Calibration and quality control reagents for immunoassays for antibodies

Abstract

Disclosed are calibration and / or quality control reagents for serological immunoassays for antibodies to microorganisms. In the reagents, antibodies specific for a microorganism antigen are complexed with anti-human Ig antibodies to form a complex. Also disclosed are kits and microfluidic devices containing the reagents, and methods of producing and using the reagents.

Description

[0001] Declaration of cross-reference / inclusion of related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 015,223, filed April 24, 2020, pursuant to 35 USC § 119(e). The entire contents of the above-cited patent application are hereby expressly incorporated herein by reference.

[0003] Statement regarding federally funded research or development

[0004] not applicable.

[0005] background

[0006] In June 2020, the U.S. Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for a laboratory-based total antibody test developed by Siemens Healthineers (Tarrytown, NY) to detect the presence of SARS-CoV-2 antibodies, including IgM and IgG in the blood. The spike protein on the surface of the SARS-CoV-2 virus enables the virus to invade and infect human cells found in various organs and blood vessels. Siemens Healthineers' total antibody COV2T assay is designed to detect antibodies against the spike protein. It is believed that these antibodies neutralize the SARS-CoV-2 virus, thereby preventing infection. Several potential vaccines developed against SARS-CoV-2 have incorporated the spike protein into their focus.

[0007] Just as new and improved COVID-19 immunoassays are needed, there is a corresponding demand for calibrators of these assays to ensure their accuracy. Specifically, there is a need for antiviral antigens / antibodies as calibrators for immunoassays, such as (but not limited to) COVID-19 immunoassays. Due to the COVID-19 pandemic and the enormous demand for these antibodies, they have become extremely expensive and difficult to obtain in the required quantities. The demand for COVID-19 immunoassay calibrators is projected to reach millions of vials per year, costing millions of dollars per gram. Therefore, the cost of such raw materials would be astronomical, and supply is also problematic. Thus, there is an urgent need for methods to minimize the actual antiviral antigens / antibodies used in calibrator products. Brief description of the attached diagram

[0009] Figure 1 The CV2T LOCI® immunoassay is schematically depicted using a calibration reagent containing antiviral antigen antibodies.

[0010] Figure 2The CV2T LOCI® immunoassay is schematically depicted using calibration reagents constructed according to this disclosure.

[0011] Detailed description

[0012] Before explaining in detail at least one embodiment of this disclosure by way of exemplary language and results, it should be understood that the application of this disclosure is not limited to the details of the construction and arrangement of the components set forth in the following description. This disclosure can have other embodiments, or can be practiced or implemented in various ways. Therefore, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary, not exhaustive. Moreover, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered limiting.

[0013] Unless otherwise defined herein, the scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. The foregoing techniques and procedures are generally practiced according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. The nomenclature and laboratory procedures and techniques used in conjunction with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0014] All patents, published patent applications, and non-patent publications mentioned in this specification indicate the level of skill of a person skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference in their entirety, to the extent that each individual patent or publication is specifically and individually indicated to be incorporated by reference.

[0015] In view of this disclosure, all the compositions, kits, devices, and / or methods disclosed herein can be prepared and performed without excessive experimentation. Although the compositions, kits, devices, and / or methods have been described in particular embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions, kits, devices, and / or methods, as well as 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 considered obvious to those skilled in the art are within the spirit, scope, and concept of this disclosure as defined by the appended claims.

[0016] As used in accordance with this disclosure, unless otherwise instructed, the following terms shall be understood to have the following meanings:

[0017] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the terms "a" or "an" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Therefore, the terms "a," "an," and "the" include plural indicators unless the context clearly indicates otherwise. Thus, for example, a 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 "multiple" means "two or more."

[0018] The term "at least one / type" will be understood to include one / type and any quantity of more than one / type, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100 / type, etc. The term "at least one / type" may be extended to 100 or 1000 or more / types, depending on the term it is attached to; furthermore, a quantity of 100 / 1000 is not considered limiting, as higher limits can also produce satisfactory results. Furthermore, the term "at least one / type of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of distinguishing two or more items and does not imply any order or sequence of importance of one item relative to another, or any order of addition.

[0019] The term "or" is used in claims to mean inclusive "and / or" unless explicitly stated to refer only to alternatives or unless the alternatives are mutually exclusive. For example, any of the following satisfy the condition "A or B": 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 both A and B are true (or exist).

[0020] As used herein, any reference to “one embodiment,” “an embodiment,” “some embodiments,” “one example,” “for example,” or “an example” means that a specific element, feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. For example, the phrases “some embodiments” or “one example” appearing in various places in the specification do not necessarily all refer to the same embodiment. Furthermore, all references to one or more embodiments or examples should be construed as non-limiting to the claims.

[0021] Throughout this application, the term "about" is used to indicate that the value includes inherent variations in the error of the composition / instrument / device used to determine the value, or variations present among study subjects. For example, but not limitingly, when using the term "about," the specified value may differ from the listed values ​​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 applicable to performing the disclosed methods and are understood by one of ordinary skill in the art.

[0022] As used in this specification and one or more claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, 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 unlisted elements or method steps.

[0023] As used herein, the term "or a combination thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or a combination thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is significant in the particular context. Continuing with this example, explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that there is generally no limit to the number of items or terms in any combination unless it is apparent from the context.

[0024] As used herein, the term "substantially" means that the subsequently described event or situation occurs completely or extensively. For example, when relating to a specific event or situation, the term "substantially" means that the subsequently described event or situation 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 closely adjacent to each other but not 100% adjacent, or that a part of one of the two items is not 100% adjacent to the other item but is closely adjacent to it.

