WO2023122562A2 - Reflex test for immunoassay for anti-sars-cov-2 antibodies - Google Patents

Abstract

Kits, microfluidics devices, and methods are disclosed for determining if a patient is positive for COVID-19 infection, in which immunoassays are performed to detect the presence of anti-SARS-CoV-2 antibodies in a patient sample. The kits, microfluidics devices, and methods incorporate the use of a reflex test within the reagent cartridge for the major anti-SARS-CoV-2 antibody assay, wherein the minor assay of the reflex test is employed when the result obtained in the major assay is above a baseline index. The reflex test (i.e., minor assay) utilizes at least one reagent that is different from the reagents utilized in the major assay.

Description

REFLEX TEST FOR IMMUNOASSAY FOR ANTI-SARS-COV-2 ANTIBODIES

CROSS REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCE STATEMENT

[0001] This application claims priority of United States Provisional application no. 63/265,707 filed 20 December 2021 , the contents of which are fully incorporated herein by reference.

FIELD

[0002] The present disclosure relates to methods, kits, and systems, and for detecting or determining if a subject is positive for COVID-19 infection based on a biological sample from a subject, and/or treating the subject.

BACKGROUND

[0003] The field of medical diagnostics utilizes many different forms of assay technologies. When a patient is suspected of being infected with a microorganism (such as, but not limited to, a bacteria or virus), an assay may be performed on a biological sample from the patient to detect antibodies directed to the microorganism that are being 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 serology assays have been employed, in which an immobilized viral/bacterial antigen and a labeled viral/bacterial antigen are often used to formulate the assay reagents. In another example, latex particle agglutination assays utilize viral/bacterial antigen- coated latex particles as a single reagent that aggregate in the presence of anti- viral/bacterial antigen antibodies in patient samples.

[0005] One example of a commercially used assay for detecting antigens and antibodies in patient samples is the Luminescent Oxygen Channeling Assay (LOCI®) technology. The LOCI® advanced chemiluminescence 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. The currently available LOCI® technology has high sensitivity and uses several reagents. In particular, the LOCI® assay requires that two of these reagents (referred to as a “chemibead” and a “sensibead”) be held by other specific binding partner assay reagents in a manner whereby the chemibead and sensibead are in close proximity to one another to achieve a signal. Upon exposure to light at a certain wavelength, the sensibead releases singlet oxygen, and if the two beads are in close proximity, the singlet oxygen is transferred to the chemibead; this causes a chemical reaction that results in the chemibead giving off 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 total antibody test developed by Siemens Healthineers (Tarrytown, NY) for the detection of the presence of total anti-SARS-CoV-2 antibodies, including IgM and IgG, in blood. This immunoassay is based on the LOCI® platform and is referred to as the CV2T LOCI® immunoassay. An EUA was issued in January 2021 for Siemens Healthineers’ SARS- CoV-2 IgG immunoassay (referred to as the CV2G LOCI® immunoassay) for the detection of the presence of SARS-CoV-2 IgG antibodies.

[0007] A spike protein on the surface of the SARS-CoV-2 virus enables the virus to penetrate and infect human cells found in multiple organs and blood vessels. The Siemens Healthineers’ Total Antibody COV2T CV2T and CV2G LOCI® immunoassays were designed to detect antibodies to the receptor binding domain (RBD) of spike protein. Some of these antibodies are believed to neutralize the SARS- CoV-2 virus and therefore prevent infection.

[0008] The accuracy and sensitivity of anti-SARS-CoV-2 antibody assays are of utmost importance to evaluate patients and eventually populations to show immunity to the virus. The specificity of the assay needs to be close to 100% with no false positives. Seroconversion of individuals is highly variant in levels and timing when antibodies are present. In some individuals, the rise of antibodies in serum or plasma is slow, and detection at those levels may be challenging. Most assays available do not detect all patients in the first seven (7) days post symptom onset; numbers can range lower than 60%. Some assays also miss samples 7-14 days post symptom onset.

[0009] In addition, due to stringent specificity requirements for anti-SARS-CoV-2 antibody assays, a relatively high signal level is utilized as the assay cutoff to reduce the incidence of reporting matrix noise or false elevation as a positive signal. However, this high signal level assay cutoff often results in misreporting of a positive signal as negative, which compromises the assay sensitivity.

[00010] Prior art of interest includes WO 2022/178591 entitled Peptides and Their Use in Diagnosis of SARS-COV-2 Infection, and WO 2022/147147 entitled Methods For Determining Sars-Cov-2 Antigen and Anti-Sars-Cov-2 Antibody In A Sample (both of which are entirely incorporated herein by reference).

[00011] There is a continuing need in the art for new and improved immunoassays for detecting the presence of anti-SARS-CoV-2 antibodies that increases the sensitivity and specificity of these assays and provides superior clinical utility over currently available assays. In particular, there is a need to differentiate between low positive signals and false signals of similar magnitudes. It is to such assays, as well as kits and microfluidics devices related to same and methods of using same, that the present disclosure is directed.

SUMMARY

[00012] The present disclosure provides methods, kits, and devices for determining if a patient is positive for COVID-19 infection and/or treating the patient. Moreover, the present disclosure provides assays, as well as kits and microfluidics devices related to immunoassays for detecting the presence of anti-SARS-CoV-2 antibodies that increases the sensitivity and specificity of these assays and provides superior clinical utility over currently available assays. In particular, the present disclosure provides a method differentiate between low positive signals and false signals of similar magnitudes.

[00013] In embodiments, the present disclosure provides a method for detecting the presence of anti-SARS-CoV-2 antibodies in a sample, the method including the steps of: (1) combining, either simultaneously or wholly or partially sequentially, a sample suspected of containing anti-SARS-CoV-2 antibodies with: (a) a composition including: a singlet oxygen-activatable chemiluminescent compound having a SARS- CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) a biotinylated SARS- CoV-2 target antigen; and (d) a composition including a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto; (2) allowing the binding of (a) and (b) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of (b) to (d), thereby resulting in the indirect binding of (a) to (d) and formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound; (3) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex; (4) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a); (5)conducting a reflex test when a result of step (4) is above a baseline index, wherein the reflex test comprises the steps of: (i) combining, either simultaneously or wholly or partially sequentially, the sample suspected of containing anti-SARS-CoV-2 antibodies with: (a) and (b) from step (1); and (c) a biotinylated anti-human immunoglobulin (Ig) antibody; (ii) allowing the binding of (a) and (c) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of the biotinylated anti-human immunoglobulin (Ig) antibody to (d), thereby resulting in the indirect binding of (a) to (d) and formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound; (iii) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex; (iv) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a); and (6) reporting the sample as positive for anti-SARS-CoV-2 antibodies if a result of (iv) is above a baseline index, or reporting the sample as negative for anti-SARS-CoV-2 antibodies if the result of (iv) is below the baseline index.

