Compositions, kits, and methods for antimicrobial serological assays using anti-human immunoglobulin antibodies

Abstract

Reagents, kits, and microfluidic devices for detecting the presence and / or concentration of antibodies to microorganisms in a human biological sample are disclosed. Methods of making and using the reagents, kits, and microfluidic devices are also disclosed. Anti-human immunoglobulin antibodies are utilized as reagents in bridging immunoassays for detecting microorganisms.

Description

[Technical field]

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

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

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

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

[0005] However, due to uncertainties associated with antibody excess (i.e., the hook effect), the need for early detection of viral / bacterial infections, and low anti-viral / bacterial antibody titers in some patient samples, there is a need to reduce the amount of sample required for testing while also enhancing the signal generated by the assay. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, there is a need in the art for new and improved assays for antibodies to microbial antigens that overcome the disadvantages and shortcomings of the prior art.To which the present disclosure is directed is such new and improved reagents, kits, microfluidic devices, and methods for detecting antibodies to microbial antigens. [Brief description of the drawings]

[0007] [Figure 1] Schematic representation of the SARS-CoV-2 Total (COV2T) assay (Siemens Healthineers, Tarrytown, NY). [Diagram 2] 1 shows a schematic diagram of one non-limiting embodiment of a serological assay format constructed in accordance with the present disclosure. [Diagram 3] 1 illustrates generally another non-limiting embodiment of a serological assay format constructed in accordance with the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Before describing at least one embodiment of the present disclosure in detail by exemplary language and results, it should be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components described in the following description. The present disclosure is capable of other embodiments or can be practiced or carried out in various ways. Therefore, the language used in this specification is intended to give the broadest possible scope and meaning; the embodiments are meant to be illustrative, not comprehensive. It should also be understood that the phraseology and terminology used in this specification are for the purpose of description and should not be regarded as limiting.

[0009] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The techniques and procedures described above are generally performed according to conventional methods well known in the art and described in various general and more specific references cited and discussed throughout this specification. The nomenclatures utilized in connection with analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the experimental procedures and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0010] All patents, published patent applications, and non-patent publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any part of this application are expressly incorporated herein by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0011] All of the compositions, kits, devices and / or methods disclosed herein can be made and carried out without undue experimentation in light of this disclosure.Although the compositions, kits, devices and / or methods are described with reference to specific embodiments, it will be clear to those skilled in the art that modifications can be made to the compositions, kits, devices and / or methods, and in the steps or sequence of steps of the methods described herein, without departing from the concept, spirit and scope of this disclosure.All such similar substitutions and modifications that are clear to those skilled in the art are deemed to be within the spirit, scope and concept of this disclosure, as defined by the appended claims.

[0012] As utilized in accordance with the present disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0013] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the term "a" or "an" may mean "one," but it is also consistent with the meanings of "one or more," "at least one," and "one or more than one." Thus, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a "compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "plurality" refers to "two or more."

[0014] Use of the term "at least one" will be understood to include one and any amount greater than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend to 100 or 1000 or more, depending on the term to which it is attached; furthermore, an amount of 100 / 1000 should not be considered limiting, since higher limits may also provide satisfactory results. Furthermore, use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. Use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is for the purpose of distinguishing between two or more items only, and is not intended to imply, for example, any permutation or order or any order of importance or addition of one item to another item.

[0015] Use of the term "or" in the claims is used to mean inclusive "and / or" unless expressly indicated to refer only to alternatives or the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and A and B are both true (or exist).

[0016] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "an example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. For example, the appearances of the phrases "in some embodiments" or "one example" in various places in the specification are not necessarily all referring to the same embodiment. Moreover, any reference to one or more embodiments or examples should not be construed as limiting the scope of the claims.

[0017] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method used to determine the value, or the variation that exists among study subjects. For example, and without limitation, when the term "about" is used, the specified value may vary by plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent from the specified value, such variations being appropriate to perform the disclosed methods and as would be understood by one of ordinary skill in the art.

[0018] As used in the specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0019] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, if order is important in the particular context. Following this example, combinations containing one or more repeats of an item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are also expressly included. Those skilled in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0020] As used herein, the term "substantially" means that the event or circumstance described thereafter occurs completely, or that the event or circumstance described thereafter occurs to a large extent or to a large extent. For example, when referring to a particular event or circumstance, the term "substantially" means that the event or circumstance described thereafter occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are within a range of proximity to each other but are not 100% adjacent to each other, or that a portion of one of the two items is not 100% adjacent to the other item but is within a range of proximity to the other item.

[0021] As used herein, the phrases "associated" and "linked" include both direct association / binding of two moieties to each other and indirect association / binding of two moieties to each other. Non-limiting examples of association / linkage include, for example, covalent binding of one moiety to another moiety by a direct bond or via a spacer group, non-covalent binding of one moiety to another moiety directly or by a specific binding pair member attached to both moieties, incorporation of one moiety into another moiety, such as by dissolving or synthesizing one moiety into another moiety, and coating one moiety onto another moiety.

[0022] The terms "analog" and "derivative" are used interchangeably herein and refer to a substance that contains in its structure the same basic carbon skeleton and carbon functional groups as a given compound, but may also contain one or more substitutions thereon. As used herein, the term "substituted" will be understood to refer to the replacement of at least one substituent on a compound with a residue R. In certain non-limiting embodiments, R may include H, hydroxyl, thiol, a halide selected from fluoride, chloride, bromide or iodide, a C1-C4 compound selected from one of the following: optionally substituted linear, branched or cyclic alkyl, and linear, branched or cyclic alkenyl, where the optional substituents are one or more of alkenyl alkyl, alkynyl alkyl, cycloalkyl, cycloalkenyl alkyl, aryl alkyl, heteroaryl alkyl, heterocyclic alkyl, optionally substituted heterocycloalkenyl alkyl, aryl cycloalkyl, and aryl heterocycloalkyl. and aryl, each of which is optionally substituted, and the optional substituents are selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocyclealkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), carboxy, and -C(O)-alkyl.

[0023] As used herein, the term "sample" will be understood to include any type of biological sample that may be utilized in accordance with the present disclosure. Examples of fluid biological samples that may be utilized include, but are not limited to, whole blood or any fraction thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.

[0024] The term "antibody" is used herein in the broadest sense to refer to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), as well as 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 replacement proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof, that exhibit the desired biological activity of analyte binding. Antibodies may be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0025] As used herein, the term "LOCI®" refers to a commercially used assay technology based on luminescent oxygen channeling assay (LOCI®) technology. LOCI® advanced chemiluminescence assays are described, for example, in U.S. Pat. No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference. Currently available LOCI® technology has high sensitivity and uses several reagents. In particular, the LOCI® assay requires that two of these reagents (called "sensibeads" and "chemibeads") are held by other specific binding partner assay reagents in a manner that the sensibeads and chemibeads are in close proximity to each other to achieve a signal. When exposed to light of a certain wavelength, the sensibeads release singlet oxygen, which is transferred to the chemibeads when the two beads are in close proximity; this triggers a chemical reaction that results in the chemibeads emitting light that can be measured at a different wavelength.

