Calibration and quality control reagents for immunoassays against antibodies

Abstract

Calibration and / or quality control reagents are disclosed for use in serological immunoassays of antibodies against microorganisms. In the agent, an antibody specific for a microbial antigen is complexed with an anti-human Ig antibody to form a complex. Also disclosed are kits and microfluidic devices containing the reagents, as well as methods of producing and using the reagents.

Description

[0001] This application is a divisional application of the application patent application entitled "Calibration and quality control reagents for immunoassays for antibodies" having an application date of April 21, 2021, and application number 202180030592.X.

[0002] This application claims the benefit under 35 USC § 119(e) of U.S. provisional application number 63 / 015,223 filed April 24, 2020. The entire contents of the above-cited patent application are hereby expressly incorporated herein by reference. TECHNICAL FIELD

[0003] The present invention relates to serological immunoassays, and in particular to calibration and / or quality control reagents for antibodies to microorganisms. BACKGROUND

[0004] 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 detecting the presence of SARS-CoV-2 antibodies, including IgM and IgG in blood. The spike protein on the surface of the SARS-CoV-2 virus enables the virus to invade and infect human cells found in multiple organs and blood vessels. The Siemens Healthineers Total Antibody COV2T assay is designed to detect antibodies to the spike protein. These antibodies are believed to neutralize the SARS-CoV-2 virus and thereby prevent infection. Multiple potential vaccines developed for SARS-CoV-2 incorporate the spike protein into their focus.

[0005] Just as new and improved COVID-19 immunoassays are needed, there is a corresponding need for calibrators for these immunoassays to ensure their accuracy. In particular, there is a need for anti-viral antigen antibodies for use in calibrators for immunoassays, such as (but not limited to) COVID-19 immunoassays. Due to the COVID-19 pandemic and the great demand for these antibodies, these anti-viral antigen antibodies have become very expensive and difficult to obtain in the required quantities. It is predicted that the demand for COVID-19 immunoassay calibrators will reach millions of vials per year, which translates into grams, costing millions of dollars. Thus, the cost of such raw materials would be an astronomical amount that would be prohibitive, and there is also a supply problem with such raw materials. Therefore, there is a great need for methods that minimize the actual anti-viral antigen antibodies utilized in the calibrator products. SUMMARY

[0006] Before one or more embodiments of the disclosure are described in detail, it is to be understood that the application of the disclosure is not limited to the details of construction and the arrangement set forth in the following description or illustrated in the accompanying drawings. The 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, but not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and not of limitation.

[0007] Unless otherwise defined, 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 nomenclature 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 synthesis and chemical analysis.

[0008] 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 disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are hereby expressly incorporated by reference in their entirety as if individually incorporated by reference.

[0009] In light of the present disclosure, all compositions, kits, devices, and / or methods disclosed herein can be prepared and performed without undue experimentation, although described in terms of specific embodiments. While the compositions, kits, devices, and / or methods have been described in terms of specific embodiments, it will be apparent to those of ordinary skill in the art that many modifications, substitutions, and alterations to the compositions, kits, devices, and / or methods, and the steps or order of steps in the methods described herein can be made without deviating from the spirit and scope of the disclosure. All such obvious substitutions and modifications are considered to be within the spirit and scope of the disclosure as defined in the following claims.

[0010] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: The use of the term “a” or “an” when used in conjunction with the term “comprising” will be understood to mean “one,” but it is also consistent with the meaning of “one or more” or “at least one.” As such the use of the term “a” or “an” herein cannot be taken to exclude the use of more than one. Use of the term “at least one” will be understood to extend to any number, including zero, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The use of the term “at least one” can extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the number of 100 / 1000 is not meant to be limiting as higher limits can 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, Z alone, as well as any combination of X, Y and Z. The use of ordinal 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 order or sequence or importance to one item over another or any added order.

[0011] The use of the term “at least one of’ will be understood to extend to any number, including zero, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The use of the term “at least one of’ can extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the number of 100 / 1000 is not meant to be limiting as higher limits can 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, Z alone, as well as any combination of X, Y and Z. The use of ordinal 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 order or sequence or importance to one item over another or any added order.

[0012] The use of the term “or” in the claims is used to mean an inclusive “and / or” unless explicitly indicated to refer to an exclusive “either / or.” For example, the phrase “A or B” satisfies the inclusive “and / or” interpretation when either A is true (or exists) and B is false (or does not exist), when A is false (or does not exist) and B is true (or exists), and when both A and B are true (or exist).

[0013] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, structure, feature, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase "in some embodiments" or "an example" in various places in the specification are not necessarily all referring to the same embodiment. Furthermore, all embodiments or examples are intended to cover equivalents and alternatives.

[0014] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, apparatus, method, or process being employed to determine the value. For example, but not by way of limitation, when the term "about" is utilized in reference to a value, that value can be varied by plus or minus 20%, or 15%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, as such variations are suitable for performing the disclosed methods and are within the capabilities of one of ordinary skill in the art.

[0015] As used in this specification and claim, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0016] The term "or its combination" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or its combination" is intended to encompass 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, it is further intended to encompass combinations that contain repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. It will be appreciated by those skilled in the art that, unless otherwise indicated, the number of items or terms in any combination is not limited to only one of each item or term.

[0017] As used herein, the term "substantially" means that the recited event or circumstance occurs exactly or the recited event or circumstance occurs over a wide range or degree. For example, when associated with a specific event or circumstance, the term "substantially" means that the recited 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" can mean that two items are 100% adjacent to each other, or that two items are in close proximity to each other but are not 100% adjacent to each other, or that a portion of one of two items is not 100% adjacent to the other item but is in close proximity to the other item.

[0018] As used herein, the phrases "associated with" and "coupled to" include both direct association / coupling and indirect association / coupling between two items. Non-limiting examples of association / coupling include covalent bonding between one item and another item, either directly or through a spacer group, non-covalent bonding between one item and another item, either directly or by means of a specific binding pair member bound to the item, such as by dissolving one item in another item or by incorporating one item into another item through synthesis, and coating one item on another item.

