WO2023056142A1

WO2023056142A1 - Lateral flow devices for duplex immunoassay for anti-sars-cov-2 antibodies and methods of use thereof

Info

Publication number: WO2023056142A1
Application number: PCT/US2022/075434
Authority: WO
Inventors: Tie Wei
Original assignee: Siemens Healthcare Diagnostics Inc.
Priority date: 2021-09-30
Filing date: 2022-08-25
Publication date: 2023-04-06

Abstract

Lateral flow devices, kits, and methods for detecting the presence and/or concentration of anti‐SARS‐CoV‐2 antibodies in a sample are disclosed. In certain non‐limiting embodiments, the lateral flow devices, kits, and methods can distinguish between anti‐SARS‐CoV‐2 antibodies generated in response to vaccination from anti‐SARS‐CoV‐2 antibodies generated in response to infection.

Description

LATERAL FLOW DEVICES FOR DUPLEX IMMUNOASSAY FOR ANTI-SARS-COV-2 ANTIBODIES AND METHODS OF USE THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS/INCOPORATION BY REFERENCE STATEMENT [0001] Not Applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] Not Applicable.

BACKGROUND

[0003] The field of medical diagnostics utilizes many different forms of assay technologies. When a patient is suspected of being infected with a microorganism (such as, but not limited to, a bacteria or virus), an assay may be performed on a biological sample from the patient to detect antibodies directed to the microorganism that are being produced by the patient's immune system.

[0004] When detection of anti-viral or anti-bacterial antigen antibodies (such as, but not limited to, IgG, IgM, and/or IgA) in patient serum and plasma is desired, bridging serology assays have been employed, in which an immobilized viral/bacterial antigen and a labeled viral/bacterial antigen are often used to formulate the assay reagents. These assays may be utilized in platforms such as (but not limited to), lateral flow assays, latex particle agglutination assays, and Luminescent Oxygen Channeling Immunoassays (LOCI® assays, Siemens Healthineers, Tarrytown, NY).

[0005] In June 2020, the U.S. Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) for a laboratory-based total antibody test developed by Siemens Healthineers (Tarrytown, NY) for the detection of the presence of total SARS-CoV-2 antibodies, including IgM and IgG, in blood. This immunoassay is based on the LOCI® platform and is referred to as the CV2T LOCI® immunoassay.

[0006] A spike protein on the surface of the SARS-CoV-2 virus enables the virus to penetrate and infect human cells found in multiple organs and blood vessels. The Siemens Healthineers' Total Antibody COV2T CV2T LOCI® immunoassay was designed to detect antibodies to the receptor binding domain (RBD) of spike protein. Some of these antibodies are believed to neutralize the SARS-CoV-2 virus and therefore prevent infection.

[0007] In addition, multiple vaccines have been developed and utilized for SARS-CoV-2 that include the spike protein within their focus, including the Pfizer/BioNTech, Moderna, Johnson & Johnson (Janssen), and Oxford-AstraZeneca COVID-19 vaccines that are in wide use. Therefore, immunized patients are now producing antibodies directed to the SARS-CoV- 2 spike protein.

[0008] Therefore, there is a need in the art for new and improved immunoassays for detecting the presence of SARS-CoV-2 antibodies that can distinguish between antibodies produced in response to vaccination from antibodies produced in response to infection, and thereby overcome the disadvantages and defects of the prior art. It is to such assays, as well as kits containing same and methods of producing and using same, that the present disclosure is directed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 schematically depicts one non-limiting embodiment of a diagnostic device constructed in accordance with the present disclosure.

[0010] FIG. 2 schematically depicts another non-limiting embodiment of a diagnostic device constructed in accordance with the present disclosure.

DETAILED DESCRIPTION

[0011] Before explaining at least one embodiment of the present disclosure in detail by way of exemplary language and results, it is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of the components set forth in the following description. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary - not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0012] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0013] All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the present disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0014] All of the articles, compositions, kits, and/or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles, compositions, kits, and/or methods have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the articles, compositions, kits, and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined by the appended claims.

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

[0016] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds. The term "plurality" refers to "two or more."

[0017] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0018] The use of the term "or" in the claims is used to mean an inclusive "and/or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0019] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0020] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition/apparatus/ device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art.

[0021] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0022] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0023] As used herein, the term "substantially" means that the subsequently described event or circumstance completely occurs or that the subsequently described event or circumstance occurs to a great extent or degree. For example, when associated with a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" may mean that two items are 100% adjacent to one another, or that the two items are within close proximity to one another but not 100% adjacent to one another, or that a portion of one of the two items is not 100% adjacent to the other item but is within close proximity to the other item.