[0026] Referring now to certain non-limiting embodiments of the present disclosure, reagents, kits, and microfluidic devices are disclosed that can be utilized in serological assays to detect antibodies against microorganisms present in human biological samples. Anti-human immunoglobulin (Ig) antibodies are utilized as part of a second reagent in various serological assay formats in combination with a first reagent containing at least one antigen against a microorganism. The anti-microbial antibodies present in the biological sample bind to the antigen of the first assay reagent, while the anti-human Ig antibodies present in the second reagent bind to the anti-microbial antibodies bound to the first assay reagent, thereby forming a detectable complex.

[0027] Bridging immunoassay formats utilizing immobilized and labeled viral antigen reagents are typically used to detect anti-viral antigen antibodies (i.e., IgG, IgM, IgA, etc.) against the SARS-CoV-2 virus in patient serum and plasma. Since one viral antigen needs to be immobilized on a bulky solid phase (such as, but not limited to, latex or magnetic particles), bridging two viral antigen molecules (at least one of which is attached to the solid phase) by a monomeric bivalent antibody (such as IgG) may generate a weak signal due to the slower Brownian motion of the solid phase antigen and the uncertainty of the orientation of the anti-viral antibody to it. As a result, there is a need to enhance the detection signal of the bridging assay, especially for samples with low anti-viral antibody titers. In addition, both binding arms of the patient's anti-viral antigen antibody may bind to multiple antigen molecules immobilized on the solid phase, thereby preventing the detection of the antibody by the labeled antigen reagent.

[0028] Certain non-limiting embodiments of the present disclosure are directed to a kit for performing a serological assay to detect the presence and / or concentration of antibodies to a microorganism in a human biological sample. The kit comprises at least two components: (a) a composition comprising at least one antigen of the microorganism bound directly or indirectly to the composition; and (b) a composition comprising an anti-human immunoglobulin antibody bound directly or indirectly to the composition, at least one of (a) and (b) further comprising a detectable molecule. The anti-microorganism antibody present in the human biological sample binds to the antigen of (a), while the anti-human immunoglobulin antibody of (b) binds to the anti-microorganism antibody bound to (a), thereby forming a detectable complex.

[0029] Non-limiting examples of samples that can be utilized in accordance with the present disclosure (and therefore, in certain non-limiting embodiments, matrix formulations can be based on) include biological samples such as, but not limited to, whole blood or any fraction thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.

[0030] The serological assays in which the reagents and kits of the present disclosure are utilized can detect human antibodies against antigens of any microorganism that one desires to detect. For example (and not by way of limitation), the microorganisms detected can be bacteria, viruses, protozoa, fungi, etc.

[0031] Non-limiting examples of bacteria that can be detected according to the present disclosure include Acinetobacter, Actinomyces, Aeromonas, Aggregatibacter, Atopobium, Bacillus, Bacteroides, Bartonella, Bifidobacterium, Borrelia, Brucella, and others. la), Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Coxiella, Eikenella, Enterobacter, Enterococcus, Escherichia, Eubacterium, Francisella rancisella, Fusobacterium, Gardnerella, Haemophilis, Helicobacter, Klebsiella, Lactobacillus, Listeria, Mobiluncus, Moraxella, Mycobacterium, Mycoplasma, Neisseria sseria, Parviomonas, Pasteurella, Porphyromonas, Prevotella, Propionibacterium, Proteus, Pseudomonas, Rickettsia, Salmonella, Serratia, Shigella, Staphylococcus,These include Streptococcus, Tannerella, Treponema, Vibrio, and Yersinia species.

[0032] Non-limiting examples of viruses that may be detected according to the present disclosure include adenovirus, astrovirus, coronavirus (such as, but not limited to, severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV)), coxsackievirus, cytomegalovirus (CMV), echovirus, encephalitis virus, enterovirus, Epstein-Barr virus (EBV), erythrovirus, hantavirus, hepatitis virus, herpes virus, human immunodeficiency virus (HIV), influenza virus, norovirus, papillomavirus, parainfluenza virus, paramyxovirus, poliovirus, rabies virus, respiratory syncytial virus (RSV), rhinovirus, rotovirus, rubella virus, measles virus, varicella zoster virus, West Nile virus, and Zika virus.

[0033] Non-limiting examples of protozoa that can be detected according to the present disclosure include Ascaris, Babesia, Cryptosporidium, Cyclospora, Entamoeba, Enterobius, Giardia, Hymenolepis, Necator, Plasmodium, Strongyloides, Taenia, Toxoplasma, and Trichomonas species, and the like.

[0034] 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, Cryptococcus, Epidermophyton, Malassezia, Microsporum, and Trichophyton species.

[0035] In specific (but non-limiting) embodiments, the microorganism detected by the serological assay is severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the virus that causes COVID-19), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), and human coronavirus HKU1 (HCoV-HKU1), HIV, Hepatitis B core total, Epstein-Barr virus, herpes virus (HSV), CMV, rubella, H. pylori, or Toxoplasma gondii.

[0036] The antigen can be any antigen derived from the microorganism to be detected. Antigens useful for detecting each of the microorganisms listed above are well known in the art and are widely available. Moreover, the selection of antigens that can be utilized according to the present disclosure is well within the understanding of those skilled in the art. Therefore, further disclosure thereof is not considered necessary.

[0037] In specific (but non-limiting) embodiments, the microorganism is SARS-CoV-2 and the antigen is any SARS-CoV-2 antigen known in the art or otherwise contemplated herein. For example (but not by way of limitation), the antigen may be derived from the nucleocapsid (N) protein, spike (S) protein, membrane (M) protein, envelope (E) protein, fusion (F) protein, etc. In specific (but non-limiting) embodiments, the antigen may be derived from the nucleocapsid protein or spike protein.

[0038] In one particular (but non-limiting) embodiment, the antigen is the receptor binding domain (RBD) of the S1 subunit of the SARS-CoV-2 spike protein. The RBD S1 antigen can be obtained from any source known in the art. For example (but not by way of limitation), certain antigens are commercially available from GenScript (Piscataway, NJ); Meridian Life Sciences, Inc. (Memphis, TN); Sino Biological US Inc. (Wayne, PA); ACRO Biosystems (Newark, DE); Biorbyt, LLC (St. Louis, MO); Icosagen, AS (San Francisco, CA); and Bios Pacific Inc. (Emeryville, CA).

[0039] The anti-human Ig antibody may specifically bind to any portion of any human immunoglobulin molecule known in the art or otherwise contemplated herein. For example (and not by way of limitation), the antibody may be directed to human IgG, IgE, IgM, IgD, and / or IgA, and / or any portion thereof (including but not limited to anti-human gamma chain, anti-human H+L, anti-human light chain, etc.). In a particular (but non-limiting) embodiment, the anti-human Ig antibody specifically binds to human IgA, human IgG, and human IgM; the use of this antibody makes the assay a total anti-viral antigen-antibody assay (i.e., a total SARS-CoV-2 antibody assay).