[0019] The terms "analog" and "derivative" are used interchangeably herein and refer to a substance that contains the same basic carbon skeleton and carbon functionality as a given compound in its structure, but can also contain one or more substitutions to it. As used herein, the term "substitution" will be understood to mean replacing at least one substituent on a compound with a residue R. In certain non-limiting embodiments, R can include H, hydroxyl, thiol, a halide selected from fluoride, chloride, bromide, or iodide, a C1-C4 compound selected from one of the following: optionally substituted straight chain, branched, or cyclic alkyl, and straight chain branched, or cyclic alkenyl, wherein the optional substituents are selected from one or more of alkenyl alkyl, alkynyl alkyl, cycloalkyl, cycloalkenyl alkyl, aryl alkyl, heteroaryl alkyl, heterocycloalkyl, optionally substituted heterocycloalkenyl alkyl, aryl cycloalkyl, and aryl heterocycloalkyl, each of which is optionally substituted, wherein the optional substituents are selected from one or more of alkenyl alkyl, alkynyl alkyl, cycloalkyl, cycloalkenyl alkyl, aryl alkyl, alkyl aryl, heteroaryl alkyl, heterocycloalkyl, optionally substituted heterocycloalkenyl alkyl, aryl cycloalkyl, and aryl heterocycloalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthiol, amino, -NH(alkyl), -NH(cycloalkyl)2, carboxyl, and -C(O))-alkyl.

[0020] The term "sample" as used herein will be understood to include any type of biological sample that can be utilized in accordance with the present disclosure. Examples of fluid biological samples that can 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, lacrimal fluid, mucus, bladder wash, semen, fecal matter, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.

[0021] The term "antibody" is used herein in the broadest sense, and refers to, for example, intact monoclonal antibodies and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and conjugates thereof (such as, but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, single-chain antibodies and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody), antibody surrogate proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof that exhibit the desired biological activity of binding to an analyte. The antibodies can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0022] The term "LOCI®" as used herein refers to the immunoassay technology based on the Luminescent Oxygen Channeling Assay (LOCI®) technology that is used commercially. The LOCI® advanced chemiluminescent assay is described, for example, in U.S. Patent 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. Specifically, the LOCI® assay requires two of these reagents, referred to as "sensibeads" and "chemibeads," to be held by other specific binding partners assay reagents in such a way that the sensibeads and chemibeads are in close proximity to each other to enable the signal. Upon exposure to light of a particular wavelength, the sensibeads release singlet oxygen, and if the two beads are in close proximity, the singlet oxygen is transferred to the chemibeads; this causes a chemical reaction that results in the chemibeads emitting light, which can be measured at a different wavelength.

[0023] Certain non-limiting embodiments of the present disclosure relate to calibration or quality control reagents for serological immunoassays for detecting antibodies against a microorganism in a human biological sample. The reagents comprise three components: (a) a matrix; (b) anti-human immunoglobulin (Ig) antibodies; and (c) human antibodies against a microorganism antigen. In the reagents, the anti-human Ig antibodies and the human anti-microorganism antibodies form complexes. The formation of these complexes increases the number of antibody prongs that are able to bind to the microorganism antigen. In other words, each anti-human Ig antibody is able to bind two human anti-microorganism antibodies (at their Fc fragments); these two human anti-microorganism antibodies each split into two prongs when they are not complexed, and become four-pronged or multi-pronged antibodies upon complexing with the anti-human Ig antibodies. Thus, these complexes are more effective at cross-linking to the microorganism antigen in an antigen bridging immunoassay.

[0024] Any calibration or quality control reagent matrix known in the art or otherwise contemplated herein can be utilized, so long as the reagent produced thereby is able to function according to the present disclosure. In certain non-limiting embodiments, the matrix can have a formulation / constitution / pH somewhat similar to that of the sample that will be measured in the immunoassay. However, in certain non-limiting embodiments, it can be desirable to avoid the use of unpurified serum or plasma-based matrices, as endogenous immunoglobulins present in such matrices can interfere with other components of the calibration or quality control reagent, as well as the complexes formed in the calibration or quality control reagent.

[0025] Non-limiting examples of samples (and, thus, in certain non-limiting embodiments, matrix formulations based thereon) that can be utilized in accordance with the present disclosure include biological samples such as, but not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, lacrimal fluid, mucus, bladder wash fluid, semen, fecal matter, pleural fluid, nasopharyngeal fluid, and combinations thereof.

[0026] In certain non-limiting embodiments, the matrix comprises human serum or plasma that is substantially depleted of human IgG. In certain non-limiting embodiments, the matrix comprises serum from a non-human source such as, but not limited to, horse, cow, goat, and the like.

[0027] In other non-limiting embodiments, the matrix comprises a water-based system that is free of other components (i.e., substantially devoid of buffers or proteins). In other non-limiting embodiments, the matrix comprises a buffer-based system alone or in combination with one or more proteins.

[0028] Non-limiting examples of buffers that can be utilized in accordance with the present disclosure include phosphate (such as, but not limited to, in a phosphate buffered saline (PBS) solution or Dulbecco's phosphate-buffered saline), Tris (such as, but not limited to, a Tris-buffered saline solution), Bis-Tris, borate (such as, but not limited to, a borate-buffered saline solution), carbonate, citrate, glycine, ACES, BES, DIPSO, HEPES, HEPPSO, MOPS, MOPSO, PIPES, PIPPS, TAPSO, TES, biobuffer, and the like, as well as any combination thereof.

[0029] Non-limiting examples of proteins that can be utilized in accordance with the present disclosure as part of the matrix include albumin (such as, but not limited to, bovine serum albumin (BSA)), casein, non-human immunoglobulin, transferrin, and the like, as well as any combination thereof.

[0030] Further, as is well known in the art, the matrix can contain additional components. For example (but not by way of limitation), the matrix can include one or more preservatives, one or more bulking agents, and / or one or more polymers. Non-limiting examples of bulking agents that can be utilized in accordance with the present disclosure include lactose, sorbitol, trehalose, sucrose, mannitol, glycine, and the like, as well as any combination thereof. Non-limiting examples of preservatives that can be utilized in accordance with the present disclosure include ProClin™ (Rohm and Haas, Philadelphia, PA), sodium azide, thiomersal, neomycin, clotrimazole, amphotericin B, MIT, streptomycin, OMADINE® (sodium pyrithione; Lonza, Basel, Switzerland), COSMOCIL® CQ (Lonza, Basel, Switzerland), and the like, as well as any combination thereof. Non-limiting examples of polymers that can be utilized in accordance with the present disclosure include polyethylene glycol (PEG), dextran, polyvinylpyrrolidone (PVP), and the like, as well as any combination thereof.

[0031] In a specific (but non-limiting) embodiment, the matrix comprises bovine serum albumin (BSA) in PBS. One non-limiting example of a matrix is 6% protease-free BSA in PBS, pH 7.4.

[0032] Serological immunoassays utilizing the calibration or quality control reagents of the present disclosure can detect human antibodies to antigens of any microorganism desired to be detected. For example (but not by way of limitation), the microorganism being detected can be a bacterium, a virus, a protozoan, a fungus, and the like.