[0025] As used herein, the phrases “associated with” and “coupled to” include both direct association / binding of two parts to each other and indirect association / binding of two parts to each other. Non-limiting examples of association / coupling include, for example, covalently binding one part to another by a direct bond or by a spacer group; non-covalently binding one part to another by a specific binding pair member that binds to said part; incorporating one part into another by dissolving one part in another or by synthesis; and coating one part onto another.

[0026] The terms “analog” and “derivative” are used interchangeably herein and refer to substances that contain the same basic carbon skeleton and carbon functionality as a given compound in their structure, but may also contain one or more substitutions thereof. As used herein, the term “substitution” will be understood to mean replacing at least one substituent on a compound with residue R. In some non-limiting embodiments, R may include H, hydroxyl, thiol, a halide selected from fluorides, chlorides, bromides, or iodides, and a C1-C4 compound selected from: optionally substituted straight-chain, branched, or cyclic alkyl groups, and straight-chain branched or cyclic alkenyl groups, wherein the optional substituents are selected from one or more alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylhexacycloalkyl groups. The group, wherein each is optionally substituted, wherein the optional substituent is selected from one or more of the following: alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl and arylheterocycloalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl)2, carboxyl and -C(O))-alkyl.

[0027] As used herein, the term "sample" will be understood to include any type of biological sample that may be utilized under this disclosure. Examples of available fluid biological samples include, but are not limited to: whole blood or any part thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.

[0028] The term "antibody" is used in the broadest sense herein and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments and conjugates (such as, but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, biantibodies, single-chain antibodies, and other antibody fragments and conjugates that retain at least a portion of the variable region of the intact antibody), antibody substitute proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. The antibody may be any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0029] As used herein, the term "LOCI®" refers to a commercially available immunoassay technique based on luminescent oxygen channel assay (LOCI®). The LOCI® advanced chemiluminescence 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. Currently available LOCI® techniques are highly sensitive and utilize several reagents. Specifically, the LOCI® assay requires two of these reagents (referred to as "sensibead" and "chemibead") to be held together by other specifically binding pair assay reagents in such a manner that the sensibead and chemibead are brought into close proximity to each other to achieve a signal. Upon exposure to light of a specific wavelength, the sensibead releases singlet oxygen, and if the two beads are brought into close proximity, the singlet oxygen is transferred to the chemibead; this triggers a chemical reaction that causes the chemibead to emit light, which can be measured at different wavelengths.

[0030] Certain non-limiting embodiments of this disclosure relate to calibration or quality control reagents for serological immunoassays used to detect antibodies against microorganisms in human biological samples. The reagents comprise three components: (a) a matrix; (b) anti-human immunoglobulin (Ig) antibodies; and (c) human antibodies against microbial antigens. In the reagents, the anti-human Ig antibodies and human antimicrobial antibodies form complexes. The formation of these complexes increases the number of antibody prongs capable of binding to microbial antigens. In other words, each anti-human Ig antibody is capable of binding two human antimicrobial antibodies (at their Fc fragment); these two human antimicrobial antibodies are present in the complex, each having two prongs when not conjugated, becoming tetra- or multi-prong antibodies upon conjugation with the anti-human Ig antibodies. Therefore, these complexes more effectively cross-link with microbial antigens in antigen-bridging immunoassays.

[0031] Any calibration or quality control reagent matrix known in the art or otherwise considered herein may be used, provided that the resulting reagents function according to this disclosure. In some non-limiting embodiments, the matrix may have a formulation / composition / pH somewhat similar to that of the sample to be measured in the immunoassay. However, in some non-limiting embodiments, it may be desirable to avoid using unpurified serum or plasma-based matrices, as endogenous immunoglobulins present in such matrices may interfere with other components of the calibration or quality control reagents, as well as complexes formed in the calibration or quality control reagents.

[0032] Non-limiting examples of samples that may be used according to this disclosure (and therefore, in some non-limiting embodiments, matrix preparations based thereon) include biological samples such as, but not limited to, whole blood or any part thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof.

[0033] In some non-limiting embodiments, the matrix comprises human serum or plasma substantially depleted of human IgG. In some non-limiting embodiments, the matrix comprises serum from a non-human source, such as (but not limited to) horse, cattle, goat, etc.

[0034] In other non-limiting embodiments, the matrix comprises a water-based system that contains no other components (i.e., substantially no buffers or proteins). In other non-limiting embodiments, the matrix comprises a buffer-based system, alone or in combination with one or more proteins.

[0035] Non-limiting examples of buffers that may be utilized according to this disclosure include phosphates (such as (but not limited to) in phosphate-buffered saline (PBS) solution or Dulbecco phosphate-buffered saline), Tris (such as, but not limited to, Tris buffered saline solution), Bis-Tris, borates (such as, but not limited to, borate buffered saline solution), carbonates, citrates, glycine, ACES, BES, DIPSO, HEPES, HEPPSO, MOPS, MOPSO, PIPES, PIPPS, TAPSO, TES, biological buffers, etc., and any combination thereof.

[0036] Non-limiting examples of proteins that may be used as part of the matrix according to this disclosure include albumins (such as, but not limited to, bovine serum albumin (BSA)), casein, non-human immunoglobulins, transferrin, and any combination thereof.