[00014] In embodiments, the present disclosure includes a kit for performing an immunoassay for determining the presence of anti-SARS-CoV-2 antibodies in a sample, the kit including:(a) a composition including: a singlet oxygen-activatable chemiluminescent compound having a SARS-CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) a biotinylated SARS-CoV-2 target antigen; and (c) a biotinylated anti-human immunoglobulin (Ig) antibody.

[00015] In embodiments, the present disclosure includes a microfluidics device for determining the presence of anti-SARS-CoV-2 antibodies in a sample, the microfluidics device including: (i) an inlet channel through which a sample is applied; (ii) at least two compartments capable of being in fluidic communication with the inlet channel and containing: (a) a composition including: a singlet oxygen-activatable chemiluminescent compound having a SARS-CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) a biotinylated SARS-CoV-2 target antigen; (c) a biotinylated anti-human immunoglobulin (Ig) antibody; and (d) a composition including a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto; and wherein each of (a), (b), (c), and (d) is disposed in at least one of the at least two compartments, and wherein (b) is disposed in a different compartment from (c).

BRIEF DESCRIPTION OF THE DRAWINGS

[00016] FIG. 1 depicts a schematic cross-sectional view of a microfluidic device suitable for use in accordance with the embodiments of the present disclosure.

[00017] FIG. 2 depicts a second schematic cross-sectional view of a microfluidic device suitable for use in accordance with the embodiments of the present disclosure. [00018] FIG. 3 depicts an exemplary block diagram of a computer system 1100.

[00019] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. The figures are not drawn to scale and may be simplified for clarity. Elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.

[00020] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. The figures are not drawn to scale and may be simplified for clarity. Elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.

DETAILED DESCRIPTION

[00021] The present disclosure relates to a method for detecting the presence of anti-SARS-CoV-2 antibodies in a sample, the method including the steps of: (1) combining, either simultaneously or wholly or partially sequentially, a sample suspected of containing anti-SARS-CoV-2 antibodies with: (a) a composition including: a singlet oxygen-activatable chemiluminescent compound having a SARS- CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) a biotinylated SARS- CoV-2 target antigen; and (d) a composition including a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto; (2) allowing the binding of (a) and (b) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of (b) to (d), thereby resulting in the indirect binding of (a) to (d) and formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound; (3) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex; (4) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a); (5)conducting a reflex test when a result of step (4) is above a baseline index, wherein the reflex test comprises the steps of: (i) combining, either simultaneously or wholly or partially sequentially, the sample suspected of containing anti-SARS-CoV-2 antibodies with: (a) and (b) from step (1); and (c) a biotinylated anti-human immunoglobulin (Ig) antibody; (ii) allowing the binding of (a) and (c) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of the biotinylated anti-human immunoglobulin (Ig) antibody to (d), thereby resulting in the indirect binding of (a) to (d) and formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound; (iii) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex; (iv) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a); and (6) reporting the sample as positive for anti-SARS-CoV-2 antibodies if a result of (iv) is above a baseline index, or reporting the sample as negative for anti-SARS-CoV-2 antibodies if the result of (iv) is below the baseline index. Advantages of the present disclosure include excellent differentiation between low positive signals from false signals of similar magnitude in immunoassays for anti-SARS-CoV-2 antibodies.

[00022] 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. [00023] 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.

[00024] 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.

[00025] 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.

Definitions

[00026] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: [00027] 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.”

[00028] 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.

[00029] 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).

[00030] 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.

[00031] 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.

[00032] 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.

[00033] 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.

[00034] 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. [00035] 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.

[00036] 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 fluidic 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.

[00037] The term “specific binding partner,” as used in particular (but not by way of limitation) herein in the terms “biotin-specific binding partner” or “target analyte-specific binding partner,” will be understood to refer to any molecule capable of specifically associating with biotin or the target analyte, respectively. 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 matrices), combinations or derivatives thereof, as well as any other molecules capable of specific binding to biotin or the target analyte, respectively.

[00038] The term “antibody” is used herein in the broadest sense and refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Such polypeptide may be naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. Non-limiting examples include 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, singlechain 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., lgG1 , lgG2, lgG3, lgG-4, lgA1 , and lgA2). In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., poly-ethylene glycol, etc.).

[00039] The term “hapten” as used herein refers to a small proteinaceous or nonprotein antigenic determinant (or “epitope”) which is capable of being recognized by a target analyte-specific binding partner, such as (but not limited to) an antibody. The term “polyhapten” as used herein will be understood to refer to a synthetic molecule that contains multiple epitopes/antigenic determinants attached thereto.

[00040] An “analyte” is a macromolecule that is capable of being recognized by an analyte-specific 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.

[00041] The term “detection agent” as used herein refers to any element, molecule, functional group, compound, fragment or moiety that is detectable. In some embodiments, a detection agent is provided or utilized alone. In some embodiments, a detection agent is provided and/or utilized in association with (e.g., joined to) another agent. Examples of detection agents include, but are not limited to: various ligands, radionuclides (e.g., ³H, ¹⁴C, ¹⁸F, ¹⁹F, ³²P, ³⁵S, ¹³⁵l, ¹²⁵l, ¹²³l, ⁶⁴Cu, ¹⁸⁷Re, ¹¹¹1n, ⁹⁰Y, ^99mTc, ¹⁷⁷Lu, ⁸⁹Zr etc.), fluorescent dyes, chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, dioxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[001] As used herein, “diagnostic test” is a step or series of steps that is or has been performed to attain information that is useful in determining whether a patient has a disease, disorder or condition and/or in classifying a disease, disorder or condition into a phenotypic category or any category having significance with regard to prognosis of a disease, disorder or condition, or likely response to treatment (either treatment in general or any particular treatment) of a disease, disorder or condition. Similarly, “diagnosis” refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have or develop a disease, disorder or condition, state, staging or characteristic of a disease, disorder or condition as manifested in the subject, information related to the nature or classification of a tumor, information related to prognosis and/or information useful in selecting an appropriate treatment or additional diagnostic testing. Selection of treatment may include the choice of a particular therapeutic agent or other treatment modality such as surgery, radiation, etc., a choice about whether to withhold or deliver therapy, a choice relating to dosing regimen (e.g., frequency or level of one or more doses of a particular therapeutic agent or combination of therapeutic agents), etc. Selection of additional diagnostic testing may include more specific testing for a given disease, disorder, or condition.