[0040] Anti-human Ig antibodies (including but not limited to anti-human IgG, anti-human IgM, and / or anti-human IgA antibodies, and antibodies that recognize two or all three human immunoglobulin antibodies) are well known in the art, widely available commercially, and have been extensively studied. For example (but not by way of limitation), some commercial sources of anti-human IgG monoclonal and / or polyclonal antibodies include Rockland Immunochemicals, Inc. (Pottstown, PA); USBiological Life Sciences (Swampscott, MA); Santa Cruz Biotechnology, Inc. (Dallas, TX); Jackson Immuno Research Labs, Inc. (West Grove, PA); Thermo Fisher Scientific (Waltham, MA); and Sigma-Aldrich Corp. (St. Louis, MO). However, this list is not comprehensive, and there are many additional commercial sources of anti-human Ig antibodies that can be utilized according to the present disclosure. Thus, further description of anti-human Ig antibodies or their characteristics is not believed to be necessary, as one of ordinary skill in the art can clearly and unambiguously identify and select a variety of anti-human Ig antibodies that can be utilized in accordance with the present disclosure.

[0041] The composition (a) comprising the microbial antigen and the composition (b) comprising the anti-human immunoglobulin antibody may have any physical and / or structural characteristics that allow for detection of the binding of the antibody to the microbial. For example (and not by way of limitation), the composition may take the form of a particle, bead, surface, or substrate. Compositions that can be utilized in serological assays are well known in the art and are commercially available. Similarly, labels that can be utilized in such compositions are well known in the art and are commercially available. Moreover, the selection of a particular composition and label for a particular serological assay format is well within the understanding of one of ordinary skill in the art. Thus, further description thereof is not considered necessary. However, for the purposes of illustration only, several different serological assay formats and particular compositions utilized therewith are provided herein below.

[0042] The kits of the present disclosure are designed for use with any serological assay format known in the art or otherwise described herein.

[0043] The first composition utilized in the serological assay format is typically designed for the isolation and / or detection of the complex formed by the crosslinking of the microbial antibody and the anti-human Ig antibody bound to the microbial antigen. In certain non-limiting embodiments, the first composition comprises an immobilization surface to which at least one microbial antigen is directly or indirectly bound; thus, the first composition provides an immobilization surface on which the complex can form, and thus is isolated from the remainder of the sample and the unbound components of the assay. However, the use of an immobilization surface as part of the first composition is for illustrative purposes only; the first composition may have any physical and / or structural characteristics that allow the isolation and / or detection of the complex formed between the antimicrobial antibody and the first and second compositions. For example (but not by way of limitation), the first composition may take the form of particles, beads, immobilized or non-immobilized surfaces or substrates, etc.

[0044] In another specific (but non-limiting) embodiment of the serological assay format, the kit can further include one or more reagents for a chemiluminescent detection system, such as (but not limited to) the Luminescent Oxygen Channeling Assay (LOCI®) format. In this specific (but non-limiting) example, the kit includes a composition comprising a chemiluminescent compound activatable by singlet oxygen (such as (but not limited to) Chemibeads) and a composition comprising a sensitizer (such as (but not limited to) Sensibeads), one of the reagents having a microbial antigen bound directly or indirectly thereto and the other reagent having an anti-human Ig antibody bound directly or indirectly thereto. In an alternative embodiment, the kit contains reagents for directly or indirectly attaching the composition to the microbial antigen and / or anti-human Ig antibody either before or during the assay.

[0045] In a specific, but non-limiting, embodiment, the first composition of the kit is further defined as comprising a singlet oxygen activatable chemiluminescent compound having at least one antigen of a microorganism bound directly or indirectly thereto. The kit further contains a second composition comprising a sensitizer capable of generating singlet oxygen in its excited state and at least one anti-human Ig antibody bound directly or indirectly to the sensitizer.

[0046] When the kit contains a second composition comprising a sensitizer and an anti-human Ig antibody, the second composition does not have to be placed in a kit that already has an anti-human Ig antibody bound to the sensitizer. That is, rather than containing a single second composition containing both the sensitizer and the anti-human Ig antibody, the kit may instead contain two reagents: (i) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner that is directly or indirectly bound to the sensitizer; and (ii) at least one anti-human immunoglobulin antibody that is biotinylated.

[0047] A chemiluminescent compound (chemiluminescer) is a compound that can be chemically activated and, as a result of such activation, emits light at a certain wavelength. For illustrative purposes and without limitation, examples of chemiluminescent materials include: olefins that can react with singlet oxygen or peroxides to form hydroperoxides or dioxetanes, which can decompose to ketones or carboxylic acid derivatives; stable dioxetanes that can decompose under the action of light; acetylenes that can react with singlet oxygen to form diketones; hydrazones or hydrazides that can form azo compounds or azocarbonyls, such as (but are not limited to) luminol; and aromatic compounds that can form endoperoxides, for example. As a result of the activation reaction, chemiluminescent materials directly or indirectly cause light emission.

[0048] In certain embodiments, a singlet oxygen activatable chemiluminescent compound can be a substance that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose with simultaneous or subsequent emission of light. A composition comprising a chemiluminescent compound can be directly excited by the activated chemiluminescent compound; alternatively, the composition can further comprise at least one fluorescent molecule that is excited by the activated chemiluminescent compound.

[0049] A sensitizer is a molecule, usually a compound, that generates a reactive intermediate, such as, for example, singlet oxygen, for activation of a chemiluminescent compound. In some non-limiting embodiments, the sensitizer is a photosensitizer. Other sensitizers that are chemically activated (e.g., by enzymes and metal salts) include, by way of example and not limitation, other substances and compositions that can generate singlet oxygen with or without activation by an external light source. For example, certain compounds have been shown to catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Non-limiting examples of other sensitizer substances and compositions include: oxides of the alkaline earth metals Ca, Sr, and Ba; d 0Derivatives of elements of groups 3A, 4A, SA, and 6A of the configuration; oxides of actinides and lanthanides; and the oxidizing agent ClO - ,BrO - , Au 3+ , IO3 - and IO4 - and in particular, molybdate, peroxomolybdate, tungstate and peroxotungstate ions, and acetonitrile. The following references, expressly incorporated herein by reference in their entireties, provide further disclosure regarding sensitizer materials and compositions that are also within the scope of this disclosure: Aubry, J. Am. Chem. Soc., 107:5844-5849 (1985); Aubry, J. Org. Chem., 54:726-728 (1989); Bohme and Brauer, Inorg. Chem., 31:3468-3471 (1992); Niu and Foo ... Bohme and Brauer, Inorg. Chem., 31:3468-3471 (1992); Niu and Foo, J. Am. Chem. Soc., 107:5844-5849 (1985); Bohme and Brauer, Inorg. Chem., 31:3468-3471 (1992); Niu and Foo, J. Am. Chem. Soc., 107:5844-5849 (1985); Bohme and Brauer, Inorg. Chem., 31:3468-3471 (1992); Niu and Foo, J. Am. Chem. Soc., 107:5844 te, Inorg. Chem., 31:3472-3476 (1992); Nardello et al., Inorg. Chem., 34:4950-4957 (1995); Aubry and Bouttemy, J. Am. Chem. Soc., 119:5286-5294 (1997); and Almeida et al., Anal. Chim. Acta, 482:99-104 (2003); the entire contents of each of which are expressly incorporated herein by reference.