[0033] Non-limiting examples of bacteria that can be detected in accordance with the present disclosure include Acinetobacter spp. Acinetobacter ), Actinomyces spp. Actinomyces ), Aeromonas spp. Aeromonas ), Aggregatibacter spp. Aggregatibacter ), Allobaculum spp. Atopobium ), Bacillus spp. Bacillus ), Bacteroides spp. Bacteroides ), Bartonella spp. Bartonella ), Bifidobacterium spp. Bifidobacterium ), Borrelia spp. Borellia ), Brucella spp. Brucella ), Campylobacter spp. Campylobacter ), Chlamydia spp. Chlamydia ), Chlamydophila spp. Chlamydophila ), Clostridium spp. Clostridium ), Corynebacterium spp. Corynebacterium ), Coxiella spp. Coxiella ), Eikenella spp. EikenellaEnterobacter spp., Enterococcus spp., Escherichia spp., Eubacterium spp., Francisella spp., Fusobacterium spp., Gardnerella spp., Haemophilus spp., Helicobacter spp., Klebsiella spp., Lactobacillus spp., Listeria spp., Mobiluncus spp., Moraxella spp., Mycobacterium spp., Mycoplasma spp., Neisseria spp., Parvimonas spp., Pasteurella spp., Porphyromonas spp., Prevotella spp., Propionibacterium spp., Proteus spp., Pseudomonas spp., Rickettsia spp., Salmonella spp., Serratia spp., Shigella spp., Staphylococcus spp., Streptococcus spp., Tannerella spp., Treponema spp., Vibrio spp., and Yersinia spp. Enterobacter Enterococcus Escherichia Eubacterium Francisella Fusobacterium Gardnerella Haemophilis Helicobacter Klebsiella Lactobacillus Listeria Mobiluncus Moraxella Mycobacterium Mycoplasma Neisseria Parviomonas Pasteurella Porphyromonas Prevotella Propionibacterium Proteus Pseudomonas Rickettsia Salmonella Serratia Shigella Staphylococcus Streptococcus Tannerella Treponema Vibrio Yersinia

[0034] Non-limiting examples of viruses that can be detected in accordance with the present disclosure include adenovirus, astrovirus, coronavirus, coxsackievirus, cytomegalovirus (CMV), echovirus, encephalitis virus, enterovirus, Epstein-Barr virus (EBV), erythrovirus, hantavirus, hepatitis virus, herpesvirus, human immunodeficiency virus (HIV), influenza virus, norovirus, papillomavirus, parainfluenza virus, paramyxovirus, poliovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rotavirus, rubella virus, rubeola virus, varicella-zoster virus, West Nile virus, and Zika virus, among others.

[0035] ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Non-limiting examples of protozoa that can be detected in accordance with the present disclosure include Ascaris, Babesia, Cryptosporidium ( Cryptosporidium ), Cyclospora ( Cyclospora ), Entamoeba ( Entamoeba ), Enterobius ( Enterobius ), Giardia ( Giardia ), Hymenolepis ( Hymenolepis ), Nectonema ( Necator ), Plasmodium ( Plasmodium ), Strongyloides ( Strongyloides ), Taenia ( Taenia ), Toxoplasma ( Toxoplasma ), and Trichomonas ( Trichomonas ) species, among others.

[0036] Non-limiting examples of fungi that can be detected in accordance with the present disclosure include yeasts, molds, and the like, including (but not limited to) Candida ( Candida ), Cryptococcus ( Cryptococcus ), Epidermophyton ( Epidermophyton ), Malassezia ( Malassezia ), Microsporum ( Microsporum ), Trichophyton ( Trichophyton ) species, among others.

[0037] In particular (but non-limiting) embodiments, the microorganism detected by immunoassay is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), HIV, Hepatitis B total core, Epstein-Barr virus, Herpes virus (HSV), CMV, Rubella virus, Helicobacter pylori ( H. pylori ), or Toxoplasma gondii ( Toxoplasma gondii ).

[0038] The antigen can be any antigen from the microorganism to be detected. Antigens useful for detection of each of the microorganisms listed above are well known in the art and widely available. Moreover, the selection of antigens that can be utilized in accordance with the present disclosure is within the ability of one of ordinary skill in the art. Accordingly, it is not deemed necessary to further disclose the same.

[0039] Human antibodies to these antigens are also widely known and commercially available. In addition, antibody manufacturers produce extremely large arrays of thousands of human anti-microbial antibodies that can be utilized in accordance with the present disclosure; see, e.g., but not limited to, antibody catalogs of manufacturers such as Roche (Basel, Switzerland), Genentech (San Francisco, CA), Aridis Pharmaceuticals LLC (San Jose, CA), MedImmune, LLC (Gaithersberg, MD), XBiotech (Austin, TX), Rockland Immunochemicals, Inc. (Pottstown, PA), US Biological 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 only a small sampling of antibody manufacturers that produce human anti-microbial antibodies, and there are many additional commercial sources of human anti-microbial antibodies that can be utilized in accordance with the present disclosure. Thus, one of ordinary skill in the art will be able to clearly and unambiguously identify and select various human anti-microbial antibodies that can be utilized in accordance with the present disclosure, and it is therefore not deemed necessary to further describe the human anti-microbial antibodies or their properties.

[0040] In a specific (but non-limiting) embodiment, the human anti-microbial antibody is a humanized anti-SARS-CoV-2 antibody, such as but not limited to, a humanized S1 antibody from GenScript (Piscataway, NJ) that is an anti-RBD, and the antigen to which the human antibody is directed is an antigen of SARS-CoV-2, such as but not limited to, at least a portion of the receptor binding domain (RBD) of the S1 subunit of the CoV spike protein.

[0041] The anti-human Ig antibodies can specifically bind to any portion of any human immunoglobulin molecule known in the art or otherwise contemplated herein. For example, but not by way of limitation, the antibodies can be directed against human IgG, IgE, IgM, IgD, and / or IgA, and / or any portion thereof (such as, but not limited to, anti-human gamma chain, anti-human H+L, anti-human light chain, etc.). Anti-human Ig antibodies are well known in the art, are widely commercially available, and have been extensively studied. For example, but not by way of limitation, some commercial sources of anti-human IgG monoclonal and / or polyclonal antibodies include Rockland Immunochemicals, Inc. (Pottstown, PA), US Biological 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 antibodies that can be utilized in accordance with the present disclosure. Thus, one of ordinary skill in the art will be able to readily and unambiguously identify and select various anti-human Ig antibodies that can be utilized in accordance with the present disclosure, and it is therefore not deemed necessary to further describe the anti-human Ig antibodies or their properties.