[002] The term “LOCI®” as used herein refers to the Luminescent Oxygen Channeling Assay technology. The LOCI® advanced chemiluminescence 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. The currently available LOCI® technology has high sensitivity and uses several reagents. In particular, the LOCI® assay requires that two of these reagents (referred to as a “sensibead” and a “chemibead”) be held by other specific binding partner assay reagents in a manner whereby the sensibead and chemibead are in close proximity to one another to achieve a signal. Upon exposure to light at a certain or predetermined wavelength, the sensibead releases singlet oxygen, and if the two beads are in close proximity, the singlet oxygen is transferred to the chemibead; this causes a chemical reaction that results in the chemibead giving off light that can be measured at a different wavelength.

[003] As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human). In some embodiments a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, or at least 80 years of age. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder or condition. In some embodiments, a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.

[004] Threshold value: As used herein, the term “threshold value” refers to a value (or values) that are used as a reference to attain information on and/or classify the results of a measurement, for example, the results of a measurement attained in an assay. A threshold value can be determined based on one or more control samples. A threshold value can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold value can be a range of values. In some embodiments, a threshold value can be a value (or range of values) reported in the relevant field (e.g., a value found in a standard table).

[005] The terms “treatment” or “treating” of a subject includes the application or administration of a compound to a subject with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term “treating” refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being. In embodiments, the term "treating" means reducing or ameliorating the progression, severity, and/or duration of COVID-19, or ameliorating one or more symptoms of COVID-19 caused by administration of one or more therapies (e.g., one or more therapeutic agents). In a particular embodiment, the term "treatment" means to ameliorate measurable physical parameters of COVID-19.

[006] As used herein, the term “therapeutically effective amount” means the amount of compound that, when administered to a subject for treating or preventing a particular disorder, disease or condition, is sufficient to effect such treatment or prevention of that disorder, disease or condition. Dosages and therapeutically effective amounts may vary for example, depending upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and any drug combination, if applicable, the effect which the practitioner desires the compound to have upon the subject and the properties of the compounds (e.g., bioavailability, stability, potency, toxicity, etc.), and the particular disorder(s) the subject is suffering from. In addition, the therapeutically effective amount that is administered intravenously may depend on the subject's blood parameters e.g., lipid profile, insulin levels, glycemia or liver metabolism. The therapeutically effective amount will also vary according to the severity of the disease state, organ function, or underlying disease or complications. Such appropriate doses may be determined using any available assays. When one or more of the compounds or therapeutic agents is to be administered to humans, a physician may for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.

[00042] Turning now to certain non-limiting embodiments of the present disclosure, provided herein are devices, reagents, kits, and methods for differentiating low positive signals from false signals of similar magnitude in immunoassays for anti-SARS-CoV- 2 antibodies.

[00043] Certain non-limiting embodiments of the present disclosure are directed to a kit for performing an immunoassay for determining the presence of anti-SARS-CoV- 2 antibodies in a sample, and for performing a reflex test as needed. The kit includes:

(a) a composition including a singlet oxygen-activatable chemiluminescent compound having a SARS-CoV-2 target antigen directly or indirectly bound thereto, and a fluorescent molecule that is excited by the activated chemiluminescent compound;

(b) a biotinylated SARS-CoV-2 target antigen; and

(c) a biotinylated anti-human immunoglobulin (Ig) antibody.

In certain particular (but non-limiting) embodiments, the kit further comprises:

(d) a composition including a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto. [00044] Each of the SARS-CoV-2 target antigen(s) utilized in (a) and (b) may comprise any portion of SARS-CoV-2 disclosed or otherwise contemplated herein or otherwise known in the art as being recognized by anti-SARS-CoV-2 antibodies produced by patients. In addition, the target antigens of (a) and (b) may be the same or different.

[00045] In certain non-limiting embodiments, all or any portion of a SARS-CoV-2 spike (S) protein may be utilized as the target antigens of (a) and/or (b) in accordance with the present disclosure. In certain non-limiting embodiments, at least one of the target antigens of (a) and (b) includes at least a portion of a SARS-CoV-2 S1 protein, such as (but not limited to) at least a portion of a receptor-binding domain (RBD) of S1 protein. In another non-limiting embodiment, at least one of the target antigens of (a) and (b) includes at least a portion of a SARS-CoV-2 S2 protein. See e.g., the world wide web at https://www.ncbi.nlm.nih.gov/Structure/SARS-CoV-2.html for related protein sequence information.

[00046] In one particular (but non-limiting) embodiment, both of the target antigens of (a) and (b) comprises at least a portion of the RBD of S1 . The RBD S1 antigen can be obtained from any source known in the art. For example (but not by way of limitation), this particular antigen is 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).

[00047] Alternatively, the target antigen of (a) and/or (b) may comprise all or any portion of another SARS-CoV-2 protein. For example, in certain non-limiting embodiments, at least one of the target antigens includes at least a portion of a SARS- CoV-2 nucleocapsid (N) protein, at least a portion of a SARS-CoV-2 membrane (M) protein, or at least a portion of a SARS-CoV-2 envelope (E) protein.

[00048] The anti-human Ig antibody of (c) may specifically bind to any portion of any human immunoglobulin molecules known in the art or otherwise contemplated herein, so long as the antibodies recognize at least one or more human immunoglobulin antibodies (i.e. , human IgG, IgE, IgM, IgD, and/or IgA, and/or any portion thereof). In a particular (but non-limiting) embodiment, the anti-human Ig antibody of (c) recognizes multiple human immunoglobulin molecules, including at least human IgG, IgM, and IgA. In another particular (but non-limiting) embodiment, the anti-human Ig antibody of (c) is an anti-human IgG antibody. [00049] Anti-human Ig antibodies and anti-human IgG antibodies are well known in the art, are widely commercially available, and have been vastly studied. For example (but not by way of limitation), a few commercial sources of anti-human Ig and 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 inclusive, and there are many additional commercial sources of anti-human Ig and IgG antibodies that can be utilized in accordance with the present disclosure. Thus, a person having ordinary skill in the art will clearly and unambiguously be able to identify and select a variety of anti-human Ig and IgG antibodies that can be utilized in accordance with the present disclosure, and as such, no further description of the anti-human Ig and IgG antibodies or the characteristics thereof is deemed necessary.

