. Chem., 31:3468-3471 (1992); Niu and Foo, J. Am. Chem. Soc., 107:5844-5849 (1985); Bohme and Brauer, Inorg. Chem., 31:3468-3471 (1992); Niu and Foo, J. Am. Chem. Soc., 107:5844 te, Inorg. Chem., 31:3472-3476 (1992); Nardello et al., Inorg. Chem., 34:4950-4957 (1995); Aubry and Bouttemy, J. Am. Chem. Soc., 119:5286-5294 (1997); and Almeida et al., Anal. Chim. Acta, 482:99-104 (2003); the entire contents of each of which are expressly incorporated herein by reference.

[0039] Also included within the scope of photosensitizers are compounds that are not true sensitizers, but which release a molecule of singlet oxygen upon excitation by heat, light, ionizing radiation, or chemical activation. Members of this class of compounds include, for example (but are not limited to), endoperoxides such as 1,4-biscarboxyethyl-1,4-naphthalene endoperoxide; 9,10-diphenylanthracene-9,10-endoperoxide; and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light by these compounds releases singlet oxygen.

[0040] A photosensitizer is a sensitizer for activating a photoactive compound, for example by the generation of singlet oxygen upon excitation with light. Photosensitizers are photoactivatable compounds, typically consisting of covalently bonded atoms, including, for example, dyes and aromatic compounds, typically having multiple conjugated double or triple bonds. The compounds are photoactivatable compounds that are 500 M or less at the excitation wavelength. -1 cm -1Greater than or equal to 5,000M -1 cm -1 Greater than or equal to 50,000M -1 cm -1 It should absorb light in the wavelength range of about 200 nm to about 1,100 nm, such as (but not limited to) about 300 nm to about 1,000 nm or about 450 nm to 950 nm, with an extinction coefficient at its absorption maximum greater than 1,100 nm. The photosensitizer should be relatively photostable and may not react efficiently with singlet oxygen. By way of illustration and not limitation, examples of photosensitizers include: acetone; benzophenone; 9-thioxanthone; eosin; 9,10-dibromoanthracene; methylene blue; metallo-porphyrins such as (but not limited to) hematoporphyrin; phthalocyanines; chlorophyll; rose bengal; and buckminsterfullerene; and derivatives of these compounds.

[0041] In particular, non-limiting examples of chemiluminescent compounds and photosensitizers that can be utilized in accordance with the present disclosure are described in U.S. Pat. No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference.

[0042] Any biotin-specific binding partner known in the art or otherwise contemplated herein can be utilized in accordance with the present disclosure. In certain non-limiting embodiments, the biotin-specific binding partner is an antibody to biotin. In other non-limiting embodiments, the biotin-specific binding partner is avidin or an analog thereof.

[0043] Any avidin analogue known in the art or otherwise contemplated herein can be utilized in accordance with the present disclosure, so long as the avidin or avidin analogue can: (1) associate with a sensitizer; (2) bind to a biotinylated analyte-specific binding partner; and (3) bind to biotin that may be present in a sample. Non-limiting examples of avidin analogues that can be utilized in accordance with the present disclosure include those disclosed in Kang et al. (J Drug Target (1995) 3:159-65), the entire contents of which are expressly incorporated herein by reference. Specific non-limiting examples of avidin analogues include avidin, streptavidin, traptavidin, neutral avidin, Neutralite avidin, Neutravidin, Lite-avidin, succinylated avidin, other forms of avidin that have been modified or engineered, esters, salts and / or derivatives of any of the above, and the like.

[0044] Any fluorescent molecule known in the art that can be excited by an activated chemiluminescent compound and emit light at a specific detectable wavelength can be utilized according to the present disclosure as the fluorescent molecules (a) and (b) (and (e), if present), so long as the signal generated by each fluorescent molecule is detectable from the signal generated by the other fluorescent molecules utilized. That is, the fluorescent molecule (a) must emit light at a wavelength sufficiently different from that emitted by the fluorescent molecule (b), so that the two signals are distinguished from each other when detected simultaneously. In one specific (but non-limiting) example, each fluorescent molecule utilized according to the present disclosure is independently selected from the group consisting of terbium, uranium, samarium, europium, gadolinium, and dysprosium. For example (but not limited to), for the generation of two or three signals that can be distinguished from each other when detected simultaneously, terbium emits light at a wavelength of about 545 nm, uranium emits light at a wavelength of about 612 nm, and samarium emits light at a wavelength of about 645 nm.

[0045] Certain non-limiting embodiments of the present disclosure are directed to a kit identical to that described hereinabove, except that the biotinylated antigens of (c) and (d) are replaced with at least one biotinylated anti-human immunoglobulin (Ig) antibody. Thus, in this kit, the reagents (a) and (b) are identical to those of the kit described above, and the kit then further comprises (c) at least one biotinylated anti-human immunoglobulin (Ig) antibody; and (d) a composition comprising a sensitizer and having a biotin-specific binding partner bound to the sensitizer (i.e., the same composition as (e) above). As with the kit described above, the first target antigen is at least a portion of the SARS-CoV-2 spike protein, and the second target antigen is at least a portion of a SARS-CoV-2 protein other than the spike protein.

[0046] Anti-human Ig antibodies can specifically bind to any part of any human immunoglobulin molecule known in the art or otherwise contemplated herein.For example (without limitation), the antibody can be directed to human IgG, IgE, IgM, IgD, and / or IgA, and / or any part thereof (including but not limited to anti-human gamma chain, anti-human H+L, anti-human light chain, etc.).Anti-human Ig antibodies (including but not limited to anti-human IgG, anti-human IgM, and / or anti-human IgA antibodies, and antibodies that recognize two or more human immunoglobulin antibodies) are well known in the art, widely available commercially, and have been extensively studied. For example (but not by way of limitation), some commercial sources of anti-human IgG monoclonal and / or polyclonal antibodies include Rockland Immunochemicals, Inc. (Pottstown, PA); USBiological Life Sciences (Swampscott, MA); Santa Cruz Biotechnology, Inc. (Dallas, TX); Jackson Immuno Research Labs, Inc. (West Grove, PA); Thermo Fisher Scientific (Waltham, MA); and Sigma-Aldrich Corp. (St. Louis, MO). However, this list is not exhaustive and there are many additional commercial sources of anti-human Ig antibodies that can be utilized in accordance with the present disclosure. Thus, further description of anti-human Ig antibodies or their characteristics is not believed to be necessary, as one of ordinary skill in the art can clearly and unambiguously identify and select various anti-human Ig antibodies that can be utilized in accordance with the present disclosure.