[0024] As used herein, the phrases "associated with" and "coupled to" include both direct association/binding of two elements/moieties to one another as well as indirect association/binding of two elements/moieties to one another. Non-limiting examples of associations/couplings include covalent binding of one moiety to another moiety either by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by means of specific binding pair members bound to the moieties, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety, for example. Association of two structural elements with one another may involve direct attachment of the two elements to one another or abutment of the two elements against one another so that liquid can flow from one element to the other element via the abutment.

[0025] The terms "analog" and "derivative" are used herein interchangeably and refer to a substance which comprises the same basic carbon skeleton and carbon functionality in its structure as a given compound, but can also contain one or more substitutions thereto. The term "substitution" as used herein will be understood to refer to the replacement of at least one substituent on a compound with a residue R.

[0026] The term "sample" as used herein will be understood to include any type of biological sample that may be utilized in accordance with the present disclosure. Examples of fluidic biological samples that may be utilized include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, combinations thereof, and the like.

[0027] The term "specific binding partner," as used in particular (but not by way of limitation) herein in the terms "biotin-specific binding partner" or "target analyte-specific binding partner," will be understood to refer to any molecule capable of specifically associating with biotin or the target analyte, respectively. For example, but not by way of limitation, the binding partner may be an antibody, a receptor, a ligand, aptamers, molecular imprinted polymers (i.e., inorganic matrices), combinations or derivatives thereof, as well as any other molecules capable of specific binding to biotin or the target analyte, respectively.

[0028] The term "antibody" is used herein in the broadest sense and refers to, for example, intact monoclonal antibodies and polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof that exhibit the desired biological activity of analyte binding (such as, but not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, 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 substitute proteins or peptides (i.e., engineered binding proteins/peptides), and combinations or derivatives thereof. The antibody can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or sub-class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl, and lgA2). [0029] The term "hapten" as used herein refers to a small proteinaceous or non-protein antigenic determinant (or "epitope") which is capable of being recognized by a target analytespecific binding partner, such as (but not limited to) an antibody. The term "polyhapten" as used herein will be understood to refer to a synthetic molecule that contains multiple epitopes/antigenic determinants attached thereto.

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

[0031] Turning now to the various non-limiting embodiments of the present disclosure, multiplex assays for the detection of multiple anti-SARS-CoV-2 antibodies in samples, as well as kits containing same and methods of production and use thereof, are disclosed. In certain non-limiting embodiments, the multiplex assays utilize a lateral flow assay format as described herein below.

[0032] Certain non-limiting embodiments of the present disclosure are directed to a lateral flow assay device for detecting the presence and/or concentration of antibodies directed to at least two target antigens of SARS-CoV-2 in a biological sample. One of the first and second target antigens comprises at least a portion of a SARS-CoV-2 spike protein, and the other of the first and second target antigens comprises at least a portion of a SARS-CoV- 2 protein other than the spike protein.

[0033] The lateral flow assay device comprises a sample pad, a conjugate pad, and a chromatographic detection pad, and may optionally comprise a support and/or an absorption pad. In addition, the lateral flow device comprises a liquid flow path extending from the sample pad through the conjugate pad to the chromatographic detection pad and through second capture sites present in the chromatographic detection pad, as described in detail herein below.

[0034] When present, the support has an upper surface, a first end, and a second end. The chromatographic detection pad may be attached to or otherwise associated with at least a portion of the upper surface of the support. The chromatographic detection pad may substantially extend from the first end to the second end of the support, whereby the sample pad and the conjugate pad (and the absorption pad, if present) are disposed on an upper surface of the chromatographic detection pad. Alternatively, the chromatographic detection pad may have a length that is less than the length of the support; in this non-limiting embodiment, the sample pad and the conjugate pad may be attached to or otherwise associated with the upper surface of the support upstream of the chromatographic detection pad (i.e., between the first end of the support and the chromatographic detection pad), while the absorption pad (if present) is attached to or otherwise associated with the upper surface of the support downstream of the chromatographic detection pad (i.e., between the chromatographic detection pad and the second end of the support).

[0035] The sample pad has an upper surface, a lower surface, a first end, and a second end. The sample pad provides a sample application site for application of a portion of a liquid sample to the lateral flow assay device. The sample pad may be attached to or otherwise associated with a portion of the upper surface of the support in a vicinity of the first end thereof, or the sample pad may be attached to or otherwise associated with a portion of the chromatographic detection pad.