[00050] In a particular (but non-limiting) embodiment, the kit may be constructed for performing an immunoassay for anti-SARS-CoV-2 antibodies using a Luminescence Oxygen Channeling Assay (LOCI®; Siemens Healthcare Diagnostics Inc., Tarrytown, NY) immunoassay format. 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. For example (but not by way of limitation), when the kit is designed for use in a LOCI® immunoassay format, the kit may include (a) a chemibead coated with the receptor binding domain (RBD) of the S1 protein of SARS-CoV-2, (b) a biotinylated RBD, and (c) a biotinylated anti-human Ig or IgG antibody; optionally, the kit may further include (d) a sensibead coated with streptavidin.

[00051] In addition to the assay components/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) that can be present in the kits 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 components/reagents present in the kits may each be in separate containers/compartments, or various components/reagents can be combined in one or more containers/compartments, depending on the cross-reactivity and stability of the components/reagents. In addition, the kit can further include a set of written instructions explaining how to use the kit.

[00052] Certain non-limiting embodiments of the present disclosure are directed to a microfluidics device that contains any of the assay reagents described or otherwise contemplated herein. For example (but not by way of limitation), certain additional nonlimiting embodiments of the present disclosure are directed to a microfluidics device that includes the reagents (a), (b), (c), and/or (d) of any of the kits described herein above.

[00053] In particular, certain non-limiting embodiments of the present disclosure are directed to a microfluidics device for determining the presence of anti-SARS-CoV-2 antibodies in a sample via any of the assays described or otherwise contemplated herein, and for performing a reflex test as needed. The microfluidics device includes: (i) an inlet channel through which a sample is applied (See e.g., FIG. 1 inlet channel between 12 and 14); and (ii) at least two compartments capable of being in fluidic communication with the inlet channel. Each of reagents (a), (b), (c), and (d) described or otherwise contemplated herein is disposed in at least one of the at least two compartments of (ii), either alone or in combination with one or more other reagents described or otherwise contemplated herein (such as, but not limited to, one or more reagents for use in the assay). The only requirement with respect to the distribution of the reagents between the at least two compartments of (ii) is that reagent (b) is disposed in a different compartment from reagent (c). Otherwise, the microfluidics device may be provided with any arrangement of compartments and distribution of the various reagents/components therebetween that allows the device to function in accordance with the present disclosure.

[00054] In certain non-limiting embodiments, (ii) contains at least three compartments, wherein reagent (b) is disposed in a first compartment, reagent (c) is disposed in a second compartment, and reagents (a) and (d) are disposed in one or more additional compartments that are separate from the first and second compartments (wherein (a) and (d) may be disposed in the same or different compartments). In this manner, the reagents of (a) and (d) can be combined with (b) to perform the major assay, and should the result of the major assay be considered “reactive” (i.e., above a baseline index), the reagents of (a) and (d) can then be combined with (c) to perform the minor assay that forms the reflex test for the major assay. See e.g., FIG. 2 including a plurality of compartments as discussed herein above.

[00055] Any of the compartments of the microfluidics device may be sealed to maintain the reagent(s) disposed therein in a substantially airtight 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 one or more compartments, as well as two or more 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 or more compartments are capable of having fluid flow therebetween upon puncture or opening of a seal formed therein or therebetween.

[00056] 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 one or more read chambers; the read chamber(s) may be any of the compartments containing the reagents described herein above, or the read chamber(s) may be in fluidic communication with said compartment(s).

[00057] The microfluidics device may further include one or more additional compartments containing other solutions, such as (but not limited to) wash solutions, calibration solutions, quality control solutions, dilution solutions, excipients, interference solutions, positive controls, negative 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.

[00058] The assay components/reagents of the kits and microfluidic devices of the present disclosure 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/microfluidics device. Alternatively, in a particular (but nonlimiting) embodiment, one or more of the reagents may be disposed in the kit or microfluidics device in the form of a single aliquot lyophilized reagent. The use of dried reagents in 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.

[00059] Turning now to the Drawings, FIG. 1 depicts a kit or microfluidic device 10 for use in accordance with the present disclosure. The device 10 is utilized in accordance with the present disclosure. The device 10 may be any type of disposable cartridge (for example but not by way of limitation, a laminate card or a molded card) or otherwise described herein, and capable of containing a microfluidic structure as described in detail herein below. In embodiments, the device 10 contains a sensor 12 and elements for producing one or more solution(s) for calibration and/or quality control. These elements include at least one cavity 14 in fluidic communication with the sensor 12. The cavity 14 contains at least one lyophilized reagent 16 (the lyophilized reagent depicts as being in bead/hemisphere form). The cavity 14 is also in fluidic communication with an activatable cavity 18 containing an excipient 20 (such as but not limited to, a liquid or a gel) for reconstitution of the lyophilized reagent 16 (wherein the fluidic communication is provided upon activation of the activatable cavity 18). The activatable cavity 18 may be, for example but not by way of limitation, a blister pack or other type of sealed cavity that is sealed to prevent contact between the excipient 20 and the lyophilized reagent 16 until use of the device 10. In use, the activatable cavity 18 may be activated such as (but not by way of limitation) by depression thereof, thus pushing the excipient 20 into the cavity 14 containing the lyophilized reagent 16 to reconstitute the lyophilized reagent 16, thereby providing a reconstituted calibration and/or quality control solution. The reconstituted calibration and/or quality control solution is then brought into contact with the sensor 12. The resultant reconstituted calibration and/or quality control solution will contain a known quantity of ions, proteins and/or gases that will serve as calibrators and/or control solutions for the sensor of interest.

[00060] In embodiments, some mixing may occur in the cavity 14 between the lyophilized reagent 16 and the excipient 20 to ensure complete reconstitution of the lyophilized reagent 16 and to ensure homogeneity of the resultant reconstituted calibration and/or quality control solution. Any method of mixing known in the microfluidics art or otherwise contemplated herein may be utilized in accordance with the presently disclosed and claimed inventive concept(s). In addition, the flow of the excipient 20 into the cavity 14 may be controlled by any method known in the art or otherwise contemplated herein; for example, but not by way of limitation, the force of activating (i.e. , depressing) the activatable cavity 18 may provide the necessary force to push the desired amount of excipient 20 into the cavity 14. Likewise, the flow of the reconstituted calibration and/or quality control solution (whether in the form of ion(s), protein(s) and/or gas(es)) from the cavity 14 and over the sensor 12 may be controlled by any method known in the art or otherwise contemplated herein; for example but not by way of limitation, the force of activating (i.e., depressing) the activatable cavity 18 may ultimately provide the necessary force to push the reconstituted calibration and/or quality control solution out of the cavity 14 and over the sensor 12. [00061] FIG. 2 depicts a device 10a, another embodiment of a microfluidics device utilized in accordance with the present disclosure. The device 10a is similar to the device 10 of FIG. 1 , except that the device 10a contains multiple cavities or compartments with multiple beads/hemispheres of lyophilized reagents, and/or other reagents discussed herein, each in contact with a different activatable cavity containing excipient. In embodiments, the device 10 contains a sensor array 12a in fluidic communication with three cavities containing lyophilized reagents: a first cavity 14a, a second cavity 22 and a third cavity 24. The first cavity 14a contains four beads/hemispheres 16a, 16a', 16a" and 16a'" of lyophilized reagent(s). The four beads/hemispheres 16a, 16a', 16a" and 16a'" may contain the same reagent or different reagents of the present disclosure. The second cavity 22 contains a plurality of beads/hemispheres of lyophilized reagent(s) (singularly represented by the reference numeral 26), while the third cavity 24 contains a plurality of beads/hemispheres of lyophilized reagent(s) (singularly represented by the reference numeral 28); the plurality of beads/hemispheres 26 may contain the same reagent or different reagents such as described herein, whereas the plurality of beads/hemispheres 28 may contain the same reagent or different reagents such as described herein.