[0047] The assay components / reagents of the compositions / kits / microfluidic devices / methods are provided in any form that enables them to function according to the present disclosure. For example, and not by way of limitation, each of the reagents is provided in liquid form and disposed in the kit in bulk and / or in single aliquot form. Alternatively, in a particular (but non-limiting) embodiment, one or more of the reagents are disposed in the kit in the form of a single aliquot of lyophilized reagent. The use of dried reagents in 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.

[0048] In addition to the assay components / 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 the artisan; therefore, further description thereof is not considered necessary. Also, each of the components / reagents present in the kit may be in a separate container / compartment, or the various components / reagents are combined in one or more containers / compartments, depending on the cross-reactivity and stability of the components / reagents. Furthermore, the kit may include a microfluidic device in which the components / reagents are arranged.

[0049] The relative amounts of the various components / reagents in the kit can be widely varied to provide components / reagents at concentrations that fully optimize the reactions required to occur during the assay method and further optimize the sensitivity of the assay. Under appropriate circumstances, one or more of the components / reagents in the kit can be provided as a dry powder, such as a lyophilized powder, and the kit can further include an excipient for dissolving the dry reagent; thus, a reagent solution having a suitable concentration for performing a method or assay according to the present disclosure can be obtained from these components. Positive and / or negative controls can also be included in the kit. Additionally, the kit can further include a set of written instructions describing how to use the kit. Kits of this nature can be used in any of the methods described or otherwise contemplated herein.

[0050] Certain further non-limiting embodiments of the present disclosure are directed to a microfluidic device comprising any of the components of the kits described hereinabove. In particular, certain non-limiting embodiments include a microfluidic device for determining the presence and / or concentration of multiple anti-SARS-CoV-2 antibodies in a sample. The microfluidic device comprises: (i) an inlet channel to which a sample is applied; and (ii) at least a first compartment capable of fluid communication with the inlet channel. The (ii) compartment contains the reagents (a)-(e) of the first kit described hereinabove (wherein (c) and (d) are biotinylated antigens), or the reagents (a)-(d) of the second kit described hereinabove (wherein (c) is at least one biotinylated anti-human Ig antibody).

[0051] Any of the singlet oxygen activatable chemiluminescent compounds, sensitizers, fluorescent molecules, target antigens, biotin-specific binding partners, and anti-human Ig antibodies described in detail herein above or otherwise contemplated herein can be utilized in the microfluidic devices of the present disclosure.

[0052] For example, in certain (but non-limiting) embodiments, the singlet oxygen activatable chemiluminescent compounds of (a) and (b) are substances that undergo a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose with simultaneous or subsequent emission of light.

[0053] In a specific (but non-limiting) embodiment, the first target antigen is at least a portion of the SARS-CoV-2 S1 protein, such as (but not limited to) its RBD.

[0054] In specific (but non-limiting) embodiments, the second target antigen is at least a portion of a SARS-CoV-2 nucleocapsid protein, at least a portion of a SARS-CoV-2 envelope protein, or at least a portion of a SARS-CoV-2 membrane protein.

[0055] In a specific (but non-limiting) embodiment, the sensitizer is a photosensitizer.

[0056] In specific (but non-limiting) embodiments, the biotin-specific binding partner of (iii) is avidin or an analogue thereof, or an antibody to biotin.

[0057] In specific (but non-limiting) embodiments, the (a) and (b) fluorescent molecules are each independently selected from the group consisting of terbium, uranium, samarium, europium, gadolinium, and dysprosium. For example (but not limited to), terbium emits at a wavelength of about 545 nm, uranium emits at a wavelength of about 612 nm, and samarium emits at a wavelength of about 645 nm.

[0058] In certain non-limiting embodiments, all of elements (a)-(e) or (a)-(d) (depending on the kit) of (ii) are present in the same compartment. In alternative non-limiting embodiments, elements (a)-(e) or (a)-(d) (depending on the kit) are divided between two or more compartments.

[0059] The device can provide any arrangement of compartments and distribution of the various components therebetween that enables the device to function in accordance with the present disclosure.

[0060] Any of the compartments of the microfluidic device are sealed to maintain the reagents disposed therein in a substantially airtight environment until the reagents are used; for example, a compartment containing a lyophilized reagent is sealed to prevent any unintentional reconstitution of the reagent. The inlet channel and the compartment, as well as the two compartments, are described as being "capable of fluid communication" with each other; this phrase indicates that each of the compartments can be sealed at any time, but that the two compartments can allow fluid to flow between them when the seal formed in or between them is broken.

[0061] The microfluidic device of the present disclosure can provide any other desired functionality known in the art or otherwise contemplated herein. For example, and not by way of limitation, the microfluidic device of the present disclosure can further include a reading chamber; the reading chamber can be any of the compartments containing reagents described herein above, or the reading chamber can be in fluid communication with said compartments. The microfluidic device can further include one or more additional compartments containing other solutions, such as (but not limited to) washing solutions, diluent solutions, excipients, interference solutions, positive controls, negative controls, quality controls, etc. These additional compartments can be in fluid communication with one or more of the other compartments. For example, the microfluidic device can further include one or more compartments containing washing solutions, which can be in fluid communication with any other compartments of the device. In another example, the microfluidic device can further include one or more compartments containing excipients for dissolving one or more dried reagents, which can be in fluid communication with any other compartments of the device. In yet a further example, the microfluidic device can include one or more compartments containing diluent solutions, which can be in fluid communication with any other compartments of the device.