[0037] Furthermore, as is well known in the art, the matrix may contain additional components. For example (but not limited to), the matrix may contain one or more preservatives, one or more fillers, and / or one or more polymers. Non-limiting examples of fillers that can be utilized according to this disclosure include lactose, sorbitol, trehalose, sucrose, mannitol, glycine, etc., and any combination thereof. Non-limiting examples of preservatives that can be utilized according to this disclosure include ProClin™ (Rohmand Haas, Philadelphia, PA), sodium azide, thimerosal, neomycin, clotrimazole, amphotericin B, MIT, streptomycin, OMADINE® (sodium pyrithione; Lonza, Basel, Switzerland), COSMOCIL® CQ (Lonza, Basel, Switzerland), etc., and any combination thereof. Non-limiting examples of polymers that can be utilized according to this disclosure include polyethylene glycol (PEG), dextran, polyvinylpyrrolidone (PVP), etc., and any combination thereof.

[0038] In a specific (but not limiting) embodiment, the matrix contains bovine serum albumin (BSA) in PBS. A non-limiting example of the matrix is ​​6% protease-free BSA in PBS, pH 7.4.

[0039] Serological immunoassays using the calibration or quality control reagents of this disclosure can detect human antibodies against antigens of any microorganism to be detected. For example (but not limitingly), the microorganism to be detected can be bacteria, viruses, protozoa, fungi, etc.

[0040] Non-limiting examples of bacteria that can be detected according to this disclosure include Acinetobacter spp. ( Acinetobacter Actinomycetes ( Actinomyces Aeromonas spp. Aeromonas Polybacterium spp. Aggregatibacter ), Atobococcus spp. Atopobium ), Bacillus spp. Bacillus ), Bacteroides ( Bacteroides ), Bartonella species ( Bartonella Bifidobacterium spp. Bifidobacterium ), genus *Treponema* ( Borellia Brucella ( ) Brucella ), Campylobacter spp. Campylobacter Chlamydia ( ) Chlamydia Chlamydia genus ( Chlamydophila Clostridium ( Clostridium Corynebacterium spp. Corynebacterium ), Coxs' body ( Coxiella ), Ekenella spp. Eikenella), Enterobacteriaceae ( Enterobacter ), Enterococcus spp. Enterococcus Escherichia coli spp. Escherichia ), Eubacterium ( Eubacterium ), Francisella spp. Francisella ), Fusobacterium spp. Fusobacterium Gardnerella spp. Gardnerella Haemophilus spp. Haemophilis ), Helicobacter spp. Helicobacter Klebsiella spp. Klebsiella Lactobacillus () Lactobacillus Listeria spp. Listeria ), Molybditis spp. Mobiluncus Moraxella spp. Moraxella ), Mycobacterium ( Mycobacterium ), Mycoplasma genus ( Mycoplasma ), Neisseria genus ( Neisseria ), Micromonas spp. Parviomonas Pasteurella ( Pasteurella ), Porphyromonas spp. Porphyromonas ), Prevotella spp. Prevotella ), Propionibacterium spp. Propionibacterium ), Proteobacteria ( Proteus ), Pseudomonas spp. ), Rickettsia species ( Pseudomonas Salmonella ( Rickettsia ), Serratia ( Salmonella ), Shigella spp. Serratia Staphylococcus spp. Shigella Streptococcus spp. Staphylococcus ), Tannerella spp. Streptococcus ), genus *Treponema* Tannerella ), Vibrio ( Treponema ) and Yersinia spp. Vibrio ) species, etc.

[0041] Non-limiting examples of viruses that can be detected according to this 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, measles virus, varicella-zoster virus, West Nile virus, and Zika virus, etc.

[0042] Non-limiting examples of protozoa that can be detected according to this disclosure include Ascaris lumbricoides, Babesia, Cryptosporidium, and others. [[ID=%53]]Yersinia Cyclospora ( Cryptosporidium ), Entamoeba ( Cyclospora ), pinworms ( Entamoeba Giardia () Enterobius ), Hymenolepis fasciculata ( Giardia ), nematodes ( Hymenolepis ), Plasmodium ( Necator ), Strongyloides ( Plasmodium ),tapeworm( Strongyloides Toxoplasma gondii ( Taenia ) and Trichomonas vaginalis ( Toxoplasma ) species, etc.

[0043] Non-limiting examples of fungi that can be detected according to this disclosure include yeasts, molds, etc., including (but not limited to) Candida spp. Trichomonas Cryptococcus ( Candida Epidermophyton ( ) Cryptococcus ), Malassezia ( Epidermophyton Microsporum ( ) Malassezia Trichophyton ( ) Microsporum ) species, etc.

[0044] In the specific (but not restrictive) implementation plan, the microorganisms detected by immunoassay are Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), HIV, Hepatitis B total core, Epstein-Barr virus, herpesvirus (HSV), CMV, rubella virus, and Helicobacter pylori. Trichophyton ) or Toxoplasma gondii ( H. pylori ).

[0045] The antigen may be any antigen derived from the microorganism to be detected. Antigens that can be used to detect each of the microorganisms listed above are well known and widely available in the art. Furthermore, the selection of antigens utilized according to this disclosure is within the capabilities of a person skilled in the art. Therefore, further disclosure is deemed unnecessary.

[0046] Human antibodies against these antigens are also widely known and commercially available. Furthermore, antibody manufacturers produce an extremely large array of thousands of human antimicrobial antibodies that can be utilized under this disclosure; see, for example (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), 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 represents only a small sample of antibody manufacturers producing human antimicrobial antibodies, and there are many other commercial sources of human antimicrobial antibodies available under this disclosure. Therefore, those skilled in the art will be able to clearly and without doubt identify and select the various human antimicrobial antibodies available under this disclosure, and thus consider it unnecessary to further describe the human antimicrobial antibodies or their characteristics.