[0050] Also included within the scope of photosensitizers are compounds that are not true sensitizers, but which release a molecule of singlet oxygen upon excitation by heat, light, ionizing radiation, or chemical activation. Members of this class of compounds include, for example (but are not limited to), endoperoxides such as 1,4-biscarboxyethyl-1,4-naphthalene endoperoxide; 9,10-diphenylanthracene-9,10-endoperoxide; and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light by these compounds releases singlet oxygen.

[0051] A photosensitizer is a sensitizer for activating a photoactive compound, for example by the generation of singlet oxygen upon excitation with light. Photosensitizers are photoactivatable compounds, typically consisting of covalently bonded atoms, including, for example, dyes and aromatic compounds, typically having multiple conjugated double or triple bonds. The compounds are photoactivatable compounds that are 500 M or less at the excitation wavelength. -1 cm -1 Greater than or equal to 5,000M -1 cm -1 Greater than or equal to 50,000M -1 cm -1 It should absorb light in the wavelength range of about 200 nm to about 1,100 nm, such as (but not limited to) about 300 nm to about 1,000 nm or about 450 nm to 950 nm, with an extinction coefficient at its absorption maximum greater than 1,100 nm. The photosensitizer should be relatively photostable and may not react efficiently with singlet oxygen. By way of illustration and not limitation, examples of photosensitizers include: acetone; benzophenone; 9-thioxanthone; eosin; 9,10-dibromoanthracene; methylene blue; metallo-porphyrins such as (but not limited to) hematoporphyrin; phthalocyanines; chlorophyll; rose bengal; and buckminsterfullerene; and derivatives of these compounds.

[0052] In particular, non-limiting examples of chemiluminescent compounds and photosensitizers that can be utilized in accordance with the present disclosure are described in U.S. Pat. No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference.

[0053] Any biotin-specific binding partner known in the art or otherwise contemplated herein can be utilized in accordance with the present disclosure. In certain non-limiting embodiments, the biotin-specific binding partner is an antibody to biotin. In other non-limiting embodiments, the biotin-specific binding partner is avidin or an analog thereof.

[0054] Any avidin analogue known in the art or otherwise contemplated herein can be utilized in accordance with the present disclosure, so long as the avidin or avidin analogue can: (1) associate with a sensitizer; (2) bind to a biotinylated analyte-specific binding partner; and (3) bind to biotin that may be present in a sample. Non-limiting examples of avidin analogues that can be utilized in accordance with the present disclosure include those disclosed in Kang et al. (J Drug Target (1995) 3:159-65), the entire contents of which are expressly incorporated herein by reference. Specific non-limiting examples of avidin analogues include avidin, streptavidin, traptavidin, neutral avidin, Neutralite avidin, Neutravidin, Lite-avidin, succinylated avidin, other forms of avidin that have been modified or engineered, esters, salts and / or derivatives of any of the above, and the like.

[0055] Any fluorescent molecule known in the art that can be excited by an activated chemiluminescent compound and emit light at a specific detectable wavelength can be utilized in accordance with the present disclosure as the (a) and (b) (and (e), if present) fluorescent molecules, so long as the signal generated by each fluorescent molecule is detectable from the signal generated by the other fluorescent molecules utilized. That is, the (a) fluorescent molecule must emit light at a wavelength sufficiently different from that emitted by the (b) fluorescent molecule, so that the two signals are distinguished from each other when detected simultaneously. In one specific (but non-limiting) example, each fluorescent molecule utilized in accordance with the present disclosure is independently selected from the group consisting of terbium, uranium, samarium, europium, gadolinium, and dysprosium. For example (but not by way of limitation), 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.

[0056] It should be noted that the attachment of antigen and anti-human immunoglobulin antibody to the other two components described hereinabove (i.e., singlet oxygen activatable chemiluminescent compound and sensitizer) is optional; in fact, the present disclosure also includes the opposite of the attachment described hereinabove. That is, certain embodiments of the present disclosure include a kit comprising: (a) a composition comprising a singlet oxygen activatable chemiluminescent compound having at least one anti-human immunoglobulin antibody directly or indirectly bound to the chemiluminescent compound; and (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and a microbial antigen directly or indirectly bound to the sensitizer. Alternatively, the kit may comprise the above (a) in combination with (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and a biotin-specific binding partner directly or indirectly bound to the sensitizer, and (c) a microbial antigen that is biotinylated.

[0057] Thus, certain non-limiting embodiments of the kit include: (a) a composition comprising a chemiluminescent compound activatable by singlet oxygen; (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state; (c) at least one anti-human immunoglobulin antibody bound directly or indirectly to the chemiluminescent compound or sensitizer; and (d) a microbial antigen bound directly or indirectly to the chemiluminescent compound or sensitizer to which (c) is not bound. Alternatively, the kit may include (a) a composition comprising a chemiluminescent compound activatable by singlet oxygen; (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and a biotin-specific binding partner bound directly or indirectly to the sensitizer; (c) at least one anti-human immunoglobulin antibody; and (d) a microbial antigen; one of (c) and (d) is bound directly or indirectly to the chemiluminescent compound, while the other of (c) and (d) is biotinylated.

[0058] All of the kits of the present disclosure can be utilized for the qualitative and / or quantitative measurement of antimicrobial antibodies.

[0059] The assay components / reagents present in the kit are provided in any form that allows them to function according to the present disclosure. For example, and not by way of limitation, each of the reagents is provided in liquid form and placed in the kit in bulk and / or in single aliquot form. Alternatively, in a particular (but non-limiting) embodiment, one or more of the reagents are placed in the kit in the form of a single aliquot of lyophilized reagent. The use of dried reagents in microfluidic devices is described in detail in U.S. Pat. No. 9,244,085 (Samproni), the entire contents of which are expressly incorporated herein by reference.

[0060] In addition to the assay components / reagents described in detail herein above, the kit may further contain other reagents for carrying out any of the specific assays described herein or otherwise contemplated. The nature of these additional reagents depends on the specific assay format, and their identification is well within the skill of the artisan; therefore, further description thereof is not considered necessary. Also, each of the components / reagents present in the kit may be in a separate container / compartment, or the various components / reagents are combined in one or more containers / compartments, depending on the cross-reactivity and stability of the components / reagents. Furthermore, the kit may include a microfluidic device in which the components / reagents are arranged.