[0062] Certain non-limiting embodiments are also directed to a method for detecting the presence and / or concentration of biotin and at least one additional target analyte in a sample, the method comprising the steps of:

[0063] In a first step, a sample suspected of containing anti-SARS-CoV-2 antibodies is combined simultaneously or in whole or in part succession with compositions (a)-(e) of a first kit described hereinabove, or with compositions (a)-(d) of a second kit described hereinabove.

[0064] In a second step, the components are incubated together to allow binding of (a) and (c) to anti-SARS-CoV-2 spike protein antibodies in the sample (i.e., anti-SARS-CoV-2-RBD antibodies if the first target antigen is the RBD of S1), allow binding of (b) and (d) to anti-SARS-CoV-2 antibodies to a second target antigen in the sample, and allow binding of (c) and (d) to (e). Indirect binding of (a) to (e) results in the formation of a spike protein complex, and indirect binding of (b) to (e) results in the formation of a second target antigen complex. In each of the spike protein complex and the second target antigen complex, the sensitizer is in close proximity to the chemiluminescent compound.

[0065] In the third step, a sensitizer is activated to generate singlet oxygen; activation of the sensitizer present in the spike protein complex and the second target antigen complex causes activation of a chemiluminescent compound present in each complex.

[0066] In a fourth step, the amount of chemiluminescence produced by the activated chemiluminescent compound in the spike protein complex is determined by measuring the amount of light emitted from the fluorescent molecule in (a), and the amount of anti-SARS-CoV-2 spike protein antibody in the sample is proportional to the amount of light emitted.

[0067] In the fifth step, the amount of chemiluminescence produced by the activated chemiluminescent compound in the second target antigen complex is determined by measuring the amount of light emitted from the fluorescent molecule in (b), and the amount of anti-SARS-CoV-2-second target antigen antibody in the sample is proportional to the amount of light emitted.

[0068] In certain (but non-limiting) embodiments, the method may further include a sixth step of determining that the anti-SARS-CoV-2 antibodies present in the sample were generated in response to vaccination based on the results of step (4) and determining that the anti-SARS-CoV-2 antibodies present in the sample were generated in response to infection based on the results of step (5).

[0069] In one specific (but non-limiting) embodiment, steps (2)-(5) may be repeated one or more times.

[0070] Any of the singlet oxygen activatable chemiluminescent compounds, sensitizers, fluorescent molecules, target antigens, biotin-specific binding partners, and anti-human Ig antibodies described in detail herein above or otherwise contemplated herein can be utilized in the methods of the present disclosure.

[0071] For example, in certain (but non-limiting) embodiments, the singlet oxygen activatable chemiluminescent compounds of (a) and (b) are substances that undergo a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose with simultaneous or subsequent emission of light.

[0072] In a specific (but non-limiting) embodiment, the first target antigen is at least a portion of the SARS-CoV-2 S1 protein, such as (but not limited to) its RBD.

[0073] In specific (but non-limiting) embodiments, the second target antigen is at least a portion of a SARS-CoV-2 nucleocapsid protein, at least a portion of a SARS-CoV-2 envelope protein, or at least a portion of a SARS-CoV-2 membrane protein.

[0074] In one specific (but non-limiting) embodiment, the sensitizer is a photosensitizer, and activation of the sensitizer in step (3) comprises irradiation with light (for example, but not limited to, irradiation at about 680 nm).

[0075] In specific (but non-limiting) embodiments, the (a) and (b) fluorescent molecules are each independently selected from the group consisting of terbium, uranium, samarium, europium, gadolinium, and dysprosium. For example (but not limited to), terbium emits at a wavelength of about 545 nm, uranium emits at a wavelength of about 612 nm, and samarium emits at a wavelength of about 645 nm.

[0076] Any sample for which it is desired to assay for the presence and / or concentration of anti-SARS-CoV-2 antibodies can be utilized as a sample according to the methods of the present disclosure. Non-limiting examples of samples include biological samples such as, but not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof. Specific non-limiting examples include lysed whole blood cells and lysed red blood cells.

[0077] As mentioned above, the various components of the method are provided in combination (either simultaneously or sequentially). When the various components of the method are added sequentially, the order of addition of the components may be changed; one skilled in the art can determine the specific desired order of addition of the different components to the assay. The simplest order of addition is, of course, to add all materials simultaneously and determine the signal generated therefrom. Alternatively, each of the components, or groups of components, can be combined sequentially. In certain embodiments, an incubation step is included after one or more additions.

[0078] In an alternative (but non-limiting) embodiment, step (1) of the method comprises first combining the sample with a biotinylated reagent (i.e., two biotinylated target antigens or at least one biotinylated anti-human Ig antibody), incubating the same, and then adding a composition comprising a chemiluminescent compound activatable by singlet oxygen and a composition comprising a sensitizer. Alternatively, step (1) of the method may comprise first combining the sample, the biotinylated reagent (i.e., two biotinylated target antigens or at least one biotinylated anti-human Ig antibody), and a composition comprising a chemiluminescent compound activatable by singlet oxygen, incubating the same, and then adding a composition comprising a sensitizer.