[0036] The conjugate pad has an upper surface, a lower surface, a first end, and a second end. The conjugate pad may be attached to or otherwise associated a portion of the sample pad in a vicinity of the second end of the sample pad, whereby a path for capillary fluid flow from the sample pad to the conjugate pad is provided. In addition, the conjugate pad comprises one or more labeled binding partners for the antibodies to be detected. For example (but not by way of limitation), the labeled binding partners may be a labeled first target antigen and a labeled second target antigen. Alternatively, the labeled binding partner may be one or more anti-human immunoglobulin antibodies. Upon contact with the liquid sample, the labeled binding partner(s) flow through the conjugate pad and into the chromatographic detection pad.

[0037] The chromatographic detection pad has an upper surface, a lower surface, a first end, and a second end. The chromatographic detection pad in a vicinity of the first end thereof is attached to or otherwise associated with a portion of the conjugate pad in a vicinity of the second end of the conjugate pad, whereby a path for capillary fluid flow from the conjugate pad and through the chromatographic detection pad is provided. The chromatographic detection pad comprises two capture sites: a first capture site is spaced downstream from the first end of the chromatographic detection pad and comprises the first target antigen directly or indirectly attached to the chromatographic detection pad. The second capture site is spaced apart from the first capture site and comprises the second target antigen directly or indirectly attached to the chromatographic detection pad.

[0038] In this manner, a complex is formed at each capture site. For example, an antibody present in the sample that is directed to the first target antigen binds to both the first target antigen attached to the chromatographic detection pad (at the first capture site) and the corresponding binding partner (such as, but not limited to, a labeled first target antigen) flowing from the conjugate pad to form a complex that is bound at the first capture site. Likewise, an antibody present in the sample that is directed to the second target antigen binds to both the second target antigen attached to the chromatographic detection pad (at the second capture site) and the corresponding binding partner (such as, but not limited to, a labeled second target antigen) flowing from the conjugate pad to form a complex that is bound at the second capture site.

[0039] In another particular (but non-limiting) embodiment, when one or more labeled anti-human Ig antibodies are utilized as the labeled binding partner disposed in the conjugate pad, the labeled anti-human Ig antibody binds to an antibody present in the sample that is directed to the first target antigen as the sample passes through the conjugate pad, and then this complex binds to the first target antigen attached to the chromatographic detection pad (at the first capture site) to form a sandwich complex that is bound at the first capture site. Likewise, the labeled anti-human Ig antibody binds to an antibody present in the sample that is directed to the second target antigen as the sample passes through the conjugate pad, and then this complex binds to the second target antigen attached to the chromatographic detection pad (at the second capture site) to form a sandwich complex that is bound at the second capture site. In this manner, a single labeled anti-human Ig antibody can bind to both of the complexes formed at the first and second capture sites and thereby allow a single labeled anti-human Ig antibody to replace the labeled first and second target antigens utilized in the previously described embodiment of the lateral flow assay device.

[0040] For the ease of description herein after, the target antigen that comprises at least a portion of the SARS-CoV-2 spike protein will be referred to as the "first" target antigen, while the target antigen that comprises at least a portion of a SARS-CoV-2 protein other than the spike protein will be referred to as the "second" target antigen. However, it will be understood that these designations are solely for the purposes of example only, and should not be construed as related to a sequence or order at which the target antigens are disposed on the lateral flow assay device. That is, the capture site that detects the SARS-CoV-2 spike protein may appear before or after the capture site that detects the SARS-CoV-2 protein other than the spike protein on the lateral flow assay device.

[0041] All or any portion of a SARS-CoV-2 spike protein may be utilized as the first target antigen in accordance with the present disclosure. In certain non-limiting embodiments, the first target antigen includes at least a portion of a SARS-CoV-2 SI protein, such as (but not limited to) at least a portion of a receptor-binding domain (RBD) of SI protein.

[0042] In one particular (but non-limiting) embodiment, the first target antigen is the receptor binding domain (RBD) of the SI subunit of SARS-CoV-2 spike protein. The RBD SI antigen can be obtained from any source known in the art. For example (but not by way of limitation), this particular antigen is commercially available from GenScript (Piscataway, NJ); Meridian Life Sciences, Inc. (Memphis, TN); Sino Biological US Inc. (Wayne, PA); ACRO Biosystems (Newark, DE); Biorbyt, LLC (St. Louis, MO); Icosagen, AS (San Francisco, CA); and Bios Pacific Inc. (Emeryville, CA).