[0061] The relative amounts of the various components / reagents in the kit can be widely varied to provide concentrations of the components / reagents that fully optimize the reactions required to occur during the assay method and further optimize the sensitivity of the assay. Under appropriate circumstances, one or more of the components / reagents in the kit can be provided as a dry powder, such as a lyophilized powder, and the kit can further include an excipient for dissolving the dry reagent; thus, a reagent solution having a suitable concentration for performing a method or assay according to the present disclosure can be obtained from these components. Non-limiting examples of other reagents that can be included in the kit include washing solutions, calibration solutions, quality control solutions, diluent solutions, excipients, interference solutions, positive controls, negative controls, and the like. Additionally, the kit can further include a set of written instructions describing how to use the kit. A kit of this nature can be used in any of the methods described or otherwise contemplated herein.

[0062] Certain non-limiting embodiments of the present disclosure are directed to microfluidic devices containing any of the serological assay reagents described herein or otherwise contemplated. For example (and not by way of limitation), certain further non-limiting embodiments of the present disclosure are directed to microfluidic devices that include any of the components of the kits described herein above.

[0063] In particular, certain non-limiting embodiments include a microfluidic device for detecting the presence and / or concentration of antibodies to a microorganism in a biological sample via a serological assay as described herein or otherwise contemplated. The microfluidic device includes (i) an inlet channel to which a biological sample is applied; and (ii) at least a first compartment that can be in fluid communication with the inlet channel. The (ii) compartment contains any of the reagents described herein or otherwise contemplated, either alone or in combination with one or more other reagents described herein or otherwise contemplated (such as, but not limited to, one or more reagents for use in a serological assay). For example (but not by way of limitation), the one or more reagents include a first and / or second composition (containing a microorganism antigen and an anti-human Ig antibody, respectively) as described herein or otherwise contemplated.

[0064] In certain non-limiting embodiments, all of the reagents in (ii) (and any additional elements described herein above) are present in the same compartment. In alternative non-limiting embodiments, the reagents are divided between two or more compartments.

[0065] A microfluidic device can provide any arrangement of compartments and distribution of various components therebetween that enables the device to function in accordance with the present disclosure.

[0066] Any of the compartments of the microfluidic device are sealed to maintain the reagents disposed therein in a substantially airtight environment until the reagents are used; for example, a compartment containing a lyophilized reagent is sealed to prevent any unintentional reconstitution of the reagent. The inlet channel and the compartment, as well as the two compartments, are described as being "capable of fluid communication" with each other; this phrase indicates that each of the compartments can be sealed at any time, but that the two compartments can allow fluid to flow between them when the seal formed in or between them is broken.

[0067] The microfluidic device of the present disclosure can provide any other desired function known in the art or otherwise contemplated herein. For example, but not by way of limitation, the microfluidic device of the present disclosure can further include a reading chamber; the reading chamber can be any of the compartments containing reagents described herein above, or the reading chamber can be in fluid communication with said compartments.

[0068] The microfluidic device may further include one or more additional compartments containing other solutions such as (but not limited to) washing solutions, calibration solutions, quality control solutions, diluent solutions, excipients, interference solutions, positive controls, negative controls, etc. These additional compartments may be in fluid communication with one or more of the other compartments. For example, the microfluidic device may further include one or more compartments containing washing solutions, which may be in fluid communication with any other compartment of the device. In another example, the microfluidic device may further include one or more compartments containing excipients for dissolving one or more dried reagents, which may be in fluid communication with any other compartment of the device. In yet a further example, the microfluidic device may include one or more compartments containing diluent solutions, which may be in fluid communication with any other compartment of the device.

[0069] Certain non-limiting embodiments of the present disclosure are directed to methods of detecting the presence and / or concentration of antibodies to a microorganism in a human biological sample. In one non-limiting embodiment, the method includes: (1) simultaneously or wholly or partially sequentially combining (a) a human biological sample suspected of containing antibodies to a microorganism, (b) a composition comprising a chemiluminescent compound activatable by singlet oxygen having at least one antigen of the microorganism bound directly or indirectly to the chemiluminescent compound, and (c) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and at least one anti-human immunoglobulin antibody bound directly or indirectly to the sensitizer; (2) allowing binding of (b) and (c) to the antibodies to the microorganism present in (a), wherein binding of (b) and (c) to the antibodies to the microorganism results in the formation of a complex in which the sensitizer is in close proximity to the chemiluminescent compound; (3) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complexes causes activation of the chemiluminescent compound present in each complex; and (4) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the complexes to determine the presence and / or concentration of antibodies to the microorganism present in the sample.

[0070] In another non-limiting embodiment, the method comprises: (1) combining simultaneously or in whole or in part sequentially: (a) a human biological sample suspected of containing antibodies to a microorganism; (b) a composition comprising a singlet oxygen activatable chemiluminescent compound having at least one antigen of the microorganism bound directly or indirectly to the chemiluminescent compound; (c) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and a biotin-specific binding partner bound directly or indirectly to the sensitizer; and (d) at least one anti-human immunoglobulin antibody that is biotinylated; and (2) combining the at least one anti-human immunoglobulin antibody with a human biological sample suspected of containing antibodies to a microorganism; (b) a composition comprising a singlet oxygen activatable chemiluminescent compound having at least one antigen of the microorganism bound directly or indirectly to the chemiluminescent compound; (c) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and a biotin-specific binding partner bound directly or indirectly to the sensitizer; and (d) at least one anti-human immunoglobulin antibody that is biotinylated; (3) allowing binding of (c) to (d) for detecting the presence and / or concentration of antibodies to the microorganism present in (a), wherein binding of (b) and (c) / (d) to the antibodies to the microorganism results in the formation of a complex in which the sensitizer is in close proximity with the chemiluminescent compound; (3) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complexes causes activation of the chemiluminescent compound present in each complex; and (4) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the complexes to determine the presence and / or concentration of antibodies to the microorganism present in the sample.

[0071] Other non-limiting embodiments of the method include: (1) simultaneously or wholly or partially sequentially combining (a) a human biological sample suspected of containing antibodies to a microorganism, (b) a composition comprising a chemiluminescent compound activatable by singlet oxygen having at least one anti-human immunoglobulin antibody directly or indirectly bound to the chemiluminescent compound, and (c) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and at least one antigen of the microorganism directly or indirectly bound to the sensitizer; (2) allowing binding of (b) and (c) to the antibodies to the microorganism present in (a), wherein binding of (b) and (c) to the antibodies to the microorganism results in the formation of a complex in which the sensitizer is in close proximity with the chemiluminescent compound; (3) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complexes causes activation of the chemiluminescent compound present in each complex; and (4) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the complexes to determine the presence and / or concentration of antibodies to the microorganism present in the sample.