[0079] Although certain embodiments of the present disclosure are described as having a LOCI® assay format, it is understood that the present disclosure is also directed to other assay formats (as well as kits, microfluidic devices, and methods for carrying out the same) in which it is desired to distinguish SARS-CoV-2 antibodies generated in response to vaccination from SARS-CoV-2 antibodies generated in response to infection. For example (and not by way of limitation), the present disclosure also includes assay formats in which different signal molecules, such as (but not limited to) different antibodies linked to different enzymes that generate signals at different wavelengths, are utilized in place of the chemiluminescent compound-containing compositions described hereinabove. EXAMPLES

[0080] Examples are provided herein 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, examples are provided merely as one of various embodiments, and are intended to be illustrative, not comprehensive. EXAMPLES

[0081] In June 2020, the U.S. Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for a laboratory-based pan-antibody test developed by Siemens Healthineers (Malvern, PA) to detect the presence of SARS-CoV-2 antibodies, including IgM, IgA, and IgG, in the blood. The spike protein on the surface of the SARS-CoV-2 virus allows the virus to enter and infect human cells found in multiple organs and blood vessels. Siemens Healthineers' pan-antibody COV2T assay is shown in Figure 1 and was designed to detect antibodies against the RBD of the spike protein. It is believed that some of these antibodies neutralize the SARS-CoV-2 virus and therefore block infection. Multiple potential vaccines in development against SARS-CoV-2 include the spike protein among their focal points.

[0082] The assay format in Figure 1 utilizes two reagents that both contain SARS-CoV-2 antigens: the first reagent contains a fluorescein-labeled receptor binding domain of the S1 subunit of the SARS-CoV-2 spike protein (S1 RBD) and preformed anti-FITC ChemiBeads, and the second reagent is formed by binding the biotinylated RBD of S1 to universal streptavidin-coated SensiBeads.

[0083] The immunoassay format of the present disclosure adds (i) a second ChemiBead having a second antigen from a different SARS-CoV-2 protein bound to the second ChemiBead and a different fluorescent molecule associated therewith, and (ii) a biotinylated second antigen to interact with the streptavidin-coated SensiBead. Thus, two LOCI® immunoassays are performed in a single reaction with the sample.

[0084] This example describes the use of a duplex immunoassay to simultaneously detect anti-nucleocapsid (NC) and anti-spike protein antibodies from SARS-CoV-2 in patient samples. A software algorithm was also developed to report whether the anti-SARS-CoV-2 antibodies present in a patient sample were generated by infection and / or vaccination, as well as the titer of each antibody.

[0085] In this example, a LOCI assay was constructed using the following reagents, as shown in FIG.

[0086] First, Universal SensiBeads were coated with streptavidin in the usual manner.

[0087] Second, two types of biotinylated SARS-CoV-2 viral antigens were generated: biotinylated nucleocapsid protein or a portion thereof (biotin-NC); and biotinylated spike protein or a portion thereof (which may be S1 or its RBD = biotin-RBD is used herein to represent this component).

[0088] Third, two types of ChemiBeads (CB) were made: CB stained with terbium (TCB) and coated with nucleocapsid protein or a portion thereof (NC-TCB); and CB stained with europium (ECB) and coated with spike protein or a portion thereof (RBD-ECB). Note that these particular combinations of dyes and antigens are merely for illustrative purposes. Any ChemiBeads dye may be utilized with any antigen protein. The only requirement is that the two ChemiBeads present have different dyes that fluoresce at different wavelengths.

[0089] Antigen-bound chemibeads can be formed in the same manner as the chemibeads utilized in all of the commercially available SARS-CoV-2 assays described above (i.e., by conjugating the fluorescein-labeled RBD of S1 to anti-FITC chemibeads), or alternatively, antigens can be directly conjugated to the surface of the chemibeads.

[0090] The fluorescent molecules of the two types of ChemiBeads (i.e., terbium and europium, or other fluorescent molecules) are different from each other and emit at different wavelengths; thus, complexes containing antibodies to a first target antigen are detected at a different wavelength than complexes containing antibodies to a second target antigen, so that assays for antibodies to both SARS-CoV-2 antigens can be performed simultaneously in the same reaction vessel. Alternatively, the two antibody assays can be performed in the same reaction vessel, but sequentially (i.e., by passing a single reaction vessel through two different detectors for signal measurements at different wavelengths).

[0091] In one non-limiting embodiment, the following reaction sequence is used. However, it will be understood that the order of addition of reagents can be easily modified and optimized by one of ordinary skill in the art. Thus, the following reaction sequence is merely for illustrative purposes and is not intended to limit the disclosed method.

[0092] Biotinylated first and second target antigens (i.e., biotin-NC and biotin-RBD) were combined with a patient sample suspected of containing anti-SARS-CoV-2 antibodies (anti-SC2 Ab). Two ChemiBeads (i.e., NC-TCB and RBD-ECB) were then added and the reaction mixture was incubated to allow sandwich formation. If anti-SARS-CoV-2 antibodies against the nucleocapsid protein (aNC-Ab) were present, the following sandwich was formed: Biotin-NC::aNC-Ab::NC-TCB. In the presence of anti-SARS-CoV-2 antibodies against the RBD of S1 (aRBD-Ab), the following sandwich was formed: Biotin:RBD::aRBD-Ab::RBD-ECB.

[0093] SensiBeads (SB) were then added in excess and the reaction mixture was incubated, such that the sandwich formed above was captured by the SensiBeads. In the presence of anti-SARS-CoV-2 antibodies (aNC-Ab) against the nucleocapsid protein, the following complex was formed: SB::biotin-NC::aNC-Ab::NC-TCB. In the presence of anti-SARS-CoV-2 antibodies against the RBD of S1 (aRBD-Ab), the following complex was formed: SB::biotin-RBD::aRBD-Ab::RBD-ECB.

[0094] Please note that SensiBeads are universal reagents with multiple biotin-specific binding partners bound to them. Thus, although the two sandwich complexes immediately above are described hereinabove as being two separate and distinct sandwich complexes, it is theoretically possible that two types of sandwiches (i.e., biotinylated target antigen::anti-SARS-CoV-2 antibody:: target analyte coated ChemiBeads) could be bound to the same SensiBeads.