[0042] Each of the anti-human immunoglobulin (Ig) antibodies and the human antibodies against microbial antigens can be present in the calibrators or quality control reagents in any concentration that allows for the formation of complexes as described herein and that allows the reagents to function according to the present disclosure. In certain non-limiting embodiments, each of the anti-human immunoglobulin (Ig) antibodies and the human antibodies against microbial antigens are present in the reagents in a concentration independently selected from about 0.001 wt%, about 0.002 wt%, about 0.003 wt%, about 0.004 wt%, about 0.005 wt%, about 0.006 wt%, about 0.007 wt%, about 0.008 wt%, about 0.009 wt%, about 0.01 wt%, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0.07 wt%, about 0.08 wt%, about 0.09 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1.0 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about 1.5 wt%, about 1.6 wt%, about 1.7 wt%, about 1.8 wt%, about 1.9 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, or higher, or a range formed by two of the above values (i.e., a range from about 0.001 wt% to about 10 wt%, a range from about 0.002 wt% to about 1 wt%, a range from about 0.001 wt% to about 0.5 wt%, etc.), or any value falling within a range of two of the above values (i.e., about 0.27 wt%).

[0043] Likewise, the human antibodies directed to microbial antigens and the anti-human immunoglobulin (Ig) antibodies can be present in the reagent in any ratio to each other that allows the reagent to function according to the present disclosure. For example (but not by way of limitation), the anti-human Ig antibodies and the human anti-microbial antibodies can be present in a ratio ranging from about 1 : 1, about 1 : 1.5, about 1 :2, about 1 :2.5, about 1 :3, about 1 :3.5, about 1 :4, about 1 :4.5, about 1 :5, about 1 :5.5, about 1 :6, about 1 :6.5, about 1 :7, about 1 :7.5, about 1 :8, about 1 :8.5, about 1 :9, about 1 :9.5, about 1 : 10, about 2:3, about 2:5, about 2:7, about 2:9, about 2: 11, about 2: 13, about 2: 15, about 2: 17, about 2: 19, or higher, or a range formed by two of the above values (i.e., a range from about 1 : 1 to about 1 : 10, a range from about 1 :2 to about 2:3, etc.), or any value falling within a range of two of the above values (i.e., about 1 : 1.7, about 1 :2.4, etc.). The ratio of each reagent can be optimized, when desired, to achieve a good balance between stability and signal. The ratio can also vary widely, depending on the particular reagents used.

[0044] Similarly, any additional components (i.e., one or more proteins, preservatives, fillers, and / or polymers) present in the matrix can each be present at any concentration that allows the reagent formed therefrom to function according to the present disclosure. For example (but not by way of limitation), each additional component is present at a concentration independently selected from about 0.001 wt%, about 0.002 wt%, about 0.003 wt%, about 0.004 wt%, about 0.005 wt%, about 0.006 wt%, about 0.007 wt%, about 0.008 wt%, about 0.009 wt%, about 0.01 wt%, about 0.02 wt%, about 0.03 wt%, about 0.04 wt%, about 0.05 wt%, about 0.06 wt%, about 0.07 wt%, about 0.08 wt%, about 0.09 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1.0 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about 1.5 wt%, about 1.6 wt%, about 1.7 wt%, about 1.8 wt%, about 1.9 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, or higher, or a range formed by two of the above values (i.e., a range from about 0.001 wt% to about 20 wt%, a range from about 0.1 wt% to about 5 wt%, a range from about 2 wt% to about 20 wt%, etc.), or any value falling within a range of two of the above values (i.e., about 13 wt%, about 23 wt%, etc.).

[0045] The calibration or quality control reagents of the present disclosure are substantially stable at a desired temperature for a desired period of time. For example, but not by way of limitation, the calibration or quality control reagents can be substantially stable for at least about 7 days, at least about 14 days, at least about 28 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 13 months, at least about 14 months, at least about 15 months, at least about 16 months, at least about 17 months, at least about 18 months, at least about 2 years, at least about 3 years, or more. In addition, the calibration reagents can be stored at room temperature (i.e., in the range of about 18 °C to about 20 °C) or stored under refrigerated or frozen conditions. When a preservative is present in the calibration or quality control reagents, the preservative utilized can be selected based on the desired specific storage conditions and storage period.

[0046] The calibration or quality control reagents of the present disclosure can be provided in any form that allows the reagents to function according to the present disclosure. For example, but not by way of limitation, the reagents can be provided in a liquid form. Alternatively, the reagents can be freeze-dried or lyophilized and provided in the form of a dry reagent.

[0047] The calibration or quality control reagents of the present disclosure can be used for qualitative or quantitative measurements.

[0048] Certain non-limiting embodiments of the present disclosure relate to kits containing any of the calibration or quality control reagents described herein or otherwise contemplated.

[0049] In certain non-limiting embodiments, the kits can further include at least one reagent for use in a serological immunoassay. For example, but not by way of limitation, the kits can further include at least one reagent containing a microbial antigen to which a human anti-microbial antibody binds.

[0050] In particular (but not limiting) embodiments, the microbe is SARS-CoV-2 and the antigen is the RBD of SARS-CoV-2 Spike SI. This antigen can be obtained from any source known in the art. For example (but not limiting), 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).

[0051] In particular (but not limiting) embodiments, the kit can further comprise one or more reagents for performing the immunoassay in a particular assay format, such as (but not limited to) the LOCI® format. For example (but not limiting), the kit can further comprise a composition comprising a singlet oxygen-activatable chemiluminescent compound (such as (but not limited to) a chemibead) and a composition comprising a sensitizing agent (such as (but not limited to) a sensibead), each having a microbe antigen directly or indirectly attached thereto (or the kit contains reagents for directly or indirectly attaching the compositions to a microbe antigen prior to or during the immunoassay).

[0052] In a specific (but not limiting) embodiment, the kit can further comprise: (i) a composition comprising: a singlet oxygen-activatable chemiluminescent compound having bound directly or indirectly thereto an antigen specifically bound by a human anti-microbial antibody of the agent, and a fluorescent molecule excited by the activated chemiluminescent compound; and (ii) a composition comprising a sensitizing agent capable of producing singlet oxygen in its excited state, and having bound directly or indirectly thereto an antigen specifically bound by a human anti-microbial antibody of the agent. In another specific (but not limiting) embodiment, the kit can further comprise: (i) a composition comprising: a singlet oxygen-activatable chemiluminescent compound having bound directly or indirectly thereto an antigen specifically bound by a human anti-microbial antibody of the agent, and a fluorescent molecule excited by the activated chemiluminescent compound; (ii) a composition comprising a sensitizing agent capable of producing singlet oxygen in its excited state, and having bound directly or indirectly thereto a biotin-specific binding partner; and (iii) an antigen specifically bound by a human anti-microbial antibody of the agent, wherein the antigen is biotinylated.