[0047] In a specific (but not limiting) embodiment, the human antimicrobial antibody is a humanized anti-SARS-CoV-2 antibody, such as (but not limited to) a humanized anti-RBD of the S1 antibody obtained from GenScript (Piscataway, NJ), and the antigen targeted by the human antibody 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.

[0048] 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 (but not limitingly), the antibody may target human IgG, IgE, IgM, IgD and / or IgA, and / or any portion thereof (such as, but not limited to, anti-human γ 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 limited to), 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. (WestGrove, PA), Thermo Fisher Scientific (Waltham, MA), and Sigma-Aldrich Corp. (St. Louis, MO). However, this list is not exhaustive, and many other commercial sources of anti-human Ig antibodies available under this disclosure exist. Therefore, those skilled in the art will be able to clearly and without doubt identify and select the various anti-human Ig antibodies available under this disclosure, and therefore consider it unnecessary to further describe the anti-human Ig antibodies or their characteristics.

[0049] Each of the said anti-human immunoglobulin (Ig) antibody and the said human antibody against the microbial antigen may be present in the calibration or quality control reagent at any concentration, wherein the concentration allows for the formation of complexes as described herein and allows the reagent to function according to this disclosure. In some non-limiting embodiments, each of the anti-human immunoglobulin (Ig) antibody and the human antibody against the microbial antigen is present in the reagent at concentrations 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%, etc. 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., from about 0.001 wt% to about 10 wt%, from about 0.002 wt% to about 1 wt%, from about 0.001 wt% to about 0.5 wt%, etc.), or any value falling within the range of two of the above values ​​(i.e., about 0.27 wt%).

[0050] Similarly, the human antibody against the microbial antigen and the anti-human immunoglobulin (Ig) antibody may be present in the reagent in any proportion between them, the proportion of which allows the reagent to function according to this disclosure. For example (but not limitingly), the anti-human Ig antibody and the human antimicrobial antibody may be present in a ratio within the following range: 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., from about 1:1). The ratio can be in the range of approximately 1:10, approximately 1:2 to approximately 2:3, etc., or any value falling within two of the above ranges (i.e., approximately 1:1.7, approximately 1:2.4, etc.). The ratio of each reagent can be optimized to achieve a good balance between stability and signal when desired. The ratio can also vary widely depending on the specific reagents used.

[0051] Similarly, any additional components present in the matrix (i.e., one or more proteins, preservatives, fillers, and / or polymers) may each be present at any concentration, which allows the reagents formed therefrom to function in accordance with this disclosure. For example (but not limitingly), each additional component is present independently at concentrations selected from the following: 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%. 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., from about 0.001 wt% to about 20 wt%). The range of wt%, from about 0.1 wt% to about 5 wt%, from about 2 wt% to about 20 wt%, etc., or any value falling within the range of two of the above values ​​(i.e., about 13 wt%, about 23 wt%, etc.).

[0052] The calibration or quality control reagents disclosed herein are substantially stable at a desired temperature for a desired time period. For example (but not limitingly), the calibration or quality control reagents may 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 longer. Furthermore, the calibration reagents may be stored at room temperature (i.e., in the range of about 18°C ​​to about 20°C) or under refrigerated or frozen conditions. When preservatives are present in the calibration or quality control reagents, the preservatives used may be selected based on the desired specific storage conditions and shelf life.

[0053] The calibration or quality control reagents described in this disclosure may be provided in any form that allows the reagents to function according to this disclosure. For example, but not limitingly, the reagents may be provided in liquid form. Alternatively, the reagents may be freeze-dried or lyophilized and provided as a dry reagent.

[0054] The calibration or quality control reagents disclosed herein can be used for qualitative or quantitative measurements.

[0055] Some non-limiting embodiments of this disclosure relate to kits containing any calibration or quality control reagents described herein or otherwise considered.

[0056] In some non-limiting embodiments, the kit may further include at least one reagent for serological immunoassay. For example, but not limited to, the kit may further include at least one reagent containing a microbial antigen bound to a human antimicrobial antibody.

[0057] In a specific (but not limiting) embodiment, the microorganism is SARS-CoV-2, and the antigen is the RBD of the SARS-CoV-2 spike S1. This antigen is available from any source known in the art. For example (but not limitingly), this specific 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).

[0058] In specific (but not limiting) embodiments, the kit may further include one or more reagents for performing an immunoassay in a specific assay form, such as (but not limited to) the LOCI® form. For example (but not limiting), the kit may further include a composition comprising a singlet oxygen-activated chemiluminescent compound (such as (but not limited to) chemibead) and a composition comprising a sensitizer (such as (but not limited to) sensibead), each having a microbial antigen directly or indirectly attached thereto (or, the kit contains reagents for directly or indirectly attaching the composition to the microbial antigen prior to or during the immunoassay).