[0095] When the reaction mixture is incubated for a sufficient time to allow the formation of the two complexes in the presence of the target antibody (as shown in Figure 3), a chemiluminescent reaction is triggered by excitation light at 680 nm to produce singlet oxygen ( 1 O 2 In the reaction mixture, one or both of (i) and (ii) can occur: (i) When the SB::biotin-NC::aNC-Ab::NC-TCB complex is present, due to spatial proximity, singlet oxygen diffuses from SensiBeads to ChemiBeads, causing a photon emission at 545 nm, which can be detected by a PMT tuned to receive light at 545 nm (this can be with a spin filter); and (ii) When the SB::biotin-RBD::aRBD-Ab::RBD-ECB complex is present, spatial proximity allows singlet oxygen to diffuse from the SensiBeads to the ChemiBeads, causing a photon emission at 612 nm, which can be detected by a PMT tuned to receive light at 612 nm (this is the current LOCI PMT detection).

[0096] Typically, two basic scenarios are detected: (ii) a signal from alone or a combination of signals from (i) and (ii). If a signal generated by the complex of (i) is detected, alone or in combination with (ii), the antibodies are due to infection. If only a signal generated by the complex of (ii) is present, the antibodies are due to vaccination.

[0097] In one non-limiting embodiment, the signals generated by the two complexes (i) and (ii) are then input into the following algorithm programmed into the instrument software: (a) If only a 612 nm signal is received, the assay detects only anti-SARS-CoV-2 RBD antibodies and the antibodies are due to vaccination. (b) If only a signal at 545 nm is received, the assay detects only anti-SARS-CoV-2 NC antibodies, and the antibodies are likely due to infection and therefore to an early stage of infection. (c) If both 612 nm and 545 nm signals are detected, then at least some of the antibodies are due to infection. For example (but not limited to), the following scenarios can be presented: 1) If the 612 nm signal is stronger than the 545 nm signal, then the patient likely has antibodies generated from infection alone or from both vaccination and infection. 2) If the 612 nm signal is not as strong or weaker than the 545 nm signal, the patient has an infection.

[0098] Under certain non-limiting conditions, it may be desirable to output information distinguishing between (b) and (c) as described above. Under other non-limiting conditions, it may be desirable to report only the "not infected" and "infected" status of the sample, distinguishing only between signals in (a) and the (b) / (c) combination, respectively; i.e., detection of any anti-SARS-CoV-2 NC antibodies in a sample should output a report of the "infected" status of the sample.

[0099] Although the use of biotinylated target antigens (i.e., biotin-NC and biotin-RBD) is described hereinabove, it will be understood that these two biotinylated reagents can be replaced with one or more biotinylated anti-human Ig antibodies as an alternative in a dual immunoassay format.

[0100] The RBD portion of the SARS-CoV-2 S protein is utilized in the above examples because neutralizing antibodies from patients bind to the RBD and block it from binding to ACE2 (a receptor) on human cell membranes; thus, immunoassays utilizing the RBD as a viral antigen will detect patient antibodies against the RBD of the spike protein. Both the Pfizer-BioNTech and Moderna COVID-19 vaccines, approved in the United States as Emergency Use Authorizations (EUAs) in 2021, are made from the entire S protein. As such, the scope of this disclosure includes the use of the entire S protein as an antigen in any of the duplex immunoassays described herein. However, due to the homology of the entire S protein to other viruses, a more specific signal may be seen by using the CoV-2 RBD as an antigen rather than the entire S protein.

[0101] Although chemi-beads containing terbium and europium are described herein above, it will be understood that combinations of fluorescent molecules that fluoresce at different wavelengths over a wider range may be used. For example, chemi-beads dyed with samarium dye (SCB) that emits photons at 645 nm can be used instead of ECB, and the PMT can be adjusted accordingly.

[0102] Furthermore, the multiplex assay of the present disclosure can be adapted to simultaneously detect antibodies against three or more SARS-CoV-2 target analytes. For each additional antibody against the target analyte to be detected, additional components, such as chemibead antigens and biotinylated antigens, are added, and the fluorescent molecules present in the additional components are different from the first and second fluorescent molecules in that they emit at wavelengths that are different and separately detectable from the first and second fluorescent molecules. In this way, antibodies against two, three, four, five, six, seven, eight, nine, ten, or more SARS-CoV-2 target antigens can be detected in a single reaction, as long as each of the chemibeads used to detect them contains different fluorescent molecules that each emit at a different and separately detectable wavelength; thus, the limiting factor of how many different antibodies can be detected in a single reaction is the number of available fluorescent molecules that function as described herein.

[0103] The dual immunoassay format of the present disclosure is useful for determining whether a patient's anti-SARS-CoV-2 antibodies are due to infection or vaccination. The assay format is also useful for determining whether vaccination is effective in uninfected individuals as well as in individuals previously infected with COVID-19. EXAMPLES

[0104] The duplex immunoassay of this example is similar to the assay format described in Example 1 above. However, this immunoassay format differs in that instead of utilizing a biotinylated target antigen, it utilizes at least one biotinylated anti-human immunoglobulin antibody to instead bind to the anti-SARS-CoV-2 antibodies in the sample to form a sandwich with each ChemiBead, and then the formed sandwich is captured by SensiBeads.

[0105] In this example, the LOCI assay was constructed using the following reagents, as shown in FIG.

[0106] First, Universal SensiBeads were obtained as described in Example 1.

[0107] Second, two types of ChemiBeads were prepared as described in Example 1 (NC-TCB and RBD-ECB).

[0108] Third, at least one biotinylated anti-human Ig antibody was generated.

[0109] In one non-limiting embodiment, the following reaction sequence is used. However, it will be understood that the order of addition of reagents can be easily modified and optimized by one of ordinary skill in the art. Thus, the following reaction sequence is merely for illustrative purposes and is not intended to limit the disclosed method.