[0043] All or any portion of another SARS-CoV-2 protein can be utilized as the second target antigen. In certain non-limiting embodiments, the second target antigen includes at least a portion of a SARS-CoV-2 nucleocapsid protein, at least a portion of a SARS-CoV-2 membrane protein, or at least a portion of a SARS-CoV-2 envelope protein.

[0044] Each of the first and second target antigens present in the conjugate pad may be labeled and disposed in the conjugate pad by any methods known in the art or otherwise contemplated herein, so long as the first and second labeled target antigens can function as described herein. For example (but not by way of limitation), a gold nanoparticle or a colored latex particle may be coated with one or both of the first and second target antigens to form a conjugate of labeled first target antigen and/or a conjugate of labeled second target antigen. [0045] Then the labeled binding partner(s) are dried or otherwise disposed on the conjugate pad such that, when the liquid biological sample flows from the sample application pad through the conjugate pad and to the chromatographic detection pad, the dried labeled binding partner(s) are mixed with the sample and solubilized so that the labeled binding partner(s) can flow through the conjugate pad into the chromatographic detection pad. Further, any antibodies present in the sample that are directed to the first or second target antigen form a complex with the solubilized labeled binding partner, wherein the complex then flows through the conjugate pad into the chromatographic pad, thereby allowing said complex to bind to the respective target antigen attached at the first or second capture site on the chromatographic detection pad.

[0046] In addition to the first and second capture sites present on the chromatographic detection pad, the lateral flow assay device may further comprise a control detectable signal to confirm that the correct procedures were used and that the assay functioned correctly. Thus, the control confirms that sample was added and mixed with and solubilized all labeled compositions dried to the conjugate pad, and that the sample and any formed complex flowed through the membrane and bound to the respective capture site. It should be understood that lateral flow assay device may further contain additional sites as well, such as (but not limited to) a reference site (the absence of a signal at which indicates the test results are invalid).

[0047] When a control detectable signal is present on the lateral flow assay device, the conjugate pad further comprises a labeled non-human Ig antibody, and the chromatographic detection pad further comprises a control capture site spaced apart from the first and second capture sites. The control capture site comprises an anti-IgG antibody directed to the labeled non-human Ig antibody directly or indirectly attached to the chromatographic detection pad. For example (but not by way of limitation), the conjugate pad may further comprise a labeled rabbit IgG, and the control capture site may comprise an anti-rabbit IgG antibody.

[0048] The chromatographic detection pad may be made of any suitable material through which the liquid biological sample may flow by capillary action. As a non-limiting example, the chromatographic detection pad may be a nitrocellulose membrane.

[0049] In addition to the sample application pad, the conjugate pad, and the chromatographic detection pad, the lateral flow assay device may further include one or more additional elements that aid in the ease, performance, and/or safety of the device. For example (but not by way of limitation), the lateral flow assay device may further include an absorption pad disposed downstream of the chromatographic detection pad that collects all liquid and waste products that flow through the chromatographic detection pad. In a particular (but non-limiting) embodiment, the absorption pad may be attached to or otherwise associated with a portion of the chromatographic detection pad in a vicinity of the second end of the chromatographic detection pad.

[0050] Certain non-limiting embodiments of the present disclosure are directed to a lateral flow assay device that is identical to the device described herein above except that the labeled first and second target antigens present in the conjugate pad are replaced with one or more labeled anti-human immunoglobulin (Ig) antibodies. When labeled anti-human Ig antibodies are utilized, said antibodies are labeled and disposed in the conjugate pad by the same methods as utilized to produce the first and second labeled target antigens, as described in detail herein above. In addition, when anti-human Ig antibodies are utilized in the conjugate pad, an antibody present in the sample that is directed to the first target antigen binds to the first target antigen attached to the chromatographic detection pad (at the first capture site) to form a complex, and the labeled anti-human Ig antibody flowing from the conjugate pad binds to this complex to form a sandwich complex that is bound at the first capture site. Likewise, an antibody present in the sample that is directed to the second target antigen binds to the second target antigen attached to the chromatographic detection pad (at the second capture site) to form a complex, and the labeled anti-human Ig antibody flowing from the conjugate pad binds to this complex to form a sandwich complex that is bound at the second capture site. In this manner, a single labeled anti-human Ig antibody can bind to both of the complexes formed at the first and second capture sites and thereby allow a single labeled anti-human Ig antibody to replace the labeled first and second target antigens utilized in the embodiment of the lateral flow assay device described herein previously.