[0053] Chemiluminescent compounds (chemiluminescent agents) are compounds that are chemically activatable and emit light of a particular wavelength as a result of such activation. By way of illustration and not limitation, examples of chemiluminescent agents include, for example: olefins capable of reacting with singlet oxygen or peroxides to form hydroperoxides or dioxetanes that can decompose into ketones or carboxylic acid derivatives; stable dioxetanes that can decompose by the action of light; alkynes that can react with singlet oxygen to form diketones; hydrazones or hydrazides that can form azo compounds or azo carbonyl compounds, such as, but not limited to, luminol; and aromatic compounds that can form endoperoxides. As a result of the activation reaction, the chemiluminescent agent directly or indirectly causes luminescence.

[0054] In certain embodiments, the singlet oxygen-activatable chemiluminescent compound can be a species that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate that can decompose and simultaneously or subsequently emit light. The composition comprising the chemiluminescent compound can be directly excited by the activated chemiluminescent compound; alternatively, the composition can further comprise at least one fluorescent molecule that is excited by the activated chemiluminescent compound.

[0055] Sensitizers are molecules, typically compounds, that generate a reactive intermediate (such as, for example, singlet oxygen) for activation of a chemiluminescent compound. In some non-limiting embodiments, the sensitizers are photosensitizers. By way of example and not limitation, other sensitizers that can be activated chemically (by, for example, enzymes and metal salts) include other substances and compositions that can generate singlet oxygen with or without activation by an external light source. For example, certain compounds have been shown to catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Non-limiting examples of other sensitizer substances and compositions include oxides of the alkaline earth metals Ca, Sr, and Ba; d 0 derivatives of the elements of Groups 3A, 4A, SA, and 6A in the Periodic Table of the Elements; oxides of the actinide and lanthanide series of elements; and oxidizing agents CI - , BrO - , Au 3+ , IO3 - , and IO4 - ; and specifically, molybdate, peroxymolybdate, tungstate, and peroxotungstate ions, and acetonitrile. The following references, which are expressly incorporated by reference herein in their entirety, provide further disclosure regarding sensitizer substances and compositions that also fall within the scope of the present disclosure: Aubry, J. Am. Chem. Soc. , 107:5844-5849 (1985); Aubry, J. Org. Chem ., 54:726-728 (1989); Böhme and Brauer, Inorg. Chem., 31:3468-3471 (1992); Niu and Foote, Inorg. Chem., 31:3472-3476 (1992); Nardello et al., Inorg. Chem., 34:4950-4957 (1995); Aubry and Bouttemy, J. Am. Chem. Soc., 119:5286-5294 (1997); and Almeida et al., Anal. Chim. Acta , 482:99-104 (2003); the entire contents of each of which are expressly incorporated herein by reference.

[0056] Also included within the scope of photosensitizers are compounds that are not true sensitizers, but will release singlet oxygen molecules upon excitation by heat, light, ionizing radiation, or chemical activation. Members of such compounds include, by way of example and not limitation, endoperoxides such as 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide; 9,10-diphenylanthracene-9,10-endoperoxide; and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating these compounds or direct absorption of light by these compounds releases singlet oxygen.

[0057] Photosensitizers are sensitizing agents used to activate photoactive compounds by, for example, generating singlet oxygen upon excitation with light. Photosensitizers are photoactivatable and include, for example, dyes and aromatic compounds, and are generally compounds composed of covalently bonded atoms, often with multiple conjugated double or triple bonds. The compounds should absorb light in the wavelength range of about 200 nm to about 1,100 nm, such as, but not limited to, in the range of about 300 nm to about 1,000 nm or in the range of about 450 nm to 950 nm, at the excitation wavelength, with an extinction coefficient at the maximum absorbance greater than 500 M -1 cm -1 , or greater than 5,000 M - 1 cm -1 , or greater than 50,000 M -1 cm -1 . Photosensitizers should be relatively photostable and can not react efficiently with singlet oxygen. By way of illustration and not limitation, examples of photosensitizers include: acetone; benzophenone; 9-thioxanthone; eosin; 9,10-dibromoanthracene; methylene blue; metalloporphyrins such as, but not limited to, hemin; phthalocyanines; chlorophyll; rose Bengal; and Buckminsterfullerene; and derivatives of these compounds.

[0058] Specific, non-limiting examples of chemiluminescent compounds and photosensitizers that can be utilized in accordance with the present disclosure are set forth in U.S. Patent No. 5,340,716 (Ullman, et al.), the entire contents of which are expressly incorporated herein by reference.

[0059] Any biotin-specific binding partner known in the art or otherwise contemplated herein can be utilized in accordance with the present disclosure. In certain non-limiting embodiments, the biotin-specific binding partner is an antibody directed against biotin. In other non-limiting embodiments, the biotin-specific binding partner is avidin or an analog thereof.

[0060] According to the present disclosure, any avidin analog known in the art or otherwise contemplated herein can be utilized, so long as the avidin or avidin analog is: (1) capable of associating with the sensitizing agent; (2) capable of binding the biotinylated analyte-specific binding partner; and (3) capable of binding biotin that can be present in the sample. Non-limiting examples of avidin analogs that can be utilized according to the present disclosure include those disclosed in Kang et al. (J Drug Target (1995) 3: 159-65), the entirety of which is expressly incorporated herein by reference. Specific non-limiting examples of avidin analogs include avidin, streptavidin, traptavidin, neutravidin, Neutralite avidin, Neutravidin, Lite-avidin, succinylated avidin, other forms of modified or genetically engineered avidin, esters, salts, and / or derivatives of any of the foregoing, etc.

[0061] Any fluorescent molecule known in the art that can be excited and emit light at a particular, detectable wavelength upon activation of the chemiluminescent compound can be utilized as the fluorescent molecule(s) of (a) and (b) (and (e), if present) according to the present disclosure, so long as the signal generated by each fluorescent molecule is detectable differently from the signal generated by the other fluorescent molecule(s) utilized. That is, the fluorescent molecule(s) of (a) must emit light at a wavelength sufficiently different from the wavelength at which the fluorescent molecule(s) of (b) emit light, such that the two signals can be distinguished from one another when detected simultaneously. In one specific (but non-limiting) example, each fluorescent molecule utilized according to the present disclosure is independently selected from terbium, uranium, samarium, europium, gadolinium, and dysprosium. For example (but non-limitingly), terbium emits light at a wavelength of about 545 nm, uranium emits light at a wavelength of about 612 nm, and samarium emits light at a wavelength of about 645 nm.