[00062] In embodiments, the cavity 14a is in fluidic communication with an activatable cavity 18a containing an excipient 20a (wherein the fluidic communication is provided upon activation of the activatable cavity 18a). The cavity 22 is in fluidic communication with an activatable cavity 30 containing an excipient 32 (wherein the fluidic communication is provided upon activation of the activatable cavity 30). The cavity or compartment 24 is in fluidic communication with an activatable cavity or compartment 34 containing an excipient 36 (wherein the fluidic communication is provided upon activation of the activatable cavity 34). The excipients 20a, 32 and 36 may be the same or different. The activatable cavities or compartments 18a/30/34 containing the excipients 20a/32/36 function in the same manner as described herein above for the activatable cavity 18 containing the excipient 20, and thereby reconstitute the beads/hemispheres 16-16'726/28 of lyophilized reagent(s). In this manner, resultant reconstituted calibration and/or quality control solution(s) containing a known quantity of ions, proteins and/or gases are provided that will serve as calibrators and/or control solutions for the sensor array 12a. It will be understood that the activatable cavities 18a, 30 and 34 may be activated at the same time (simultaneously), or the activation of the activatable cavities 18a, 30 and 34 may be staggered.

[00063] In embodiments, one or more microfluidics devices 10 and 10a are provided and configured for methods of the present disclosure or as kits of the present disclosure. In embodiments microfluidic device for determining the presence of anti- SARS-CoV-2 antibodies in a sample includes: (i) an inlet channel through which a sample is applied; (ii) at least two compartments capable of being in fluidic communication with the inlet channel and containing: (a) a composition including: a singlet oxygen-activatable chemiluminescent compound having a SARS-CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) a biotinylated SARS-CoV-2 target antigen; (c) a biotinylated anti-human immunoglobulin (Ig) antibody; and (d) a composition including a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto; and wherein each of (a), (b), (c), and (d) is disposed in at least one of the at least two compartments, and wherein (b) is disposed in a different compartment from (c). In embodiments, the SARS-CoV-2 target antigens of (a) and (b) are the same. In embodiments, at least one of the target antigens of (a) and (b) is at least a portion of a SARS-CoV-2 spike protein. In embodiments, at least one of the target antigens comprises a receptor-binding domain (RBD) of S1 protein. In embodiments, at least one of the target antigens of (a) and (b) is 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. In embodiments, the antibody of (c) is an anti-human IgG antibody. In embodiments, (ii) contains at least three compartments, wherein (b) is disposed in a first compartment, (c) is disposed in a second compartment, and (a) and (d) are disposed in one or more additional compartments.

[00064] The relative amounts of the various components/reagents in the kits and microfluidics devices of the present disclosure can vary widely to provide for concentrations of the components/reagents that substantially optimize the reactions that need to occur during the assay methods and further to optimize substantially the sensitivity and specificity of an assay. Under appropriate circumstances, one or more of the components/reagents in the kit/microfluidics device can be provided as a dry powder, such as a lyophilized powder, and the kit/microfluidics device 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 components.

[00065] In addition to the various reagents discussed herein above, additional reagents/components may also be present in the kits and microfluidics devices of the present disclosure. For example (but not by way of limitation), positive and/or negative controls may also be included with the kit/microfluidics device. Kits/microfluidics devices of this nature can be used in any of the methods described or otherwise contemplated herein.

[00066] Certain non-limiting embodiments of the present disclosure are directed to a method for detecting the presence of anti-SARS-CoV-2 antibodies in a sample, the method including the steps of: (1) combining, either simultaneously or wholly or partially sequentially, a sample suspected of containing anti-SARS-CoV-2 antibodies with reagents (a), (b), and (d) discussed in detail herein above; (2) allowing the binding of (a) and (b) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of (b) to (d), thereby resulting in the indirect binding of (a) to (d), wherein the indirect binding of (a) to (d) results in the formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound; (3) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex; and (4) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a).

[00067] Then, when a result of step (4) is above a baseline index, steps (5) and (6) are performed: (5) conducting a reflex test including the steps of: (i) combining, either simultaneously or wholly or partially sequentially, the sample suspected of containing anti-SARS-CoV-2 antibodies with reagents (a), (c), and (d) discussed in detail herein above; (ii) allowing the binding of (a) and (c) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of (c) to (d), thereby resulting in the indirect binding of (a) to (d), wherein the indirect binding of (a) to (d) results in the formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound; (iii) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex; (iv) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a); and (6) reporting the sample as positive for anti-SARS-CoV-2 antibodies if a result of (iv) is above a baseline index, or reporting the sample as negative for anti-SARS-CoV-2 antibodies if the result of (iv) is below the baseline index.

[00068] For example (but not by way of limitation), when the method utilizes a LOCI® immunoassay format, step (1) combines (a) a chemibead coated with the receptor binding domain (RBD) of the S1 protein of SARS-CoV-2, (b) a biotinylated RBD, and (d) a sensibead coated with streptavidin. If a result above baseline index is obtained with these reagents, then a reflex test as outlined in step (5) is performed using (a) the chemibead coated with RBD, (c) a biotinylated anti-human Ig or IgG antibody, and (d) the sensibead coated with streptavidin.

[00069] Any sample for which an assay for the presence and/or concentration of anti-SARS-CoV-2 antibodies is desired can be utilized as the sample in accordance with the methods of the present disclosure. Non-limiting examples of samples include a biological sample 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, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellularfluid, 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.