[0110] At least one biotinylated anti-human Ig antibody (anti-hIg Ab) was combined with a patient sample suspected of containing anti-SARS-CoV-2 antibodies (anti-SC2 Ab). Two ChemiBeads (i.e., NC-TCB and RBD-ECB) were then added and the reaction mixture was incubated to allow sandwich formation. If anti-SARS-CoV-2 antibodies against the nucleocapsid protein (aNC-Ab) were present, the following sandwich was formed: Biotin-anti-hIg Ab::aNC-Ab::NC-TCB. In the presence of anti-SARS-CoV-2 antibodies against the RBD of S1 (aRBD-Ab), the following sandwich was formed: Biotin-anti-hIg Ab::aRBD-Ab::RBD-ECB.

[0111] SensiBeads (SB) were then added in excess and the reaction mixture was incubated, such that the sandwich formed above was captured by the SensiBeads. In the presence of anti-SARS-CoV-2 antibodies (aNC-Ab) against the nucleocapsid protein, the following complex was formed: SB::biotin-anti-hIg Ab::aNC-Ab::NC-TCB. In the presence of anti-SARS-CoV-2 antibodies against the RBD of S1 (aRBD-Ab), the following complex was formed: SB::biotin-anti-hIg Ab::aRBD-Ab::RBD-ECB.

[0112] It should be noted that SensiBeads are universal reagents with multiple biotin-specific binding partners bound to them. Thus, although the two sandwich complexes immediately above are described hereinabove as being two separate and distinct sandwich complexes, it is theoretically possible that two types of sandwiches (i.e., biotinylated anti-human Ig antibody::anti-SARS-CoV-2 antibody::chemibeads coated with target analyte) could be bound to the same SensiBeads.

[0113] When the reaction mixture is incubated for a sufficient time to allow the formation of the two complexes in the presence of the target antibody (as shown in Figure 5), a chemiluminescent reaction is triggered by excitation light at 680 nm to produce singlet oxygen ( 1 O 2 In the reaction mixture, one or both of (i) and (ii) can occur: (i) when the SB::biotin-anti-hIg Ab::aNC-Ab::NC-TCB complex is present, due to spatial proximity, singlet oxygen diffuses from the SensiBeads to the ChemiBeads, causing a photon emission at 545 nm, which can be detected by a PMT tuned to receive light at 545 nm (this can be with a spin filter); and (ii) When the SB::biotin-anti-hIg Ab::aRBD-Ab::RBD-ECB complex is present, spatial proximity allows singlet oxygen to diffuse from the SensiBeads to the ChemiBeads, causing a photon emission at 612 nm, which can be detected by a PMT tuned to receive light at 612 nm (this is the current LOCI PMT detection).

[0114] The signals generated by the two complexes (i) and (ii) are then input into the following algorithm programmed into the instrument software: (a) If only a 612 nm signal is received, the assay detects only anti-SARS-CoV-2 RBD antibodies and the antibodies are due to vaccination. (b) If only a signal at 545 nm is received, the assay detects only anti-SARS-CoV-2 NC antibodies, and the antibodies are likely due to infection and therefore to an early stage of infection. (c) If both 612 nm and 545 nm signals are detected, the following scenarios: 1) If the signal at 612 nm is stronger than the signal at 545 nm, then the patient has antibodies generated from both vaccination and infection. 2) If the 612 nm signal is not as strong or weaker than the 545 nm signal, the patient has an infection.

[0115] Although chemi-beads containing terbium and europium are described herein above, it will be understood that combinations of fluorescent molecules that fluoresce at different wavelengths over a wider range can be used. For example, chemi-beads dyed with samarium dye (SCB) that emits photons at 645 nm can be used instead of ECB, and the PMT can be adjusted accordingly.

[0116] Thus, in accordance with the present disclosure, compositions, kits, and devices, as well as methods for making and using them, are provided that fully meet the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth hereinabove, it is apparent 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 are within the spirit and broad scope of the present disclosure.

Claims

1. 1. A kit for performing a multiplex assay utilizing a chemiluminescence detection system for determining the presence and / or concentration of multiple anti-SARS-CoV-2 antibodies in a sample, the kit comprising: (a) a singlet oxygen activatable chemiluminescent compound having a first target antigen bound directly or indirectly to the chemiluminescent compound; A fluorescent molecule excited by an activated chemiluminescent compound and a composition comprising: (b) a singlet oxygen activatable chemiluminescent compound having a second target antigen bound directly or indirectly to the chemiluminescent compound; a fluorescent molecule excited by the activated chemiluminescent compound, the fluorescent molecule being different from the fluorescent molecule (a) and emitting light at a wavelength different from that of the fluorescent molecule (a); and a composition comprising: (c) a biotinylated first target antigen; (d) a biotinylated second target antigen; (e) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, the composition having a biotin-specific binding partner bound directly or indirectly to the sensitizer; Including, The kit, wherein the first target antigen is at least a portion of a SARS-CoV-2 spike protein and the second target antigen is at least a portion of a SARS-CoV-2 protein other than the spike protein.

2. 1. A kit for performing a multiplex assay utilizing a chemiluminescence detection system for determining the presence and / or concentration of multiple anti-SARS-CoV-2 antibodies in a sample, the kit comprising: (a) a singlet oxygen activatable chemiluminescent compound having a first target antigen bound directly or indirectly to the chemiluminescent compound; A fluorescent molecule excited by an activated chemiluminescent compound and a composition comprising: (b) a singlet oxygen activatable chemiluminescent compound having a second target antigen bound directly or indirectly to the chemiluminescent compound; a fluorescent molecule excited by the activated chemiluminescent compound, the fluorescent molecule being different from the fluorescent molecule (a) and emitting light at a wavelength different from that of the fluorescent molecule (a); and a composition comprising: (c) at least one biotinylated anti-human immunoglobulin (Ig) antibody; (d) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, the composition having a biotin-specific binding partner bound directly or indirectly to the sensitizer; Including, The kit, wherein the first target antigen is at least a portion of a SARS-CoV-2 spike protein and the second target antigen is at least a portion of a SARS-CoV-2 protein other than the spike protein.