[0072] In yet another non-limiting embodiment, the method includes the steps of (1) simultaneously or in whole or in part sequentially combining: (a) a human biological sample suspected of containing antibodies to a microorganism; (b) a composition comprising a singlet oxygen activatable chemiluminescent compound having at least one anti-human immunoglobulin antibody bound directly or indirectly to the chemiluminescent compound; (c) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and a biotin-specific binding partner bound directly or indirectly to the sensitizer; and (d) at least one antigen of the microorganism that has been biotinylated; and (2) forming a mixture of (c) / (d). (3) allowing binding of (c) to (d) for detecting the presence and / or concentration of antibodies to the microorganism present in (a), wherein binding of (b) and (c) / (d) to the antibodies to the microorganism results in the formation of a complex in which the sensitizer is in close proximity with the chemiluminescent compound; (3) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the complexes causes activation of the chemiluminescent compound present in each complex; and (4) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the complexes to determine the presence and / or concentration of antibodies to the microorganism present in the sample.

[0073] Any antimicrobial antibody that allows for detection via the assay formats described herein or otherwise contemplated is detected by the serological assays of the present disclosure. In a specific (but non-limiting) embodiment, the antibody is an anti-viral antigen antibody against the SARS-CoV-2 virus.

[0074] Some non-limiting assay embodiments utilize signal producing system (sps) members including, for example, a sensitizer, such as a photosensitizer, and a chemiluminescent molecular composition, each of which has either a microbial antigen or an anti-human immunoglobulin antibody directly or indirectly attached thereto (or can have an antigen or antibody directly or indirectly bound thereto during the assay); in these assay embodiments, activation of the sensitizer produces a product that activates the chemiluminescent composition, thereby producing a detectable signal related to the amount of bound human anti-microbial antibody that is detected. An exemplary (but non-limiting) embodiment of an assay platform on which the present disclosure can be based is the Luminescence Oxygen Channeling Assay (LOCI®; Siemens Healthcare Diagnostics Inc., Tarrytown, NY). The LOCI® assay is described, for example, in U.S. Pat. No. 5,340,716 (Ullman et al.), the entire contents of which are expressly incorporated herein by reference.

[0075] Any of the microbial antigens described in detail herein above or otherwise contemplated herein, anti-human immunoglobulin antibodies, chemiluminescent compounds activatable by singlet oxygen, sensitizers, fluorescent molecules, and biotin or analogs thereof can be utilized in the methods of the present disclosure.

[0076] For example, in one particular (but non-limiting) embodiment, a singlet oxygen activatable chemiluminescent compound is a substance that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate species that can decompose with simultaneous or subsequent emission of light.

[0077] In one specific (but non-limiting) embodiment, the sensitizer is a photosensitizer, and activation of the sensitizer in step (3) comprises irradiation with light (for example, but not limited to, irradiation at about 680 nm).

[0078] Any sample that is desired to be assayed for the presence of antibodies against microorganisms can be used as a sample according to the method of the present disclosure.Non-limiting examples of samples include, but are not limited to, biological samples such as whole blood or any part thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.Specific non-limiting examples include lysed whole blood cells and lysed red blood cells.In a specific (but non-limiting) example, the sample is from a human.

[0079] As mentioned above, the various components of the method are provided in combination (either simultaneously or sequentially). When the various components of the method are added sequentially, the order of addition of the components may be changed; those skilled in the art can determine the specific desired order of addition of different components to the assay. The simplest order of addition is, of course, to add all materials simultaneously and determine the signal generated therefrom. Alternatively, each of the components, or groups of components, can be combined sequentially. In certain embodiments, one or more steps (such as, but not limited to, one or more incubation steps and / or one or more washing steps) are included after one or more additions.

[0080] In an alternative (but non-limiting) embodiment, step (1) of the method utilizing a chemiluminescent detection system comprises first combining the sample with a composition comprising a biotinylated anti-human immunoglobulin antibody and a sensitizer, and incubating it before adding a composition comprising a chemiluminescent compound activatable by singlet oxygen and a microbial antigen. Alternatively, step (1) of the method may comprise first combining the sample with a composition comprising a chemiluminescent compound activatable by singlet oxygen, and incubating it before adding a composition comprising a sensitizer. In this latter embodiment, the biotinylated anti-human immunoglobulin antibody is added before or after the incubation step. It should be noted that these different orders of addition are also applicable when the antigen is biotinylated and the anti-human immunoglobulin antibody is associated with a chemiluminescent compound.

[0081] Although the above embodiments are described with respect to the detection of antibodies in human samples, it will be understood that the scope of the present disclosure is not limited to use with human samples. Rather, any of the embodiments described hereinabove can be adapted to the detection of antibodies against microorganisms in biological samples from other mammals. Thus, the present disclosure also includes compositions, kits, devices and methods for detecting anti-microbial antibodies in biological samples of non-human mammals by replacing the anti-human immunoglobulin antibodies utilized in all of the above-described embodiments with anti-mammalian immunoglobulin antibodies corresponding to the species from which the biological sample is obtained. EXAMPLES

[0082] Examples are provided herein below.However, it should be understood that the present disclosure is not limited in its application to the specific experiments, results and experimental procedures disclosed herein.Rather, examples are provided merely as one of various embodiments, and are intended to be illustrative, not comprehensive.

[0083] In June 2020, the U.S. Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for a laboratory-based pan-antibody test developed by Siemens Healthineers (Malvern, PA) to detect the presence of SARS-CoV-2 antibodies, including IgM, IgA, and IgG, in the blood. The spike protein on the surface of the SARS-CoV-2 virus allows the virus to enter and infect human cells found in multiple organs and blood vessels. Siemens Healthineers' pan-antibody COV2T assay is shown in Figure 1 and was designed to detect antibodies against the spike protein. It is believed that some of these antibodies neutralize the SARS-CoV-2 virus and therefore block infection. Multiple potential vaccines in development against SARS-CoV-2 include the spike protein among their focal points.

[0084] The assay format of FIG. 1 utilizes two reagents that both contain SARS-CoV-2 antigens: the first reagent contains a preformed anti-FITC chemibead with a fluorescein-labeled receptor binding domain of the S1 subunit of the SARS-CoV-2 spike protein (RBD of S1), and the second reagent contains a streptavidin-coated SensiBead that is bound to the biotin-RBD of S1. Note that the biotinylated RBD of the S1 antigen is produced as a separate reagent from the universal streptavidin-coated SensiBead and can be combined to produce the second assay reagent at any time before or during the assay.

[0085] However, bridging of two viral antigen molecules (i.e., the two RBDs of the S1 antigen shown in Figure 1) by monomeric bivalent antibodies (such as IgG) present in biological samples may generate weak signals due to slower Brownian motion and uncertainty in the orientation of the anti-viral antibodies when attached to the first antigen. Furthermore, considering the presence of multiple antigen molecules on each ChemiBead, it is possible that both binding arms of a patient's anti-RBD antibodies may bind to the surface of a single ChemiBead. As a result, there is a need to provide a mechanism to enhance the detection signal of the bridging assay, especially for samples with low anti-viral antibody titers, and also to detect anti-SARS-CoV-2 antibodies with both binding arms bound to the surface of a single ChemiBead.