[0051] When anti-human Ig antibody(ies) is utilized as the labeled binding partner in the conjugate pad, the anti-human Ig antibodies may specifically bind to any portion of any human immunoglobulin molecules known in the art or otherwise contemplated herein. For example (but not by way of limitation), the antibodies may be directed to human IgG, IgE, IgM, IgD, and/or IgA, and/or any portion thereof (such as, but not limited to, anti-human gamma chain, anti-human H+L, anti-human light chain, and the like). Anti-human Ig antibodies (including, but not limited to, anti-human IgG, anti-human IgM, and/or anti-human IgA antibodies, as well as antibodies that recognize two or more human immunoglobulin antibodies) are well known in the art, are widely commercially available, and have been vastly studied. For example (but not by way of limitation), a few commercial sources of anti-human IgG monoclonal and/or polyclonal antibodies include Rockland Immunochemicals, Inc. (Pottstown, PA); USBiological Life Sciences (Swampscott, MA); Santa Cruz Biotechnology, Inc. (Dallas, TX); Jackson Immuno Research Labs, Inc. (West Grove, PA); Thermo Fisher Scientific (Waltham, MA); and Sigma-Aldrich Corp. (St. Louis, MO). However, this list is not inclusive, and there are many additional commercial sources of anti-human Ig antibodies that can be utilized in accordance with the present disclosure. Thus, a person having ordinary skill in the art will clearly and unambiguously be able to identify and select a variety of anti-human Ig antibodies that can be utilized in accordance with the present disclosure, and as such, no further description of the anti-human Ig antibodies or the characteristics thereof is deemed necessary.

[0052] Certain non-limiting embodiments of the present disclosure include kits that include one or more of the lateral flow assay devices described or otherwise contemplated herein. In addition to the devices described in detail herein above, the kits may further contain other reagent(s) for conducting any of the particular assays described or otherwise contemplated herein. For example (but not by way of limitation), the kit may include one or more positive and/or negative control solutions. In addition, the kit can further include a set of written instructions explaining how to use the kit. A kit of this nature can be used in any of the methods described or otherwise contemplated herein.

[0053] Certain non-limiting embodiments of the present disclosure include methods for detecting the presence and/or concentrations of multiple anti-SARS-CoV-2 antibodies in a sample. In the method, a biological sample suspected of containing SARS-CoV-2 antibodies is applied to the sample application pad of any of the lateral flow devices described or otherwise contemplated herein, and the sample is allowed to flow through the lateral flow assay device from the sample application pad through the conjugate pad and through the chromatographic detection pad (and to the absorption pad, if present). Then it is determined whether antibodies to the first target antigen are present in the biological sample based on the result at the first capture site, and it is determined whether antibodies to the second target antigen are present in the biological sample based on the result observed at the second capture site. [0054] In a particular (but non-limiting) embodiment, the method further comprises the step of determining that anti-SARS-CoV-2 antibodies present in the sample were generated in response to infection based on the result at the capture site containing the target antigen that comprises at least a portion of a SARS-CoV-2 protein other than the spike protein.

[0055] In a particular (but non-limiting) embodiment, the method may further comprise the step of determining that anti-SARS-CoV-2 antibodies present in the sample were generated in response to vaccination if a result is observed at the capture site containing the target antigen that comprises at least a portion of a SARS-CoV-2 spike protein but not observed at the capture site containing the target antigen that comprises a SARS-CoV-2 protein other than the spike protein.

[0056] Any sample for which an assay for the presence and/or concentration of anti-SARS- CoV-2 antibodies is desired can be utilized as the sample in accordance with the methods of the present disclosure. Non-limiting examples of samples include a biological sample such as, but not limited to, whole blood or any portion thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof. Particular non-limiting examples include lysed whole blood cells and lysed red blood cells.

[0057] While particular embodiments of the present disclosure are described as having the lateral flow assay format, it is to be understood that the present disclosure is also directed to other assay formats (and kits, microfluidics devices, and methods of performing same) for which distinction between SARS-CoV-2 antibodies generated in response to vaccination are distinguished from SARS-CoV-2 antibodies generated in response to infection is desired.

EXAMPLES

[0058] Examples are provided hereinbelow. However, the present disclosure is to be understood to not be limited in its application to the specific experimentation, results, and laboratory procedures disclosed herein. Rather, the Examples are simply provided as one of various embodiments and are meant to be exemplary, not exhaustive.