3. The kit of claim 1 or 2, wherein the first target antigen comprises a receptor binding domain (RBD) of the S1 protein.

4. 3. The kit of claim 1 or 2, wherein the second target antigen is at least a portion of a SARS-CoV-2 nucleocapsid protein.

5. 3. The kit of claim 1 or 2, wherein the second target antigen is at least a portion of a SARS-CoV-2 membrane protein or at least a portion of a SARS-CoV-2 envelope protein.

6. The singlet oxygen activatable chemiluminescent compounds of each of (a) and (b) are substances that undergo a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose with simultaneous or subsequent emission of light; The sensitizer is a photosensitizer; and (e) the biotin-specific binding partner is selected from the group consisting of avidin, an analogue of avidin, and an antibody against biotin; The kit of claim 1, wherein the kit comprises at least one of the following:

7. The singlet oxygen activatable chemiluminescent compounds of each of (a) and (b) are substances that undergo a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose with simultaneous or subsequent emission of light; The sensitizer is a photosensitizer; and (d) the biotin-specific binding partner is selected from the group consisting of avidin, an analogue of avidin, and an antibody to biotin; The kit of claim 2, wherein the kit is at least one of:

8. 3. The kit of claim 1 or 2, wherein the fluorescent molecules (a) and (b) are each independently selected from the group consisting of terbium, uranium, samarium, europium, gadolinium, and dysprosium.

9. 1. A microfluidic device for determining the presence and / or concentration of a plurality of anti-SARS-CoV-2 antibodies in a sample, the microfluidic device comprising: (i) an inlet channel through which the sample is applied; (ii) capable of fluid communication with the inlet channel; (a) a singlet oxygen activatable chemiluminescent compound having a first target antigen bound directly or indirectly to the chemiluminescent compound; A fluorescent molecule excited by an activated chemiluminescent compound and a composition comprising: (b) a singlet oxygen activatable chemiluminescent compound having a second target antigen bound directly or indirectly to the chemiluminescent compound; a fluorescent molecule excited by the activated chemiluminescent compound, the fluorescent molecule being different from the fluorescent molecule (a) and emitting light at a wavelength different from that of the fluorescent molecule (a); and a composition comprising: (c) a biotinylated first target antigen; (d) a biotinylated second target antigen; (e) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, the composition having a biotin-specific binding partner bound directly or indirectly to the sensitizer; At least a first compartment may contain Including, A microfluidic device, wherein the first target antigen is at least a portion of a SARS-CoV-2 spike protein and the second target antigen is at least a portion of a SARS-CoV-2 protein other than the spike protein.

10. 1. A microfluidic device for determining the presence and / or concentration of a plurality of anti-SARS-CoV-2 antibodies in a sample, the microfluidic device comprising: (i) an inlet channel through which the sample is applied; (ii) capable of fluid communication with the inlet channel; (a) a singlet oxygen activatable chemiluminescent compound having a first target antigen bound directly or indirectly to the chemiluminescent compound; A fluorescent molecule excited by an activated chemiluminescent compound and a composition comprising: (b) a singlet oxygen activatable chemiluminescent compound having a second target antigen bound directly or indirectly to the chemiluminescent compound; a fluorescent molecule excited by the activated chemiluminescent compound, the fluorescent molecule being different from the fluorescent molecule (a) and emitting light at a wavelength different from that of the fluorescent molecule (a); and a composition comprising: (c) at least one biotinylated anti-human immunoglobulin (Ig) antibody; (d) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, the composition having a biotin-specific binding partner bound directly or indirectly to the sensitizer; At least a first compartment may contain Including, A microfluidic device, wherein the first target antigen is at least a portion of a SARS-CoV-2 spike protein and the second target antigen is at least a portion of a SARS-CoV-2 protein other than the spike protein.

11. The microfluidic device of claim 9 or 10, wherein the first target antigen comprises a receptor binding domain (RBD) of S1 protein.

12. The microfluidic device of claim 9 or 10, wherein the second target antigen is at least a portion of a SARS-CoV-2 nucleocapsid protein.

13. The microfluidic device of claim 9 or 10, wherein the second target antigen is at least a portion of a SARS-CoV-2 membrane protein or at least a portion of a SARS-CoV-2 envelope protein.

14. The microfluidic device of claim 9 , wherein (a) to (e) are present in the same compartment.

15. The microfluidic device of claim 9, wherein (a)-(e) are divided between two or more compartments.

16. The microfluidic device of claim 10 , wherein (a) to (d) are present in the same compartment.

17. The microfluidic device of claim 10, wherein (a)-(d) are divided between two or more compartments.

18. The singlet oxygen activatable chemiluminescent compounds of each of (a) and (b) are substances that undergo a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose with simultaneous or subsequent emission of light; The sensitizer is a photosensitizer; and (e) the biotin-specific binding partner is selected from the group consisting of avidin, an analogue of avidin, and an antibody against biotin; The microfluidic device of claim 9 , wherein the at least one of

19. The singlet oxygen activatable chemiluminescent compounds of each of (a) and (b) are substances that undergo a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose with simultaneous or subsequent emission of light; The sensitizer is a photosensitizer; and (d) the biotin-specific binding partner is selected from the group consisting of avidin, an analogue of avidin, and an antibody to biotin; The microfluidic device of claim 10, wherein the at least one of

20. 11. The microfluidic device of claim 9 or 10, wherein the fluorescent molecules (a) and (b) are each independently selected from the group consisting of terbium, uranium, samarium, europium, gadolinium, and dysprosium.