[0086] Figure 2 shows one embodiment of an assay format constructed in accordance with the present disclosure that addresses these issues. In this assay format, a biotinylated anti-human immunoglobulin antibody was added to the reaction mixture in place of the biotinylated viral antigen (S1 biotin-RBD) in Figure 1 to measure total anti-RBD antibodies in patient samples. In this example, the antibody utilized was a commercially available affinity-purified goat polyclonal antibody against human IgG / M / A obtained from Thermo Fisher Scientific (Rockford, IL; catalog number A18849).

[0087] This method provides an alternative means of measuring total anti-RBD antibodies (including anti-RBD IgG, IgM, and IgA) in Figure 1 as well as a bridging assay format (i.e., RBD-coated ChemiBeads (CB) and RBD-coated SensiBeads (SB)). The advantage of this approach is that it overcomes the problem of steric hindrance due to anti-RBD antibodies binding to two antigen molecules located on two different beads, and also eliminates the potential problem that anti-RBD antibodies cannot bridge ChemiBeads and SensiBeads if both binding arms of the patient's anti-RBD antibody can bind to the ChemiBeads surface.

[0088] Table 1 contains data demonstrating the detection of anti-SARS-CoV-2 antibodies in COVID-19 positive samples using the assay format of the present disclosure (labeled "Biotin anti-human AMG" in Table 1, as shown in Figure 2), compared to the detection of anti-SARS-CoV-2 antibodies in COVID-19 positive samples using the assay format of Figure 1 (labeled "Biotin-RBD of S1" in Table 1).

[0089] [Table 1]

[0090] Please note that the term "anti-human AMG" as used in Table 1 refers to an anti-human antibody that specifically binds to all of IgA, IgM, and IgG, and therefore provides detection of total anti-SARS-CoV2 antibodies in COVID-19 patient samples (where "total" refers to the sum of IgG, IgM, and IgA).

[0091] As can be seen in Table 1, positive results for anti-SARS-CoV-2 antibodies were detected in 9 of the 15 COVID-19 positive samples tested using the assay format of Figure 1. In contrast, positive results for anti-SARS-CoV-2 antibodies were detected in all 15 COVID-19 positive samples tested using the assay format of the present disclosure (shown in Figure 2). Additionally, both assay formats provided negative results for all 7 COVID-19 negative samples tested.

[0092] Thus, the present disclosure provides compositions, kits, microfluidic devices, and methods that are effective in detecting antibodies against microorganisms in biological samples. In particular (but not by way of limitation), this example demonstrates that the compositions, kits, microfluidic devices, and methods of the present disclosure are highly effective in detecting anti-SARS-CoV-2 antibodies in biological samples.

[0093] As discussed in detail hereinabove, it should be noted that the attachment of antigen and anti-human immunoglobulin antibody to the other two components of FIG. 2 (i.e., Chemibeads and Sensibeads) is optional; in fact, the present disclosure also includes the opposite of the attachment described hereinabove. For example, FIG. 3 shows another non-limiting embodiment of an assay format constructed according to the present disclosure. Except for the two compounds bound to Chemibeads and Sensibeads being swapped, the assay format of FIG. 3 is identical to the assay format of FIG. 2. That is, the assay format of FIG. 3 utilizes Chemibeads having at least one anti-human immunoglobulin antibody bound directly or indirectly to the Chemibeads, and Sensibeads having an antigen of a microorganism bound directly or indirectly to a sensitizer (wherein the Sensibeads can have a biotin-specific binding partner bound directly or indirectly to it, and the antigen is biotinylated for subsequent attachment to Sensibeads).

[0094] Thus, in accordance with the present disclosure, compositions, kits, and devices, as well as methods for making and using them, are provided that fully meet the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth hereinabove, it is apparent that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that are within the spirit and broad scope of the present disclosure.

Claims

1. 1. A kit for carrying out a serological assay to detect the presence and / or concentration of antibodies to a microorganism in a human biological sample utilizing a chemiluminescence detection system, comprising: (a) a composition comprising a singlet oxygen activatable chemiluminescent compound having a microbial antigen directly or indirectly bound to the chemiluminescent compound; (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and at least one anti-human immunoglobulin antibody bound directly or indirectly to the sensitizer; The microorganism is a virus, The virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), At least one anti-human immunoglobulin antibody specifically binds to all of human IgA, human IgG, and human IgM; The kit.

2. 1. A kit for carrying out a serological assay to detect the presence and / or concentration of antibodies to a microorganism in a human biological sample utilizing a chemiluminescence detection system, comprising: (a) a composition comprising a singlet oxygen activatable chemiluminescent compound having a microbial antigen directly or indirectly bound to the chemiluminescent compound; (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and a biotin-specific binding partner bound directly or indirectly to the sensitizer; (c) at least one anti-human immunoglobulin antibody, which is biotinylated; The microorganism is a virus, The virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At least one anti-human immunoglobulin antibody specifically binds to all of human IgA, human IgG, and human IgM; The kit.

3. For detecting the presence and / or concentration of antibodies to microorganisms in a human biological sample 1. A microfluidic device comprising: (i) an inlet channel to which a human biological sample is applied; (ii) capable of fluid communication with the inlet channel; (a) a composition comprising a singlet oxygen activatable chemiluminescent compound having a microbial antigen directly or indirectly bound to the chemiluminescent compound; (b) at least one composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and at least one anti-human immunoglobulin antibody capable of binding directly or indirectly to the sensitizer; and at least one first section including The microorganism is a virus The virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) at least one anti-human immunoglobulin antibody specifically binds to all of human IgA, human IgG, and human IgM; The microfluidic device.

4. (b) further comprising: A composition comprising a sensitizer capable of generating singlet oxygen in its excited state and a biotin-specific binding partner that is directly or indirectly bound to the sensitizer; and At least one anti-human immunoglobulin antibody, which is biotinylated. The microfluidic device of claim 3 , wherein:

5. The microfluidic device of claim 3 , wherein (a) and (b) are in the same compartment.

6. The microfluidic device of claim 3 , wherein (a) and (b) are in two different compartments.

7. 1. A method for detecting the presence and / or concentration of antibodies to a microorganism in a human biological sample, comprising: （１） (a) a human biological sample suspected of containing antibodies to a microorganism; (b) a composition comprising a singlet oxygen activatable chemiluminescent compound having at least one antigen of a microorganism bound directly or indirectly to the chemiluminescent compound; and (c) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and at least one anti-human immunoglobulin antibody bound directly or indirectly to the sensitizer. simultaneously or sequentially; (2) allowing (b) and (c) to bind to antibodies to the microorganism present in (a), wherein the binding of (b) and (c) to the antibodies to the microorganism results in the formation of a complex in which the sensitizer is in close proximity to the chemiluminescent compound; (3) activating a sensitizer to generate singlet oxygen, where activation of the sensitizer present in the conjugates causes activation of a chemiluminescent compound present in each conjugate; and (4) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the complex to determine the presence and / or concentration of antibodies to the microorganism present in the sample; The microorganism is a virus The virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (c) at least one anti-human immunoglobulin antibody specifically binds to all of human IgA, human IgG, and human IgM; The method.