Example 1

[0059] In this Example, the use of a duplex immunoassay to simultaneously detect anti- nucleocapsid (NC) and anti-spike protein antibodies from SARS-CoV-2 in a patient's sample is described. The duplex immunoassay has been developed to report if the anti-SARS-CoV-2 antibodies present in a patient's sample were generated due to infection and/or vaccination. In the duplex immunoassay, antibodies to two target antigens of SARS-CoV-2 are detected; one of the antigens comprises at least a portion of a SARS-CoV-2 spike protein, and the other antigen comprises at least a portion of a SARS-CoV-2 protein other than the spike protein. As the Pfizer/BioNTech and Moderna mRNA vaccines primarily used in the US at the time of filing the subject application include mRNA encoding the SARS-CoV-2 spike protein, the use of these two target antigens allows for the determination of whether the SARS-CoV-2 antibodies present in a patient's sample result from infection and/or vaccination.

[0060] FIG. 1 illustrates a first non-limiting embodiment of a diagnostic lateral flow device constructed in accordance with the present disclosure. The lateral flow assay device is indicated by reference numeral 10 and includes a support 12, a sample pad 14, a conjugate pad 16, and a chromatographic detection pad 18, and optionally an absorption pad 20.

[0061] The support 12 has an upper surface 30, a first end 32, and a second end 34. The sample pad 14 has an upper surface 36, a first end 40, and a second end 42. The conjugate pad 16 has a first end 48 and a second end 50. The chromatographic detection pad 18 has an upper surface 58, a first end 62, and a second end 64.

[0062] The chromatographic detection pad 18 may be attached to or otherwise associated with at least a portion of the upper surface 30 of the support 12. FIG. 1 depicts the chromatographic detection pad 18 as having a length that extends substantially from the first end 32 to the second end 34 of the support 12, and the sample pad 14, the conjugate pad 16, and the absorption pad 20 as being disposed upon the upper surface 58 of the chromatographic detection pad 18. However, this depiction is for purposes of example only; it will be understood that the chromatographic detection pad 18 may have a length that is less than the length of the support 12, whereby at least a portion of the sample pad 14, and conjugate pad 16, and/or the absorption pad 20 is attached to or otherwise associated with the support 12.

[0063] The sample pad 14 is attached to or otherwise associated with the upper surface 58 of the chromatographic detection pad 18 in a vicinity of the first end 62 of the chromatographic detection pad 18, and the upper surface 36 of the sample pad 14 forms a sample application site for application of a liquid sample to the lateral flow assay device 10. The conjugate pad 16 is attached to or otherwise associated with the upper surface 58 of the chromatographic detection pad 18 between the attachment point of the sample pad 14 and the second end 64 of the chromatographic detection pad 18 and is attached to or otherwise associated with a portion of the sample pad 14 in a vicinity of the second end 42 of the sample pad 14, whereby a path for capillary fluid flow from the sample pad 14 to the conjugate pad 16 is provided. The chromatographic detection pad 18 in a vicinity of the first end 62 thereof is associated with a portion of the conjugate pad 16 in a vicinity of the second end 50 of the conjugate pad 16, whereby a path for capillary fluid flow from the conjugate pad 16 and through the chromatographic detection pad 18 is provided. When present, the absorption pad 20 is attached to or otherwise associated with the chromatographic detection pad 18 in a vicinity of the second end 64 of the chromatographic detection pad 18, whereby a path for capillary fluid flow from the chromatographic detection pad 18 to the absorption pad 20 is provided.

[0064] Therefore, the lateral flow device 10 comprises a liquid flow path that extends from the sample pad 14 through the conjugate pad 16 and through the chromatographic detection pad 18 (and further to the absorption pad 20, if present).

[0065] The conjugate pad has a labeled first target antigen 52 and a labeled second target antigen 54 dried thereon and/or otherwise disposed therein.

[0066] The chromatographic detection pad 18 includes a first capture site 66 and a second capture site 70 spaced apart from the first capture site 66. The first capture site 66 is spaced downstream from the first end 62 of the chromatographic detection pad 18, and the first capture site 66 comprises first target antigen 68 directly or indirectly attached to the chromatographic detection pad 18. The second capture site 70 is spaced downstream from the first capture site 66 and comprises second target antigen 72 directly or indirectly attached to the chromatographic detection pad 18. In addition, the liquid flow path that extends from the sample pad 14 through the conjugate pad 16 to the chromatographic detection pad 18 extends through both of the first and the second capture sites 66 and 70, respectively.