[0059] In a specific (but not limiting) embodiment, the kit may further comprise: (i) a composition comprising: a singlet oxygen-activated chemiluminescent compound having an antigen specifically bound to a human antimicrobial antibody of the reagent, which is directly or indirectly bound thereto, and a fluorescent molecule excited by the activated chemiluminescent compound; and (ii) a composition comprising a sensitizer capable of generating singlet oxygen in its excited state and having an antigen specifically bound to a human antimicrobial antibody of the reagent, which is directly or indirectly bound thereto. In another specific (but not limiting) embodiment, the kit may further comprise: (i) a composition comprising: a singlet oxygen-activated chemiluminescent compound having an antigen specifically bound to a human antimicrobial antibody of the reagent, which binds thereto directly or indirectly, 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 that binds thereto directly or indirectly; and (iii) an antigen specifically bound to a human antimicrobial antibody of the reagent, wherein the antigen is biotinylated.

[0060] Chemiluminescent compounds (chemiluminescent agents) are chemically activatable compounds that emit light of a specific wavelength as a result of such activation. Examples of chemiluminescent agents, by way of example and not limitation, include, for instance: alkenes 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 under light; alkynes capable of reacting with singlet oxygen to form diketones; hydrazones or hydrazides capable of forming azo compounds or azocarbonyl compounds, such as (but not limited to) luminol; and aromatic compounds capable of forming internal peroxides. As a result of the activation reaction, chemiluminescent agents directly or indirectly induce luminescence.

[0061] In some embodiments, the singlet oxygen-activated chemiluminescent compound can be a substance that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate, which can decompose and emit light simultaneously or subsequently. A composition containing a chemiluminescent compound can be directly excited by the activated chemiluminescent compound; or, the composition may further contain at least one fluorescent molecule excited by the activated chemiluminescent compound.

[0062] A sensitizer is a molecule, typically a compound, that generates a reactive intermediate (such as, for example, singlet oxygen) for activating a chemiluminescent compound. In some non-limiting embodiments, the sensitizer is a photosensitizer. Other sensitizers that can be chemically activated (by, for example, enzymes and metal salts) by way of example, and not limitation, 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 alkaline earth metals Ca, Sr, and Ba; d 0 Derivatives of elements in groups 3A, 4A, SA, and 6A in the configuration; oxides of actinides and lanthanides; and the oxidizing agent CIO. - BrO - Au 3+ IO3 - and IO4 - Specifically, molybdate, peroxymolybdate, tungstate, and peroxytungstate ions, and acetonitrile. The following references (which are expressly incorporated herein by reference in their entirety) provide further disclosure regarding sensitizing substances and compositions that also fall within the scope of this disclosure: Aubry, J. Toxoplasma gondii , 107:5844-5849 (1985); Aubry, Am. Chem. Soc. ., 54:726-728 (1989); Böhme and Brauer, J. Org. 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, Inorg. Chem., 119:5286-5294 (1997); and Almeida et al., J. Am. Chem. Soc., Anal. Chim. , 482:99-104 (2003); the full contents of each are hereby expressly incorporated by reference.

[0063] The scope of photosensitizers also includes compounds that are not true sensitizers, but which release singlet oxygen molecules upon excitation by heat, light, ionizing radiation, or chemical activation. Members of this class include, for example (but not limited to), internal peroxides, such as 1,4-dicarboxyethyl-1,4-naphthalene internal peroxide; 9,10-diphenylanthracene-9,10-internal peroxide; and 5,6,11,12-tetraphenylnaphthalene-5,12-internal peroxide. Heating these compounds or direct absorption of light by these compounds releases singlet oxygen.

[0064] A photosensitizer is a sensitizer used to activate a photoactive compound by, for example, photoexcitation to generate singlet oxygen. Photosensitizers are photoactivated and include, for example, dyes and aromatic compounds, and are typically compounds composed of covalently bonded atoms, usually having multiple conjugated double or triple bonds. The compound 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, and at the excitation wavelength, have an extinction coefficient greater than 500 M at its maximum absorbance. -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 should not react efficiently with singlet oxygen. Examples of photosensitizers, by way of example and not limitation, include: acetone; benzophenone; 9-thioxanthone; eosin; 9,10-dibromoanthracene; methylene blue; metalloporphyrins such as (but not limited to) hematoporphyrin; phthalocyanine; chlorophyll; rose red; and barkminsterfullerene; and derivatives of these compounds.

[0065] Specific, non-limiting examples of chemiluminescent compounds and photosensitizers utilized in this disclosure are described in U.S. Patent No. 5,340,716 (Ullman, et al.), the entire contents of which are expressly incorporated herein by reference.

[0066] Any biotin-specific binding partner known in the art or otherwise contemplated herein may be used in accordance with this disclosure. In some non-limiting embodiments, the biotin-specific binding partner is an antibody against biotin. In other non-limiting embodiments, the biotin-specific binding partner is an anti-biotin protein or an analogue thereof.

[0067] According to this disclosure, any avidin analogue known in the art or otherwise considered herein may be used, provided that the avidin or avidin analogue: (1) is capable of associating with a sensitizer; (2) is capable of binding a biotinylated analyte-specific binding partner; and (3) is capable of binding biotin that may be present in the sample. Non-limiting examples of avidin analogues that may be used according to this 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, streptoavidin, traptavidin, neutral avidin, 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.

[0068] 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 used according to this disclosure as the fluorescent molecule for (a) and (b) (and (e), if present), provided that the signal generated by each fluorescent molecule can be detected differently from the signal generated by the other fluorescent molecules used. That is, the fluorescent molecule of (a) must emit light at a wavelength sufficiently different from the wavelength emitted by the fluorescent molecule of (b) so that the two signals can be distinguished from each other when detected simultaneously. In a specific (but not limiting) example, each fluorescent molecule used according to this disclosure is independently selected from terbium, uranium, samarium, europium, gadolinium, and dysprosium. For example (but not limiting), 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.