[0062] The assay components / reagents present in the kit can be provided in any form that allows them to function according to the present disclosure. For example, but non-limitingly, each reagent can be provided in liquid form and disposed within the kit in bulk and / or single aliquot form. Alternatively, in one specific (but non-limiting) embodiment, one or more of the reagents can be disposed in the kit in the form of a single aliquot lyophilized reagent. The use of dry reagents in microfluidic devices is described in detail in U.S. Patent No. 9,244,085 (Samproni), the entirety of which is expressly incorporated herein by reference.

[0063] In addition to the assay components / reagents described in detail above, the kits can further contain other reagents used in performing any particular assay described or otherwise contemplated herein. The nature of these additional reagents will depend on the particular assay format, and their identification is well within the skill of those of ordinary skill in the art; thus, further description thereof is not believed to be necessary. Moreover, the components / reagents present in the kits can 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 kits can include microfluidic devices in which the components / reagents are disposed.

[0064] The relative amounts of the various components / reagents in the kits can vary widely to provide concentrations of components / reagents that substantially optimize the reactions that need to occur during the assay methods, and further substantially optimize the sensitivity of the assays. Where appropriate, one or more of the components / reagents in the kits can be provided as a dry powder, such as a lyophilized powder, and the kits can further include excipients for reconstituting the dry reagents; in this manner, reagent solutions having the appropriate concentrations for performing methods or assays according to the present disclosure can be obtained from these components. Non-limiting examples of other reagents that can be included in the kits include wash solutions, dilution solutions, excipients, interference solutions, positive controls, negative controls, and the like. Moreover, the kits can further include a set of written instructions that explain how to use the kits. Kits of this nature can be used in any of the methods described or otherwise contemplated herein.

[0065] Certain non-limiting embodiments of the present disclosure relate to microfluidic devices that contain any of the calibration or quality control reagents described or otherwise contemplated herein. For example, but not by way of limitation, certain additional non-limiting embodiments of the present disclosure relate to microfluidic devices that include components of any of the kits described above.

[0066] In particular, certain non-limiting embodiments include microfluidic devices for detecting antibodies to a microorganism in a sample via a serological immunoassay described or otherwise contemplated herein. The microfluidic devices include: (i) an inlet channel through which a sample is applied; and (ii) at least one first compartment that is capable of fluid communication with the inlet channel. The compartment of (ii) contains any of the calibration or quality control reagents described or otherwise contemplated herein, 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 a serological immunoassay. For example, but not by way of limitation, the one or more reagents can include LOCI® immunoassay reagents described above or otherwise contemplated herein.

[0067] In certain non-limiting embodiments, all of the elements of (ii) (and any additional elements as described above) are present in the same compartment. In alternative non-limiting embodiments, the elements are separated between two or more compartments.

[0068] The microfluidic device can provide any arrangement of compartments and distribution of various components therebetween that allows the device to function according to the present disclosure.

[0069] Any compartment of the microfluidic device can be sealed to maintain reagents disposed therein in a substantially air-tight environment until they are used; for example, a compartment containing lyophilized reagents can be sealed to prevent any inadvertent reconstitution of the reagents. An inlet channel and a compartment, and two compartments can be described as "capable of being in fluid communication with each other"; this phrase indicates that each of the compartments described can still be sealed, but that the two compartments are capable of having fluid flow therebetween upon piercing the seal formed therein or therebetween.

[0070] The microfluidic device of the present disclosure can be provided with any other desirable feature known in the art or otherwise contemplated herein. For example, and without limitation, the microfluidic device of the present disclosure can further comprise a read chamber; the read chamber can be any compartment containing a reagent described above, or the read chamber can be in fluid communication with the compartment. The microfluidic device can further comprise one or more additional compartments containing other solutions, such as (but not limited to) wash solutions, dilution solutions, excipients, interference solutions, positive controls, negative controls, and the like. These additional compartments can be in fluid communication with one or more of the other compartments. For example, the microfluidic device can further comprise one or more compartments containing wash solutions, and these compartments can be capable of being in fluid communication with any other compartment of the device. In another example, the microfluidic device can further comprise one or more compartments containing excipients for dissolving one or more dry reagents, and the compartments can be capable of being in fluid communication with any other compartment of the device. In yet another further example, the microfluidic device can comprise one or more compartments containing dilution solutions, and the compartments can be capable of being in fluid communication with any other compartment of the device.

[0071] Certain non-limiting embodiments of the present disclosure relate to a method of producing any calibration or quality control reagent for a serological immunoassay for antibodies to a microorganism in a human biological sample, as described or otherwise contemplated herein. In the method, human antibodies to a microorganism antigen are combined with anti-human Ig antibodies in a matrix, and incubated under conditions in which the anti-human Ig antibodies form a complex with one or more human antibodies to a microorganism antigen.

[0072] The incubation step can be performed at any temperature and for any time period that allows for the formation of one or more complexes described herein. For example, but not by way of limitation, the incubation step can be performed at a temperature ranging from about 10 °C to about 50 °C, such as, but not limited to, a range from about 18 °C to about 40 °C, and specifically (but not by way of limitation) about 37 °C, and for a time period of about 30 seconds, about 60 seconds, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours or more, and ranges formed by any two of the above values (i.e., a range from about 1 minute to about 5 hours, a range from about 30 minutes to about 2 hours, etc.), or any value falling between two of the above values (i.e., about 53 minutes, about 15 hours, etc.).

[0073] Further, the method of producing any of the calibration or quality control reagents can contain one or more additional steps to form the reagents described herein.

[0074] Certain non-limiting embodiments of the present disclosure relate to methods of calibrating serological immunoassays for antibodies to microorganisms in human biological samples. In the methods, an immunoassay is performed using any of the calibration or quality control reagents described herein or otherwise contemplated.

[0075] Any human anti-microbial antibody capable of being detected via an assay format described herein or otherwise contemplated can be detected by the immunoassays of the present disclosure. In particular (but non-limiting) embodiments, the antibody is an anti-viral antigen antibody to the SARS-Covid2 virus.