[00070] The measurement, analysis/comparison, and determination steps of the methods of the present disclosure may be performed by any methods known in the art or otherwise contemplated herein. For example (but not by way of limitation), the method may be performed in a diagnostic instrument that contains a processor that calculates the results of steps (4), (5)(iv), and/or (6) and uses algorithms to perform the determining and reporting steps.

[00071] It will also be understood that the scope of the present disclosure is not limited to performing step (5) only after a specific result is obtained in step (4). That is, for ease of measurement and to minimize the process time for the assays, steps (i)- (iv) of step (5) may be performed simultaneously with steps (1)-(4) (or partially sequentially therewith); however, the results of step (5) may not need to be factored into the reporting step if the result obtained in step (4) is conclusively above a positive (or negative) reporting threshold.

[00072] Certain additional non-limiting embodiments of the present disclosure are directed to a system for performing any of the methods disclosed or otherwise contemplated herein. The system includes a diagnostic instrument as described herein below or otherwise contemplated herein, in combination with one or more of any of the microfluidics devices disclosed or otherwise contemplated herein. Any of the steps described herein may be performed, for example but not by way of limitation, by a user. However, as used herein, the term “user” is not limited to use by a human being; rather, the term “user” may comprise (for example, but not by way of limitation) a computer, a server, a website, a processor, a network interface, a human, a user terminal, a virtual computer, combinations thereof, and the like.

[00073] The various embodiments of the present disclosure (i.e., compositions, kits, microfluidics devices, etc.) may be utilized with any diagnostic instrument that is capable of (or has been modified to be capable of) functioning in accordance with the methods described herein. In certain, non-limiting embodiments, the instrument may be a point of care instrument. The diagnostic instrument may be a system or systems that are able to embody and/or execute the logic of the methods/processes described herein. Logic embodied in the form of software instructions and/or firmware may be executed on any appropriate hardware. For example, logic embodied in the form of software instructions and/or firmware may be executed by one or more components on a dedicated system or systems, on a personal computer system, on a distributed processing computer system, and/or the like. In some embodiments, the entire logic may be implemented in a stand-alone environment operating on an instrument (such as, but not limited to, a point of care instrument). In other embodiments, the logic may be implemented in a networked environment such as a distributed system in which multiple instruments collect data that is transmitted to a centralized computer system for analyzing the data and supplying the results of the analysis to the instruments. Each element of the instrument may be partially or completely network-based or cloud based, and may or may not be located in a single physical location.

[00074] Circuitry used herein includes (but is not limited to) analog and/or digital components, or one or more suitably programmed processors (e.g., microprocessors) and associated hardware and software, or hardwired logic. Also, “components” may perform one or more functions. The term “component” may include hardware, such as but not limited to, a processor (e.g., microprocessor), an application specific integrated circuit (ASIC), field programmable gate array (FPGA), a combination of hardware and software, and/or the like.

[00075] Software utilized herein may include one or more computer readable medium (i.e., computer readable instructions) that when executed by one or more components cause the component to perform a specified function. It should be understood that the algorithms described herein may be stored on one or more nontransient memory. Non-limiting exemplary non-transient memory may include random access memory, read only memory, flash memory, and/or the like. Such non-transient memory may be electrically based, optically based, and/or the like.

[00076] Referring now to FIG. 3 a block diagram of a computer system 1100 that can be used in the operations described above and methods of the present disclosure, according to one embodiment is shown. The system 1100 includes a processor 1110, a memory 1120, a storage device 1130 and an input/output device 1140. Each of the components 1110, 1120, 1130 and 1140 are interconnected using a system bus 1150. The system may include analyzing equipment 1160 for measuring one or more analytes of the present disclosure in a sample or for performing a diagnostic assay. The processor 1110 is capable of processing instructions for execution within the system 1100. In one embodiment, the processor 1110 is a single-threaded processor. In another embodiment, the processor 1110 is a multi-threaded processor. The processor 1110 is capable of processing instructions stored in the memory 1120 or on the storage device 1130, including for receiving or sending information through the input/output device 1140. The memory 1120 stores information within the system 1100. In one embodiment, the memory 1120 is a computer-readable medium. In one embodiment, the memory 1120 is a volatile memory unit. In another embodiment, the memory 1120 is a non-volatile memory unit. In embodiments, the storage device 1130 is capable of providing mass storage for the system 1100. In one embodiment, the storage device 1130 is a computer-readable medium. The input/output device 1140 provides input/output operations for the system 1100. In one embodiment, the input/output device 1140 includes a keyboard and/or pointing device. Preferably, methods of the present disclosure are performed in a system 1100. For example, a computer program product can include instructions that cause a processor 1110 to perform the steps of a method of the present disclosure.

[00077] Additionally, non-transitory computer readable media containing executable instructions that when executed cause a processor to perform operations including a method as provided herein are provided. For example, a non-transitory computer readable medium containing executable instructions that when executed cause a processor to perform operations including a method for detecting the presence of anti- SARS-CoV-2 antibodies in a sample, the method including the steps of: (1 ) combining, either simultaneously or wholly or partially sequentially, a sample suspected of containing anti-SARS-CoV-2 antibodies with: (a) a composition including: a singlet oxygen-activatable chemiluminescent compound having a SARS-CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) a biotinylated SARS-CoV-2 target antigen; and (d) a composition including a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto; (2) allowing the binding of (a) and (b) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of (b) to (d), thereby resulting in the indirect binding of (a) to (d) and formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound; (3) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex; (4) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a); (5) conducting a reflex test when a result of step (4) is above a baseline index, wherein the reflex test comprises the steps of: (i) combining, either simultaneously or wholly or partially sequentially, the sample suspected of containing anti-SARS-CoV-2 antibodies with: (a) and (b) from step (1); and (c) a biotinylated anti-human immunoglobulin (Ig) antibody; (ii) allowing the binding of (a) and (c) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of the biotinylated anti-human immunoglobulin (Ig) antibody to (d), thereby resulting in the indirect binding of (a) to (d) and formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound; (iii) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex; (iv) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a); and (6) reporting the sample as positive for anti-SARS-CoV-2 antibodies if a result of (iv) is above a baseline index, or reporting the sample as negative for anti-SARS-CoV-2 antibodies if the result of (iv) is below the baseline index.