[0067] In use of the lateral flow assay device 10, when a biological sample is applied to the sample application site of the sample pad 14, the biological sample flows from the sample pad 14 through the conjugate pad 16 and through the chromatographic detection pad 18. When the biological sample contains an antibody 82 against the first SARS-CoV-2 target antigen, the antibody 82 binds to the first labeled target antigen 52 in the conjugate pad 16 to form a complex, and then this complex binds to the first target antigen 68 attached to the first capture site 66. The presence of the label in the sandwich complex formed at the first capture site 66 allows for detection of the antibody 82 present in the biological sample that is directed against the first target antigen.

[0068] Likewise, when the biological sample contains an antibody 84 against the second SARS-CoV-2 target antigen, the antibody 84 binds to the second labeled target antigen 54 in the conjugate pad 16 to form a complex, and then this complex binds to the second target antigen 72 attached to the second capture site 70. The presence of the label in the sandwich complex formed at the second capture site 70 allows for detection of the antibody 84 present in the biological sample that is directed against the second target antigen.

[0069] In certain non-limiting embodiments, the lateral flow assay device 10 further includes a control capture site 90 on the chromatographic detection pad 18. The control capture site 90 is typically located downstream of both the first and second capture sites 66 and 70, respectively, so as to ensure that the procedures were performed correctly and the biological sample properly flowed through the sample pad 14, the conjugate pad 16, and the chromatographic detection pad 18 and passed all capture sites located thereon. The conjugate pad 16 will be provided with a labeled compound/composition that will bind to a compound/composition that is directly or indirectly attached to the control capture site 90, thereby ensuring that the biological sample solubilized the dried components present in the conjugate pad 16, and that the biological sample and the solubilized components flowed through the conjugate pad 16 and into the chromatographic detection pad 18.

[0070] In a particular (but non-limiting) embodiment, the conjugate pad 16 contains a labeled non-human IgG antibody 94 (such as, but not limited to, a labeled rabbit IgG), and the chromatographic detection pad 18 contains an anti-IgG antibody 92 (such as, but not limited to, an anti-rabbit IgG antibody) directly or indirectly attached thereto at the control capture site 90.

Example 2

[0071] The duplex immunoassay of this Example is similar to the assay format described above in Example 1. However, this immunoassay format differs in that, instead of utilizing both first and second target antigens as the labeled components in the assay, a single labeled anti-human immunoglobulin antibody is utilized instead.

[0072] FIG. 2 illustrates a second non-limiting embodiment of a diagnostic lateral flow device constructed in accordance with the present disclosure. The lateral flow assay device is indicated by reference numeral 10a and is identical to the lateral flow assay device 10 of FIG. 1, except as described herein below.

[0073] Like the lateral flow assay device 10, the lateral flow assay device 10a of FIG. 2 includes a support 12a, a sample pad 14a, a conjugate pad 16a, and a chromatographic detection pad 18a, and optionally an absorption pad 20a. The lateral flow assay device 10a also includes the same liquid flow path that extends from the sample pad 14a through the conjugate pad 16a and through the chromatographic detection pad 18a (and further to the absorption pad 20a, if present). In addition, the chromatographic detection pad 18a includes first and second capture sites 66a and 70a with first and second target antigens 68a and 72a, respectively, directly or indirectly attached to the chromatographic detection pad 18a as described herein above with respect to the lateral flow assay device 10 of Example 1; the chromatographic detection pad 18a also optionally includes a control capture site 90a for monitoring the performance of the assay.

[0074] The lateral flow assay device 10a differs from the lateral flow assay device 10 in that, instead of using labeled first and second target antigens, the conjugate pad 16a of the lateral flow assay device 10a has a labeled anti-human immunoglobulin (Ig) antibody 110 dried thereon and/or otherwise disposed therein.

[0075] In use of the lateral flow assay device 10a, when a biological sample is applied to the sample application site of the sample pad 14a, the biological sample flows from the sample pad 14a through the conjugate pad 16a and through the chromatographic detection pad 18a. When the biological sample contains an antibody 82 against the first SARS-CoV-2 target antigen, the labeled anti-human Ig antibody 110 binds to the antibody 82 to form a complex, and then this complex binds to the first target antigen 68a attached to the first capture site 66a. The presence of the label in the sandwich complex formed at the first capture site 66a allows for detection of the antibody 82 present in the biological sample that is directed against the first target antigen.