[0076] In some non-limiting assay embodiments, a signal production system (sps) member is utilized that includes a sensitizing agent, such as, for example, a photosensitizer, and a chemiluminescence- fluorescence molecular composition, and each of which has a microbial antigen directly or indirectly attached thereto (or is capable of having an antigen directly or indirectly attached thereto during the immunoassay); in these assay embodiments, activation of the sensitizing agent results in activation of the chemiluminescence composition, thereby generating a detectable signal that is related to the amount of bound human anti-microbial antibody that is detected. One exemplary (but non-limiting) embodiment of an assay platform that can be the basis of the present disclosure is the Luminescence Oxygen Channeling Assay (LOCI®; Siemens Healthcare Diagnostics Inc., Tarrytown, NY). The LOCI® assay is described, for example, in U.S. Patent No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference.

[0077] In particular (but non-limiting) assay embodiments, the assay is performed by incubating the calibration or quality control reagent with two or more reagents used in the LOCI® format. For example (but not by way of limitation), the calibration or quality control reagent can be incubated with: a singlet oxygen-activatable chemiluminescence compound (such as, but not limited to, a chemibead) and a composition comprising a sensitizing agent (such as, but not limited to, a sensibead), each of which has a microbial antigen directly or indirectly bound thereto. In one particular (but non-limiting) embodiment, the calibration or quality control reagent is combined, simultaneously or partially or wholly sequentially, with: (i) a composition comprising a singlet oxygen-activatable chemiluminescence compound having directly or indirectly bound thereto an antigen that is specifically bound by a human anti-microbial antibody of the reagent, and a fluorescence molecule that is excited by the activated chemiluminescence compound; and (ii) a composition comprising a sensitizing agent that is capable of generating singlet oxygen in its excited state, and having directly or indirectly bound thereto an antigen that is specifically bound by a human anti-microbial antibody of the reagent. In another particular (but non-limiting) embodiment, the calibration or quality control reagent is combined, simultaneously or partially or wholly sequentially, with: (i) a composition comprising a singlet oxygen-activatable chemiluminescence compound having directly or indirectly bound thereto an antigen that is specifically bound by a human anti-microbial antibody of the reagent, and a fluorescence molecule that is excited by the activated chemiluminescence compound; (ii) a composition comprising a sensitizing agent that is capable of generating singlet oxygen in its excited state, and having directly or indirectly bound thereto a biotin-specific binding partner; and (iii) a biotinylated antigen that is specifically bound by a human anti-microbial antibody of the reagent.

[0078] In a second step, the components are incubated together to allow the chemiluminescent compound-containing composition and the sensitizer-containing composition to bind to the human anti-microbial antibody present in the complex of the calibration or quality control reagent, thereby resulting in the formation of a complex / aggregate in which the sensitizer is brought into close proximity with the chemiluminescent compound.

[0079] In a third step, the sensitizer is activated to generate singlet oxygen, wherein the activation of the sensitizer present in the complex / aggregate results in the activation of the chemiluminescent compound present in each complex / aggregate.

[0080] In a fourth step, the amount of chemiluminescence generated by the chemiluminescent compound activated in the complex / aggregate is determined by measuring the amount of light emitted by the fluorescent molecule to determine the amount of human anti-microbial antibody in the calibration or quality control reagent.

[0081] Any of the microbial antigens, singlet oxygen-activatable chemiluminescent compounds, sensitizers, fluorescent molecules, and biotin or analogs thereof described in detail above or otherwise contemplated herein can be utilized in the methods of the present disclosure.

[0082] For example, in certain specific (but non-limiting) embodiments, the singlet oxygen-activatable chemiluminescent compound is a species that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose and simultaneously or subsequently emit light.

[0083] In specific (but non-limiting) embodiments, the sensitizer is a photosensitizer, and the activation of the sensitizer in step (3) comprises irradiation with light (such as, but not limited to, irradiation at about 680 nm).

[0084] As mentioned above, the various components of the method are provided in combination (simultaneously or sequentially). When the various components of the method are added sequentially, the order of addition of the components can be varied; the particular desired order of addition of the different components to the assay can be determined by one of ordinary skill in the art. The simplest order of addition is, of course, to add all of the species simultaneously and to assay the signal produced thereby. Alternatively, each component, or group of components, can be combined sequentially. In certain embodiments, an incubation step can be included following one or more additions.

[0085] While specific embodiments of the present disclosure are described as having the LOCI® assay format, it should be understood that the present disclosure also relates to any other immunoassay format (and kits, microfluidic devices, and methods of practicing the same) capable of detecting human antibodies to microbial antigens. Thus, the disclosure of this specific assay format is for purposes of example only and should not be construed as limiting the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0086] Figure 1 A CV2T LOCI® immunoassay format using a calibration reagent comprising anti-viral antigen antibody is schematically depicted.

[0087] Figure 2 A CV2T LOCI® immunoassay format using a calibration reagent constructed according to the present disclosure is schematically depicted. DETAILED DESCRIPTION

[0088] Examples are provided below. However, it is to be understood that this application is not limited to the particular experimental, results and laboratory procedures disclosed herein. Rather, the examples are provided merely as an exemplification of various embodiments and are not meant to be exhaustive or to limit the scope of the application.

[0089] In this example, anti-human IgG antibody is used for anti-viral antigen (AVAg) antibody in the complexed calibrator base matrix. The complexed AVAg antibody, which when uncomplexed splits into two arms, becomes a four-armed or multi-armed antibody upon complexing with the anti-human IgG antibody. Such complexes, which contain two or more AVAg antibodies complexed with anti-human Ig antibody, are able to cross-link viral antigen more efficiently in an antigen bridging assay.

[0090] For example, as shown in Figure 1 In a CV2T LOCI® assay format, a calibrator containing only AVAg antibody is bound to chemibeads and sensibeads in a 1 : 1 ratio. Specifically, anti-FITC chemibeads prepped with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein S1 subunit labeled with fluorescein and streptavidin-coated sensibeads that bind to the RBD of biotinylated S1 are mixed with a calibrator containing AVAg antibody. To generate a signal, one arm of the AVAg antibody must bind to the chemibead and the other arm of the same AVAg antibody must bind to the sensibead. Upon incubation of the chemibeads and sensibeads with the calibrator, the mixture is excited at 680 nm, which causes the sensibeads to release singlet oxygen. The close proximity of the chemibeads and sensibeads (by binding to the same antibody) causes the singlet oxygen to be transferred to the chemibeads, and this initiates a chemical reaction that results in the chemibeads emitting light that can be measured at 612 nm.