Treatment

[00078] In embodiments, the present disclosure includes a method of treating a subject in need thereof by determining that the subject is a subject in need thereof (has COVID-19), and subsequently treating the subject. For examples, the methods of the present disclosure can be applied to diagnose a patient as positive for COVID- 19, followed by administering a therapeutic agent to the subject in need thereof in a therapeutically effective amount. For example, a physician may administer a therapeutically effective amount of any drug, therapeutic agent or biologic suitable for treating COVI D-19 subsequent to diagnosis in accordance with the present disclosure. One non-limiting example of an agent suitable for treating COVID-19 includes VEKLURY® brand remdesivir (100 MG for injection antiviral medication for treatment of patients hospitalized with COVID-19). See also, e.g., the international patent application WO 2017/049060 A1 , and US US20210395345 entitled Methods for treating or preventing sars-cov-2 infections and covid-19 with anti-sars-cov-2 spike glycoprotein antibodies. The therapeutically effective amount of agent provided may be determined by a physician based on the subject’s response, comorbidities, and the like. Additional embodiments

[00079] Embodiments of the present disclosure include a kit for performing an immunoassay for determining the presence of anti-SARS-CoV-2 antibodies in a sample, the kit including: (a) a composition including: a singlet oxygen-activatable chemiluminescent compound having a SARS-CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) a biotinylated SARS-CoV-2 target antigen; (c) a biotinylated anti-human immunoglobulin (Ig) antibody and (d) a composition including a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto. In embodiments, the SARS-CoV-2 target antigens of (a) and (b) are the same. In embodiments, at least one of the target antigens of (a) and (b) is at least a portion of a SARS-CoV-2 spike protein. In embodiments, the at least one of the target antigens comprises a receptorbinding domain (RBD) of S1 protein.

[00080] Embodiments of the present disclosure include a kit for performing an immunoassay for determining the presence of anti-SARS-CoV-2 antibodies in a sample, the kit including: (a) a composition including: a singlet oxygen-activatable chemiluminescent compound having a SARS-CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) a biotinylated SARS-CoV-2 target antigen; (c) a biotinylated anti-human immunoglobulin (Ig) antibody and (d) a composition including a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto, wherein at least one of the target antigens of (a) and (b) is 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. In embodiments, the antibody of (c) is an anti-human IgG antibody.

[00081] In embodiments, the present disclosure includes a microfluidics device for determining the presence of anti-SARS-CoV-2 antibodies in a sample, the microfluidics device including: (i) an inlet channel through which a sample is applied; ii) at least two compartments capable of being in fluidic communication with the inlet channel and containing: (a) a composition including: a singlet oxygen-activatable chemiluminescent compound having a SARS-CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) a biotinylated SARS-CoV-2 target antigen; (c) a biotinylated anti-human immunoglobulin (Ig) antibody; and (d) a composition including a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto; and wherein each of

(a), (b), (c), and (d) is disposed in at least one of the at least two compartments, and wherein (b) is disposed in a different compartment from (c), wherein the SARS-CoV-2 target antigens of (a) and (b) are the same. In embodiments, at least one of the target antigens of (a) and (b) is at least a portion of a SARS-CoV-2 spike protein. In embodiments, at least one of the target antigens comprises a receptor-binding domain (RBD) of S1 protein. In embodiments, the at least one of the target antigens of (a) and

(b) is 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. In embodiments, the antibody of (c) is an anti-human IgG antibody. In embodiments, (ii) contains at least three compartments, wherein (b) is disposed in a first compartment, (c) is disposed in a second compartment, and (a) and (d) are disposed in one or more additional compartments.

[00082] In embodiments, the present disclosure includes a method for detecting the presence of anti-SARS-CoV-2 antibodies in a sample, the method including the steps of: (1) combining, either simultaneously or wholly or partially sequentially, a sample suspected of containing anti-SARS-CoV-2 antibodies with: (a) a composition including: a singlet oxygen-activatable chemiluminescent compound having a SARS- CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound; (b) a biotinylated SARS-CoV-2 target antigen; and (d) a composition including a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto; (2) allowing the binding of (a) and (b) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of (b) to (d), thereby resulting in the indirect binding of (a) to (d) and formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound; (3) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex; (4) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a); (5) conducting a reflex test when a result of step (4) is above a baseline index, wherein the reflex test comprises the steps of: (i) combining, either simultaneously or wholly or partially sequentially, the sample suspected of containing anti-SARS-CoV-2 antibodies with: (a) and (b) from step (1); and (c) a biotinylated anti-human immunoglobulin (Ig) antibody; (ii) allowing the binding of (a) and (c) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of the biotinylated anti-human immunoglobulin (Ig) antibody to (d), thereby resulting in the indirect binding of (a) to (d) and formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound; (iii) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex; (iv) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a); and (6) reporting the sample as positive for anti-SARS-CoV-2 antibodies if a result of (iv) is above a baseline index, or reporting the sample as negative for anti-SARS-CoV-2 antibodies if the result of (iv) is below the baseline index, wherein the sample is a biological sample selected from the group consisting of whole blood or any portion thereof, urine, saliva, sputum, cerebrospinal fluid, 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. In embodiments, the SARS- CoV-2 target antigens of (a) and (b) are the same. In embodiments, the at least one of the target antigens of (a) and (b) is at least a portion of a SARS-CoV-2 spike protein. In embodiments, the least one of the target antigens comprises a receptor-binding domain (RBD) of S1 protein. In embodiments, at least one of the target antigens of (a) and (b) is 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. In embodiments, the antibody of (c) utilized in step (5)(i) is an anti-human IgG antibody.

EXAMPLE

[00083] An Example is 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 Example is simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.

[00084] The present Example is directed to reagents and methods for differentiating low positive signals from false signals of similar magnitude in immunoassays for anti- SARS-CoV-2 antibodies.

[00085] Prior to the present disclosure, reflex testing for initial values in the grey zone (i.e. , reactive but not positive) is often used. However, the common approach for reflex testing is merely to repeat the test a few times. While this approach may bring the sub-cutoff results up to the positive zone, it does not solve the issue for low positive samples that are clearly reactive but well below the cutoff index.

[00086] In the present disclosure, a new approach for a reflex test is provided that involves re-testing the sample with a reagent different from that used for the initial test. The approach employs the principle that two different sets of reagents often do not show false signals for the same patients, but both inheritably show signals for true analyte due to specific antibody-antigen (Ab-Ag) binding. As a result, if both show greater than reactive signals, the probability that the signal is truly positive is significantly higher than the probability that the signal is truly negative, even when the positive signals are weak and well below the cutoff index. Conversely, if one reagent is positive and the other is negative, the “positive” signal is more than likely false. The present disclosure identified that the reagents utilized in the two LOCI® COVID-19 antibody assays, CV2T and CV2G, provide a pair of reagents that can be utilized in this type of approach (where one assay provides the major assay and the other assay provides the minor assay or reflex test). The reagents used in these two assays are similar, with the exception that biotin-RBD is used in the CV2T assay and biotin-anti- human-IgG antibody is used in the CV2G assay. Therefore, the reagent cartridge utilized for the major LOCI® assay can be modified to have one reagent well contain biotin-conjugate from the other assay for the reflex test (and thus allow for both LOCI® assays to be performed).