[0069] The assay components / reagents present in the kit may be provided in any form that allows them to function according to this disclosure. For example, but not limitingly, each reagent may be provided in liquid form and presented in the kit in bulk and / or as a single aliquot. Alternatively, in a particular (but not limiting) embodiment, one or more reagents may be presented in the kit as a single aliquot lyophilized reagent. The use of dried reagents in microfluidic devices is described in detail in U.S. Patent No. 9,244,085 (Samproni), the entire contents of which are expressly incorporated herein by reference.

[0070] In addition to the assay components / reagents described in detail above, the kit may further contain other reagents for performing any specific assay described herein or otherwise considered. The properties of these additional reagents will depend on the specific assay, and their identification is entirely within the skill of a person skilled in the art; therefore, further description is deemed unnecessary. Furthermore, the components / reagents present in the kit may each be in separate containers / compartments, or various components / reagents may be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the components / reagents. Additionally, the kit may include a microfluidic device in which the components / reagents are disposed.

[0071] The relative amounts of the various components / reagents in the kit can be widely varied to provide concentrations that substantially optimize the reactions that need to occur during the assay and further substantially optimize the sensitivity of the assay. Where appropriate, one or more components / reagents in the kit can be provided as dry powders, such as lyophilized powders, and the kit may further include excipients for dissolving the dry reagents; in this way, a reagent solution having an appropriate concentration for performing the method or assay according to this disclosure can be obtained from these components. Non-limiting examples of other reagents that may be included in the kit include washing solutions, dilution solutions, excipients, interfering solutions, positive controls, negative controls, etc. Furthermore, the kit may further include a set of written instructions explaining how to use the kit. Kits of this nature can be used in any method described herein or otherwise considered.

[0072] Some non-limiting embodiments of this disclosure relate to microfluidic devices containing any calibration or quality control reagents described herein or otherwise considered. For example (but not limitingly), some other non-limiting embodiments of this disclosure relate to microfluidic devices that include components of any of the kits described above.

[0073] Specifically, certain non-limiting embodiments include a microfluidic device for detecting antibodies against microorganisms in a sample via a serological immunoassay described herein or otherwise considered. The microfluidic device includes: (i) an inlet channel through which a sample is applied; and (ii) at least one first compartment in fluid communication with said inlet channel. The compartment of (ii) contains any calibration or quality control reagent described herein or otherwise considered, alone or in combination with one or more other reagents described herein or otherwise considered (such as, but not limited to, one or more reagents for serological immunoassays). For example (but not limitingly), said one or more reagents may include the LOCI® immunoassay reagents described above or otherwise considered herein.

[0074] In some non-limiting embodiments, all elements of (ii) (and any additional elements as described above) reside in the same compartment. In alternative non-limiting embodiments, the elements are separated between two or more compartments.

[0075] The microfluidic device can provide any arrangement of compartments and distribution of various components therein, which allows the device to function according to this disclosure.

[0076] Any compartment of a microfluidic device can be sealed to maintain the reagents disposed therein in a substantially hermetically sealed environment until they are used; for example, a compartment containing lyophilized reagents can be sealed to prevent any unintentional remodeling of the reagents. An inlet channel and a compartment, as well as two compartments, can be described as “capable of fluid communication with each other”; this phrase indicates that each compartment can still be sealed, but after puncturing the seal formed therein or between them, the two compartments are able to have fluid flow between them.

[0077] The microfluidic device of this disclosure may provide any other desired features known in the art or otherwise contemplated herein. For example, but not limitingly, the microfluidic device of this disclosure may further include a reading chamber; said reading chamber may be any compartment containing the reagents described above, or said reading chamber may be in fluid communication with said compartment. The microfluidic device may further include one or more additional compartments containing other solutions, such as (but not limited to) washing solutions, diluents, excipients, interfering 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, and these compartments 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 drying reagents, and said compartments may be in fluid communication with any other compartment of the device. In yet another further example, the microfluidic device may include one or more compartments containing diluents, and said compartments may be in fluid communication with any other compartment of the device.

[0078] Certain non-limiting embodiments of this disclosure relate to methods for producing any calibration or quality control reagent for the serological immunoassay of antibodies against microorganisms in human biological samples, as described herein or otherwise contemplated. In said method, a human antibody against a microbial antigen is combined with an anti-human Ig antibody in a matrix and incubated under conditions where a complex is formed between the anti-human Ig antibody and one or more human antibodies against the microbial antigen.

[0079] The incubation step can be performed at any temperature and for any duration, allowing the formation of one or more complexes described herein. For example, but not limited to, the incubation step can be performed at a temperature ranging from about 10°C to about 50°C (such as, but not limited to, from about 18°C ​​to about 40°C, and specifically (but not limited to), about 37°C) and for the following durations: 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, etc. Approximately 50 minutes, approximately 55 minutes, approximately 1 hour, approximately 1.5 hours, approximately 2 hours, approximately 2.5 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 12 hours, approximately 18 hours, approximately 24 hours or longer, and or a range formed by any two of the above values ​​(i.e., a range from approximately 1 minute to approximately 5 hours, a range from approximately 30 minutes to approximately 2 hours, etc.), or any value falling between two of the above values ​​(i.e., approximately 53 minutes, approximately 15 hours, etc.).

[0080] Furthermore, any method for producing calibration or quality control reagents may include one or more additional steps to form the reagents described herein.