[0091] In contrast, as shown in Figure 2As shown in FIG. 2, the complexes of AVAg antibodies bound to anti-human IgG antibodies are formed using the calibrators generated according to the present disclosure. These complexes present in the calibrators can contain two AVAg antibodies bound to each anti-human IgG antibody, thereby providing reagents within the calibrators that contain at least four tines, where each tine can bind to an anti-viral antigen (i.e., in the case of the CV2T assay, the RBD of S1). When this calibrator is incubated with chemibeads and sensibeads from the CV2T LOCI® assay, each complex containing four tines can bind up to four antigens, each present on a chemibead or sensibead, and thereby allow the formation of aggregates containing two or more chemibeads and / or two or more sensibeads. The formation of these aggregates greatly increases the chance of sensibeads being in close enough proximity to chemibeads for the time of exposure to the excitation wavelength, and thereby exponentially increases the emitted light from the various chemibeads present in the aggregates upon exposure to the excitation wavelength. Thus, the signal generated by the calibrator reagents of the present disclosure can produce a signal that is about 20 to about 80 times stronger (or more) than the signal generated by the calibrator reagents of the prior art. Figure 1 The signal generated by the calibrator utilized in FIG. 2 is about 20 to about 80 times stronger (or more) than the signal generated by the calibrator utilized in FIG. 1.

[0092] The calibrator was generated as follows: BSA was chosen as the calibrator matrix due to the abundance of endogenous antibodies in human serum / plasma (although other materials / samples that do not contain human serum and plasma can also be used). A humanized chimeric anti-RBD Ig antibody (GenScript, Piscataway, NJ) was mixed with one of two different anti-human monoclonal antibodies: 4H5 (Siemens Healthcare Diagnostics, Tarrytown, NY) and 2F3 (Thermo Fisher Scientific, Waltham, MA) in a PBS (pH 7.4) buffer at 6% protease-free BSA as the base matrix, where the anti-human Ig antibody to AVAg antibody ratio was about 1 :2. The mixture was incubated at about 37 °C for at least one hour prior to storage.

[0093] Monoclonal antibody 4H5 was generated by immunizing mice with human IgG, and this monoclonal antibody specifically binds to the constant domain of the IgG heavy chain. For production, a hybridoma cell line was grown in cell culture, and the culture supernatant was purified using protein A affinity chromatography. This monoclonal antibody has shown specificity in exclusive binding to human IgG.

[0094] In a specific sample amplification study, the calibrator reagents generated above were used to contain Figure 1 and Figure 2in the chemibead and sensibead CV2T LOCI® assays. As shown in Table 1, in this viral antigen bridging immunoassay, each of the two calibrator reagents, which contain complexes formed by multi-armed AVAg antibodies, enhanced the assay signal by about 20 to 25 fold compared to the assay signal seen when the calibrator reagents contained non-complexed AVAg antibodies.

[0095] Table 1

[0096] With this high level of signal enhancement, the amount of anti-viral antigen antibody required to produce the calibrator or quality control reagents is greatly reduced, thereby resulting in at least a 10 to 20 fold cost savings.

[0097] Thus, in accordance with the present disclosure, compositions, kits and devices and methods for their production and use have been provided which fully satisfy the objects and advantages hereinbefore described. Although the present disclosure has been described in conjunction with the specific figures, experiments, results, and language above, it is clear 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

1. A calibration or quality control reagent for use in a serological immunoassay for detecting antibodies to a microorganism in a human biological sample, the reagent comprising: (a) a matrix; (b) an anti-human immunoglobulin (Ig) antibody; and (c) a human antibody to an antigen of the microorganism; and wherein a complex of (b) and (c) is formed in the reagent.

2. The calibration or quality control reagent of claim 1, wherein the matrix comprises BSA in PBS.

3. The calibration or quality control reagent of claim 1, wherein the matrix comprises at least one additional component selected from the group consisting of a polymer, a preservative, a bulking agent, and combinations thereof.

4. The calibration or quality control reagent of claim 1, wherein the microorganism is a virus.

5. The calibration or quality control reagent of claim 4, wherein the virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

6. The calibration or quality control reagent of claim 5, wherein the antigen of SARS-CoV-2 to which the antibody of (c) is directed is at least a portion of the receptor binding domain (RBD) of the S1 subunit of the CoV spike protein.

7. The calibration or quality control reagent of claim 1, wherein (b) and (c) are present in a ratio ranging from about 1:1 to about 1:

10.

8. The calibration or quality control reagent of claim 1, wherein (b) is present in the reagent at a concentration ranging from about 0.01 wt% to about 10 wt%.

9. The calibration or quality control reagent of claim 1, wherein (c) is present in the reagent at a concentration ranging from about 0.01 wt% to about 10 wt%.

10. A kit comprising: at least one reagent of claim 1.

11. The kit of claim 10, further comprising at least one reagent for the serological immunoassay.

12. The kit of claim 10, further comprising at least one composition comprising an antigen of the microorganism.

13. The kit of claim 12, wherein the antigen comprises at least a portion of the receptor binding domain (RBD) of the S1 subunit of the CoV spike protein.

14. The kit of claim 11, further comprising: (i) a composition comprising: a singlet oxygen-activatable chemiluminescent compound having directly or indirectly bound thereto an antigen specifically bound by the human antibody of (c) of the reagent; and a fluorescent molecule excited by the activated chemiluminescent compound; and (ii) a composition comprising a sensitizing agent capable of producing singlet oxygen in its excited state and having directly or indirectly bound thereto an antigen specifically bound by the human antibody of (c) of the reagent.

15. The kit of claim 11, further comprising: (i) a composition comprising: a singlet oxygen-activatable chemiluminescent compound having directly or indirectly bound thereto an antigen specifically bound by the human antibody of (c) of the reagent; and a fluorescent molecule excited by the activated chemiluminescent compound; ​ ​ (ii) 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 (iii) a biotinylated antigen that specifically binds to the human antibody of (c) of the reagent.

16. A microfluidic device comprising at least one reagent of claim 1.

17. A method of producing a calibration or quality control reagent for a serological immunoassay for antibodies to a microorganism in a human biological sample, the method comprising: incubating human antibodies to an antigen of the microorganism with anti-human Ig antibodies in a matrix, thereby forming a complex between the anti-human Ig antibodies and one or more human antibodies to an antigen of the microorganism.

18. The method of claim 17, wherein the microorganism is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

19. A method of calibrating a serological immunoassay for antibodies to a microorganism in a human biological sample, the method comprising: performing the immunoassay using a calibration or quality control reagent comprising anti-human Ig antibodies complexed with human antibodies to an antigen of the microorganism.

20. The method of claim 19, wherein the microorganism is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

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