8. 8. The method of claim 7, wherein the human biological sample is selected from the group consisting of whole blood or any fraction thereof, urine, saliva, sputum, cerebrospinal fluid, skin, intestinal fluid, intraperitoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.

9. 1. A method for detecting the presence and / or concentration of antibodies to a microorganism in a human biological sample, comprising: （１） (a) a human biological sample suspected of containing antibodies to a microorganism; (b) a composition comprising a singlet oxygen activatable chemiluminescent compound having at least one antigen of a microorganism bound directly or indirectly to the chemiluminescent compound; (c) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and a biotin-specific binding partner that is directly or indirectly bound to the sensitizer; and (d) at least one anti-human immunoglobulin antibody, which is biotinylated; simultaneously or sequentially; (2) allowing binding of (c) to (d) to form (c) and (d) and binding of (b) and (c) and (d) to antibodies to the microorganism present in (a), wherein the binding of (b) and (c) and (d) to the antibodies to the microorganism results in the formation of a complex in which the sensitizer is in close proximity to the chemiluminescent compound; (3) activating a sensitizer to generate singlet oxygen, where activation of the sensitizer present in the conjugates causes activation of a chemiluminescent compound present in each conjugate; and (4) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the complex to determine the presence and / or concentration of antibodies to the microorganism present in the sample; The microorganism is a virus The virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (c) at least one anti-human immunoglobulin antibody specifically binds to all of human IgA, human IgG, and human IgM; The method.

10. 10. The method of claim 9, wherein the human biological sample is selected from the group consisting of whole blood or any fraction thereof, urine, saliva, sputum, cerebrospinal fluid, skin, intestinal fluid, peritoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.

11. 1. A kit for carrying out a serological assay to detect the presence and / or concentration of antibodies to a microorganism in a human biological sample utilizing a chemiluminescence detection system, comprising: (a) a composition comprising a singlet oxygen activatable chemiluminescent compound having at least one anti-human immunoglobulin antibody bound directly or indirectly to the chemiluminescent compound; (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and a microbial antigen bound directly or indirectly to the sensitizer; The microorganism is a virus The virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), The at least one anti-human immunoglobulin antibody is selected from the group consisting of human IgA, human IgG, and human Binds specifically to all IgMs. The kit.

12. 1. A kit for carrying out a serological assay to detect the presence and / or concentration of antibodies to a microorganism in a human biological sample utilizing a chemiluminescence detection system, comprising: (a) a composition comprising a singlet oxygen activatable chemiluminescent compound having at least one anti-human immunoglobulin antibody bound directly or indirectly to the chemiluminescent compound; (b) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and a biotin-specific binding partner bound directly or indirectly to the sensitizer; (c) a microbial antigen, which is biotinylated; The microorganism is a virus The virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), At least one anti-human immunoglobulin antibody specifically binds to all of human IgA, human IgG, and human IgM; The kit.

13. 1. A microfluidic device for detecting the presence and / or concentration of antibodies to a microorganism in a human biological sample, comprising: (i) an inlet channel to which a human biological sample is applied; (ii) capable of fluid communication with the inlet channel; (a) a composition comprising a singlet oxygen activatable chemiluminescent compound having at least one anti-human immunoglobulin antibody bound directly or indirectly to the chemiluminescent compound; (b) at least one composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and a microbial antigen capable of binding directly or indirectly to the sensitizer; and at least one first section including The microorganism is a virus The virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (a) at least one anti-human immunoglobulin antibody specifically binds to all of human IgA, human IgG, and human IgM; The microfluidic device.

14. (b) further comprising: A composition comprising a sensitizer capable of generating singlet oxygen in its excited state and a biotin-specific binding partner that is directly or indirectly bound to the sensitizer; and Biotinylated microbial antigens The microfluidic device of claim 13 , wherein the microfluidic device is defined as:

15. The microfluidic device of claim 13 , wherein (a) and (b) are in the same compartment.

16. The microfluidic device of claim 13 , wherein (a) and (b) are in two different compartments.

17. 1. A method for detecting the presence and / or concentration of antibodies to a microorganism in a human biological sample, comprising: （１） (a) a human biological sample suspected of containing antibodies to a microorganism; (b) a composition comprising a singlet oxygen activatable chemiluminescent compound having at least one anti-human immunoglobulin antibody directly or indirectly bound to the chemiluminescent compound; and (c) combining, simultaneously or sequentially, a sensitizer capable of generating singlet oxygen in its excited state and a composition comprising at least one antigen of a microorganism bound directly or indirectly to the sensitizer; (2) allowing (b) and (c) to bind to antibodies to the microorganism present in (a), wherein the binding of (b) and (c) to the antibodies to the microorganism results in the formation of a complex in which the sensitizer is in close proximity to the chemiluminescent compound; (3) activating a sensitizer to generate singlet oxygen, where activation of the sensitizer present in the conjugates causes activation of a chemiluminescent compound present in each conjugate; and (4) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the complex to determine the presence and / or concentration of antibodies to the microorganism present in the sample; The microorganism is a virus The virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) at least one anti-human immunoglobulin antibody specifically binds to all of human IgA, human IgG, and human IgM; The method.

18. 18. The method of claim 17, wherein the human biological sample is selected from the group consisting of whole blood or any fraction thereof, urine, saliva, sputum, cerebrospinal fluid, skin, intestinal fluid, intraperitoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.

19. 1. A method for detecting the presence and / or concentration of antibodies to a microorganism in a human biological sample, comprising: （１） (a) a human biological sample suspected of containing antibodies to a microorganism; (b) a composition comprising a singlet oxygen activatable chemiluminescent compound having at least one anti-human immunoglobulin antibody bound directly or indirectly to the chemiluminescent compound; (c) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state, and a biotin-specific binding partner that is directly or indirectly bound to the sensitizer; and (d) at least one antigen of the microorganism, which is biotinylated; simultaneously or sequentially; (2) allowing (c) to bind to (d) to form (c) and (d) and (b) and (c) and (d) to bind to antibodies against the microorganism present in (a), the binding of (b) and (c) and (d) to an antibody against the microorganism results in the formation of a complex in which the sensitizer is in close proximity to the chemiluminescent compound; and (3) activating a sensitizer to generate singlet oxygen, where activation of the sensitizer present in the conjugates causes activation of a chemiluminescent compound present in each conjugate; and (4) determining the amount of chemiluminescence produced by the activated chemiluminescent compound in the complex to determine the presence and / or concentration of antibodies to the microorganism present in the sample; The microorganism is a virus The virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), (b) at least one anti-human immunoglobulin antibody specifically binds to all of human IgA, human IgG, and human IgM; The method.

20. 20. The method of claim 19, wherein the human biological sample is selected from the group consisting of whole blood or any fraction thereof, urine, saliva, sputum, cerebrospinal fluid, skin, intestinal fluid, peritoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.

Images