[00087] For example (but not by way of limitation), one reagent well in the CV2T reagent cartridge is modified to include the biotin-anti-human-IgG antibody reagent from the CV2G LOCI® assay for the reflex test. A gray zone software is then provided for the reflex test. When the initial results in the major assay are above a baseline index and thus considered “reactive,” the instrument gray zone software uses the following algorithm to report final results. [00088] The following example is a reflex test for CV2T. Reagents for two separate assays are present in the same reagent cartridge: the major assay of CV2T is used to test all patient samples, and the minor assay of CV2G is only used when the initial patient result is reactive or in the gray zone. Both major and minor assays are calibrated during calibration. The minor assay is calibrated using a calibrator spiked with antibody at the “reactive” level, which is clearly above the dead zero but well below the assay cutoff index. If the initial CV2T assay (i.e., the major assay) result is reactive, the test is repeated using the reagents containing biotin-anti-human-IgG antibody or biotin-anti-human Ig antibody (i.e., the minor assay):

(a) if the reflex test result is > reactive, then report the patient as positive;

(b) if the reflex test result is < reactive, then report the patient as negative. [00089] Use of the reflex test approach described herein will significantly enhance the sensitivity of COVID-19 antibody assays while maintaining the assay specificity. [00090] 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 kit for performing an immunoassay for determining the presence of anti- SARS-CoV-2 antibodies in a sample, the kit comprising:

(a) a composition comprising: a singlet oxygen-activatable chemiluminescent compound having a SARS-CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound;

(b) a biotinylated SARS-CoV-2 target antigen; and

(c) a biotinylated anti-human immunoglobulin (Ig) antibody.

2. The kit of claim 1 , further comprising:

(d) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto.

3. The kit of claim 1 , wherein the SARS-CoV-2 target antigens of (a) and (b) are the same.

4. The kit of claim 1 , wherein at least one of the target antigens of (a) and (b) is at least a portion of a SARS-CoV-2 spike protein.

5. The kit of claim 4, wherein at least one of the target antigens comprises a receptor-binding domain (RBD) of S1 protein.

6. The kit of claim 1 , wherein at least one of the target antigens of (a) and (b) is 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.

7. The kit of claim 1 , wherein the antibody of (c) is an anti-human IgG antibody.

8. A microfluidics device for determining the presence of anti-SARS-CoV-2 antibodies in a sample, the microfluidics device comprising:

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

(ii) at least two compartments capable of being in fluidic communication with the inlet channel and containing:

(a) a composition comprising: a singlet oxygen-activatable chemiluminescent compound having a SARS-CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound;

(b) a biotinylated SARS-CoV-2 target antigen;

(c) a biotinylated anti-human immunoglobulin (Ig) antibody; and

(d) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto; and wherein each of (a), (b), (c), and (d) is disposed in at least one of the at least two compartments, and wherein (b) is disposed in a different compartment from (c).

9. The microfluidics device of claim 8, wherein the SARS-CoV-2 target antigens of (a) and (b) are the same.

10. The microfluidics of claim 8, wherein at least one of the target antigens of (a) and (b) is at least a portion of a SARS-CoV-2 spike protein.

11 . The microfluidics device of claim 10, wherein at least one of the target antigens comprises a receptor-binding domain (RBD) of S1 protein.

12. The microfluidics device of claim 8, wherein at least one of the target antigens of (a) and (b) is 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.

13. The microfluidics device of claim 8, wherein the antibody of (c) is an anti-human IgG antibody.

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14. The microfluidics device of claim 8, wherein (ii) contains at least three compartments, wherein (b) is disposed in a first compartment, (c) is disposed in a second compartment, and (a) and (d) are disposed in one or more additional compartments.

15. A method for detecting the presence of anti-SARS-CoV-2 antibodies in a sample, the method comprising the steps of:

(1) combining, either simultaneously or wholly or partially sequentially, a sample suspected of containing anti-SARS-CoV-2 antibodies with:

(a) a composition comprising: a singlet oxygen-activatable chemiluminescent compound having a SARS-CoV-2 target antigen directly or indirectly bound thereto; and a fluorescent molecule that is excited by the activated chemiluminescent compound;

(b) a biotinylated SARS-CoV-2 target antigen; and

(d) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner directly or indirectly bound thereto;

(2) allowing the binding of (a) and (b) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of (b) to (d), thereby resulting in the indirect binding of (a) to (d) and formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound;

(3) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex;

(4) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a);

(5) conducting a reflex test when a result of step (4) is above a baseline index, wherein the reflex test comprises the steps of: (i) combining, either simultaneously or wholly or partially sequentially, the sample suspected of containing anti-SARS- CoV-2 antibodies with:

(a) and (b) from step (1); and

(c) a biotinylated anti-human immunoglobulin (Ig) antibody;

(ii) allowing the binding of (a) and (c) to anti-SARS-CoV-2 antibodies in the sample and allowing the binding of the biotinylated antihuman immunoglobulin (Ig) antibody to (d), thereby resulting in the indirect binding of (a) to (d) and formation of a complex in which the sensitizer is brought into close proximity to the chemiluminescent compound;

(iii) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complex causes the activation of the chemiluminescent compound present in the complex;

(iv) determining the amount of chemiluminescence generated by the activated chemiluminescent compound in the complex by measuring the amount of light emitted by the fluorescent molecule of (a); and

(6) reporting the sample as positive for anti-SARS-CoV-2 antibodies if a result of (iv) is above a baseline index, or reporting the sample as negative for anti-SARS-CoV-2 antibodies if the result of (iv) is below the baseline index.

16. The method of claim 15, wherein the sample is a biological sample selected from the group consisting of whole blood or any portion thereof, urine, saliva, sputum, cerebrospinal fluid, 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.

17. The method of claim 15, wherein the SARS-CoV-2 target antigens of (a) and (b) are the same.

18. The method of claim 15, wherein at least one of the target antigens of (a) and (b) is at least a portion of a SARS-CoV-2 spike protein.

19. The method of claim 18, wherein at least one of the target antigens comprises a receptor-binding domain (RBD) of S1 protein.

20. The method of claim 15, wherein at least one of the target antigens of (a) and (b) is 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.

21. The method of claim 15, wherein the antibody of (c) utilized in step (5)(i) is an anti-human IgG antibody.

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