21. 1. A method for detecting the presence and / or concentration of a plurality of anti-SARS-CoV-2 antibodies in a sample, the method comprising: (1) Samples suspected to contain SARS-CoV-2 antibodies: (a) a singlet oxygen activatable chemiluminescent compound having a first target antigen bound directly or indirectly to the chemiluminescent compound; A fluorescent molecule excited by an activated chemiluminescent compound A composition comprising: (b) a singlet oxygen activatable chemiluminescent compound having a second target antigen bound directly or indirectly to the chemiluminescent compound; a fluorescent molecule excited by the activated chemiluminescent compound, the fluorescent molecule being different from the fluorescent molecule (a) and emitting light at a wavelength different from that of the fluorescent molecule (a); A composition comprising: (c) a biotinylated first target antigen; (d) a biotinylated second target antigen; and (e) combining simultaneously or in whole or in part sequentially with a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner bound directly or indirectly to the sensitizer, The first target antigen is at least a portion of the SARS-CoV-2 spike protein, and the second target antigen is at least a portion of a SARS-CoV-2 protein other than the spike protein. a process, which is at least a part of (2) allowing binding of (a) and (c) to anti-SARS-CoV-2 spike protein antibodies in the sample, allowing binding of (b) and (d) to anti-SARS-CoV-2 antibodies to a second target antigen in the sample, and allowing binding of (c) and (d) to (e), where the indirect binding of (a) to (e) results in the formation of a spike protein complex and the indirect binding of (b) to (e) results in the formation of a second target antigen complex, where in each of the spike protein complex and the second target antigen complex a sensitizer is in proximity to a chemiluminescent compound; (3) activating a sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the spike protein complex and the second target antigen complex causes activation of a chemiluminescent compound present in each complex; and (4) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the spike protein complex by measuring the amount of light emitted from the fluorescent molecule of (a), wherein the amount of anti-SARS-CoV-2 spike protein antibody in the sample is proportional to the amount of light emitted; and (5) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the second target antigen complex by measuring the amount of light emitted from the fluorescent molecule of (b), wherein the amount of anti-SARS-CoV-2-second target antigen antibody in the sample is proportional to the amount of light emitted; A method comprising:

22. 1. A method for detecting the presence and / or concentration of a plurality of anti-SARS-CoV-2 antibodies in a sample, the method comprising: (1) Samples suspected to contain SARS-CoV-2 antibodies: (a) a singlet oxygen activatable chemiluminescent compound having a first target antigen bound directly or indirectly to the chemiluminescent compound; A fluorescent molecule excited by an activated chemiluminescent compound A composition comprising: (b) a singlet oxygen activatable chemiluminescent compound having a second target antigen bound directly or indirectly to the chemiluminescent compound; a fluorescent molecule excited by the activated chemiluminescent compound, the fluorescent molecule being different from the fluorescent molecule (a) and emitting light at a wavelength different from that of the fluorescent molecule (a); A composition comprising: (c) at least one biotinylated anti-human immunoglobulin (Ig) antibody; (d) combining simultaneously or in whole or in part sequentially with a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner bound directly or indirectly to the sensitizer, the first target antigen is at least a portion of a SARS-CoV-2 spike protein and the second target antigen is at least a portion of a SARS-CoV-2 protein other than the spike protein; (2) allowing binding of (a) and (c) to anti-SARS-CoV-2 spike protein antibodies in the sample, allowing binding of (b) and (c) to anti-SARS-CoV-2 antibodies to a second target antigen in the sample, and allowing binding of (c) to (d), where the indirect binding of (a) to (d) results in the formation of a spike protein complex and the indirect binding of (b) to (d) results in the formation of a second target antigen complex, where in each of the spike protein complex and the second target antigen complex, a sensitizer is in proximity to a chemiluminescent compound; (3) activating a sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the spike protein complex and the second target antigen complex causes activation of a chemiluminescent compound present in each complex; and (4) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the spike protein complex by measuring the amount of light emitted from the fluorescent molecule of (a), wherein the amount of anti-SARS-CoV-2 spike protein antibody in the sample is proportional to the amount of light emitted; and (5) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the second target antigen complex by measuring the amount of light emitted from the fluorescent molecule of (b), wherein the amount of anti-SARS-CoV-2-second target antigen antibody in the sample is proportional to the amount of light emitted; A method comprising:

23. The method further comprises: (6) determining that the anti-SARS-CoV-2 antibodies present in the sample were generated in response to vaccination based on the results of step (4), and determining that the anti-SARS-CoV-2 antibodies present in the sample were generated in response to infection based on the results of step (5).

24. The method of claim 21 or 22, further comprising repeating steps (2) to (5).

25. 23. The method of claim 21 or 22, wherein the first target antigen comprises the receptor binding domain (RBD) of the S1 protein.

26. 23. The method of claim 21 or 22, wherein the second target antigen is at least a portion of a SARS-CoV-2 nucleocapsid protein.

27. 23. The method of claim 21 or 22, wherein the second target antigen is at least a portion of a SARS-CoV-2 membrane protein or at least a portion of a SARS-CoV-2 envelope protein.

28. The singlet oxygen activatable chemiluminescent compounds of each of (a) and (b) are substances that undergo a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose with simultaneous or subsequent emission of light; (e) the sensitizer is a photosensitizer, and activation of the sensitizer in step (3) comprises irradiation with light; and (e) the biotin-specific binding partner is selected from the group consisting of avidin, an analogue of avidin, and an antibody against biotin; 22. The method of claim 21, wherein the at least one of

29. The singlet oxygen activatable chemiluminescent compounds of each of (a) and (b) are substances that undergo a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose with simultaneous or subsequent emission of light; (d) the sensitizer is a photosensitizer, and activation of the sensitizer in step (3) comprises irradiation with light; and (d) the biotin-specific binding partner is selected from the group consisting of avidin, an analogue of avidin, and an antibody to biotin; 23. The method of claim 22, wherein the at least one of

30. The sample is a biological sample selected from the group consisting of whole blood or any fraction thereof, urine, saliva, sputum, cerebrospinal fluid, skin, intestinal fluid, peritoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.

23. The method according to item 21 or 22.

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