[0081] Certain non-limiting embodiments of this disclosure relate to methods for calibrating serological immunoassays for antibodies against microorganisms in human biological samples. In these methods, the immunoassay is performed using any calibration or quality control reagents described herein or otherwise considered.

[0082] Any human antimicrobial antibody that can be detected by the immunoassays of this disclosure or by any assay form described herein or otherwise contemplated. In a particular (but not limiting) embodiment, the antibody is an antiviral antigen antibody against SARS-Covid2 virus.

[0083] In some non-limiting assay embodiments, a signal generation system (SPS) member is utilized, comprising a sensitizer, such as a photosensitizer, and a chemiluminescent-fluorescent molecular composition, each having a microbial antigen directly or indirectly attached thereto (or capable of enabling the antigen to directly or indirectly attach thereto during an immunoassay); in these assay embodiments, activation of the sensitizer results in activation of the product of the chemiluminescent composition, thereby generating a detectable signal correlated with the amount of bound human antimicrobial antibody detected. An exemplary (but non-limiting) embodiment of an assay platform that may serve as the basis of this disclosure is the luminescent oxygen channel 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.

[0084] In a specific (but not limiting) assay embodiment, the assay is performed by incubating the calibration or quality control reagent with two or more reagents for use in LOCI® form. For example (but not limiting), the calibration or quality control reagent may be incubated with a singlet oxygen-activated chemiluminescent compound (such as (but not limited to) chemibead) and a composition containing a sensitizer (such as (but not limited to) sensibead), each having a microbial antigen that binds directly or indirectly thereto. In a specific (but not limiting) embodiment, the calibration or quality control reagent is simultaneously, partially or entirely, sequentially combined with: (i) a composition containing a singlet oxygen-activated chemiluminescent compound having an antigen that binds directly or indirectly thereto to a human antimicrobial antibody specifically bound to the reagent, and a fluorescent molecule excited by the activated chemiluminescent compound; and (ii) a composition containing a sensitizer capable of generating singlet oxygen in its excited state and having an antigen that binds directly or indirectly thereto to a human antimicrobial antibody specifically bound to the reagent. In another specific (but non-limiting) embodiment, the calibration or quality control reagent is simultaneously or partially or entirely combined with: (i) a composition comprising a singlet oxygen-activated chemiluminescent compound having an antigen specifically bound to a human antimicrobial antibody of the reagent, which binds thereto directly or indirectly, 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 that binds thereto directly or indirectly; and (iii) a biotinylated antigen specifically bound to a human antimicrobial antibody of the reagent.

[0085] In the second step, the components are incubated together to allow the composition containing the chemiluminescent compound and the composition containing the sensitizer to bind to the human antimicrobial 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 to the chemiluminescent compound.

[0086] In the third step, the sensitizer is activated to generate singlet oxygen, wherein activation of the sensitizer present in the complex / aggregate results in activation of the chemiluminescent compound present in each complex / aggregate.

[0087] In the 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 molecules, in order to determine the amount of human antimicrobial antibody in the calibration or quality control reagent.

[0088] Any microbial antigens, singlet oxygen-activated chemiluminescent compounds, sensitizers, fluorescent molecules, and biotin or analogues thereof may be used in the methods described in detail above or otherwise considered herein.

[0089] For example, in some specific (but not limiting) embodiments, the singlet oxygen-activated chemiluminescent compound is a substance that undergoes a chemical reaction with singlet oxygen to form a metastable intermediate that can decompose and emit light simultaneously or subsequently.

[0090] In a specific (but not limiting) implementation, the sensitizer is a photosensitizer, and the activation of the sensitizer in step (3) includes irradiation with light (such as, but not limited to, irradiation at about 680 nm).

[0091] As mentioned above, the various components of the method are provided in combination (simultaneously or sequentially). The order in which the components are added can be varied when they are added sequentially; those skilled in the art can determine the specific desired order in which different components are added to the assay. The simplest order of addition is, of course, to add all substances simultaneously and measure the signal generated therefrom. Alternatively, each component, or a group of components, can be combined sequentially. In some embodiments, an incubation step may be included after one or more additions.

[0092] Although specific embodiments of this disclosure are described as having a LOCI® assay, it should be understood that this disclosure also relates to any other immunoassays (and kits, microfluidic devices, and methods thereof) capable of detecting human antibodies against microbial antigens. Therefore, the disclosure of this specific assay is for illustrative purposes only and should not be construed as limiting the scope of this disclosure. Example

[0093] Examples are provided below. However, it should be understood that the application of this disclosure is not limited to the specific experiments, results, and laboratory procedures disclosed herein. Rather, the embodiments are provided merely as one of various implementations and are intended to be exemplary rather than exhaustive.

[0094] In this embodiment, anti-human IgG antibody is used to construct anti-viral antigen (AVAg) antibody in the composite calibrator matrix. The composite AVAg antibody (which separates into two forked arms when not composited) becomes a tetra-forked or multi-forked antibody after being composited with the anti-human IgG antibody. Such complexes (containing two or more AVAg antibodies composited with anti-human Ig antibodies) can more effectively cross-link viral antigens in antigen bridging assays.

[0095] For example, such as Acta As shown, in the CV2T LOCI® assay format, a calibrator containing only AVAg antibody is bound to chemibead and sensibead in a 1:1 ratio. Specifically, a pre-prepared anti-FITC chemibead containing the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein S1 subunit labeled with fluorescein, a streptoacidin-coated sensibead that binds to the biotinylated S1 RBD, and a calibrator containing AVAg antibody are mixed. To generate a signal, one fork arm of the AVAg antibody must bind to the chemibead, and the other fork arm of the same AVAg antibody must bind to the sensibead. After incubation with the calibrator, the mixture is excited at 680 nm, which causes the sensibead to release singlet oxygen. The close proximity of chemibead and sensibead (through binding to the same antibody) causes singlet oxygen to be transferred to chemibead, and this triggers a chemical reaction that causes chemibead to emit light that can be measured at 612 nm.

[0096] In comparison, such as Figure 1As shown, a calibrator produced according to this disclosure is used to form a complex of AVAg antibodies that bind to anti-human IgG antibodies. These complexes present in the calibrator may contain two AVAg antibodies that bind to each anti-human IgG antibody, thus providing a reagent within the calibrator containing at least four forked arms, each forked arm capable of binding to an antiviral antigen (i.e., the RBD of S1 in the case of the CV2T assay). When the calibrator is incubated with chemibead and sensibead from the CV2TLOCI® assay, each complex containing four forked arms can bind up to four antigens, each present on either the chemibead or the sensibead, thereby allowing the formation of aggregates containing two or more chemibeads and / or two or more sensibeads. The formation of these aggregates greatly increases the chance that the sensibead will be sufficiently close to the chemibead during exposure to the excitation wavelength, and thus exponentially increases the emission light emitted by the various chemibeads present in the aggregates after exposure to the excitation wavelength. Therefore, the signal generated by the calibrator of this disclosure can produce a higher efficiency than... Figure 2 The calibrators used in the process generate signals that are approximately 20 to 80 times stronger.

[0097] The calibrators were 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 without human serum and plasma can also be used). Using 6% protease-free BSA in PBS (pH 7.4) buffer as the base matrix, 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), with an anti-human Ig antibody to AVAg antibody ratio of approximately 1:2. The mixture was incubated at approximately 37°C for at least one hour before storage.

[0098] 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, hybridoma cell lines were grown in cell cultures, and the culture supernatant was purified using protein A affinity chromatography. This monoclonal antibody has shown specificity in its exclusive binding to human IgG.

[0099] In specific sample amplification studies, the calibration reagents generated above are used for samples containing... Figure 1 Figure 1-2 (注:原文中的“Yersinia ”疑似拼写错误,我按照原样翻译为“Yersinia ” ,你可根据实际情况修改。)In the CV2T LOCI® assays of chemibead and sensibead, as shown in Table 1, each of the two calibration reagents (which contain a complex formed by a multi-branched AVAg antibody) enhanced the assay signal by approximately 20 to 25 times compared to the assay signal observed when the calibration reagent contained a non-complexed AVAg antibody.

[0100] Table 1

[0101]

[0102] This high level of signal enhancement greatly reduces the amount of antiviral antigen antibodies required to generate calibration or quality control reagents, resulting in cost savings of at least 10 to 20 times.

[0103] Therefore, compositions, kits, and devices, as well as methods of their manufacture and use, have been provided according to this disclosure to fully satisfy the purposes and advantages described above. Although this disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth above, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be readily apparent. Therefore, it is intended to cover all such alternatives, modifications, and variations that fall within the spirit and broad scope of this disclosure.

Claims

1. A reagent kit comprising: At least one calibration or quality control reagent for a serological immunoassay to detect antibodies against a microorganism in a human biological sample, wherein the microorganism is SARS-CoV-2, the reagent comprising: (a) A matrix comprising a buffer-based system in combination with one or more proteins; (b) Anti-human immunoglobulin (Ig) antibody; and (c) A human antibody against an antigen of the said microorganism, wherein the antibody in (c) targets an antigen of SARS-CoV-2 that is at least a portion of the receptor-binding domain of the S1 subunit of the CoV spike protein; and (b) and (c) are present in a 1:2 ratio, wherein a complex of (b) and (c) is formed in the reagent; At least one reagent for the serological immunoassay, and (i) Composition I, comprising: A chemiluminescent compound that can be activated by singlet oxygen, having an antigen that is specifically bound to a human antibody of (c) of the reagent, either directly or indirectly; and Fluorescent molecules excited by activated chemiluminescent compounds; and (ii) Composition II, comprising a sensitizer capable of generating singlet oxygen in its excited state and having an antigen specifically bound to a human antibody of (c) of the reagent, which binds directly or indirectly thereto.

2. The kit of claim 1, wherein the matrix comprises BSA in PBS.

3. The kit of claim 1, wherein the matrix comprises at least one additional component selected from polymers, preservatives, fillers, and combinations thereof.

4. The kit of claim 1, wherein (b) is present in the reagent at a concentration ranging from 0.01 wt% to 10 wt%.

5. The kit of claim 1, wherein (c) is present in the reagent at a concentration ranging from 0.01 wt% to 10 wt%.

6. The kit of claim 1, further comprising at least one composition containing an antigen of the said microorganism.

7. The kit of claim 6, wherein the antigen comprises at least a portion of the receptor-binding domain of the S1 subunit of the CoV spike protein.

8. The kit of claim 1, wherein: (i) The composition I comprises: A chemiluminescent compound that can be activated by singlet oxygen, having an antigen that is specifically bound to a human antibody of (c) of the reagent, either directly or indirectly; and Fluorescent molecules excited by activated chemiluminescent compounds; and (ii) The composition II comprises a sensitizer capable of generating singlet oxygen in its excited state and having a biotin-specific binding partner that binds directly or indirectly thereto; and The kit further comprises: (iii) A biotinylated antigen that specifically binds to the human antibody of (c) of the reagent.

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