[0076] Likewise, when the biological sample contains an antibody 84 against the second SARS-CoV-2 target antigen, the labeled anti-human Ig antibody 110 binds to the antibody 84 to form a complex, and then this complex binds to the second target antigen 72a attached to the second capture site 70a. The presence of the label in the sandwich complex formed at the second capture site 72a allows for detection of the antibody 84 present in the biological sample that is directed against the first target antigen.

[0077] Thus, in accordance with the present disclosure, there have been provided compositions, kits, and devices, as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

What is claimed is:

1. A lateral flow assay device for detecting the presence and/or concentration of antibodies directed to at least two target antigens of SARS-CoV-2 in a biological sample, the lateral flow assay device comprising: a sample pad having an upper surface, a lower surface, a first end, and a second end, wherein the sample pad provides a sample application site for application of a portion of a liquid sample to the lateral flow assay device; a conjugate pad having an upper surface, a lower surface, a first end, and a second end, wherein the conjugate pad is associated with a portion of the sample pad in a vicinity of the second end of the sample pad, whereby a path for capillary fluid flow from the sample pad to the conjugate pad is provided, and wherein the conjugate pad comprises at least one labeled binding partner for SARS- CoV-2 antibodies in the biological sample, wherein the at least one labeled binding partner comprises:

(i) a labeled first target antigen and a labeled second target antigen; or

(ii) a labeled anti-human Ig antibody; a chromatographic detection pad having an upper surface, a lower surface, a first end, and a second end, wherein the chromatographic detection pad in a vicinity of the first end thereof is associated with a portion of the conjugate pad in a vicinity of the second end of the conjugate pad, whereby a path for capillary fluid flow from the conjugate pad and through the chromatographic detection pad is provided, the chromatographic detection pad comprising: a first capture site spaced downstream from the first end of the chromatographic detection pad, the first capture site comprising the first target antigen directly or indirectly attached to the chromatographic detection pad; and a second capture site spaced apart from the first capture site, the second capture site comprising the second target antigen directly or indirectly attached to the chromatographic detection pad; and wherein one of the first and second target antigens comprises at least a portion of a SARS-CoV-2 spike protein, and the other of the first and second target antigens comprises at least a portion of a SARS-CoV-2 protein other than the spike protein; and wherein the lateral flow device comprises a liquid flow path extending from the sample pad through the conjugate pad to the chromatographic detection pad and through both of the first and the second capture sites.

2. The device of claim 1, wherein one of the first and second target antigens comprises a receptor-binding domain (RBD) of SI protein.

3. The device of claim 1, wherein one of the first and second target antigens is at least a portion of a SARS-CoV-2 nucleocapsid protein.

4. The device of claim 1, wherein one of the first and second target antigens is at least a portion of a SARS-CoV-2 membrane protein or at least a portion of a SARS-CoV-2 envelope protein.

5. The device of claim 1, wherein the at least one labeled binding partner of the conjugate pad comprises a labeled first target antigen and a labeled second target antigen.

6. The device of claim 1, wherein the at least one labeled binding partner of the conjugate pad is a labeled anti-human Ig antibody.

7. The device of claim 1, wherein the at least one labeled binding partner is labeled with a gold nanoparticle.

8. The device of claim 1, wherein the chromatographic detection pad is a nitrocellulose membrane.

9. The device of claim 1, wherein the chromatographic detection pad further comprises a control capture site spaced apart from the first and second capture sites.

10. The device of claim 9, wherein the conjugate pad further comprises a labeled nonhuman Ig antibody, and wherein the control capture site comprises an anti-IgG antibody directly or indirectly attached to the chromatographic detection pad, wherein the anti-IgG antibody is directed to the labeled non-human Ig antibody.

11. The device of claim 1, further comprising an absorption pad associated with a portion of the chromatographic detection pad in a vicinity of the second end of the chromatographic detection pad.

12. A method for detecting the presence and/or concentrations of multiple anti-SARS- CoV-2 antibodies in a sample, the method comprising the steps of:

(1) applying a biological sample suspected of containing SARS-CoV-2 antibodies to the lateral flow device of any one of claims 1-11;

(2) allowing the sample to flow through the lateral flow assay device;

(3) determining whether antibodies to the first target antigen are present in the biological sample based on the result at the first capture site; and

(4) determining whether antibodies to the second target antigen are present in the biological sample based on the result observed at the second capture site.

13. The method of claim 12, further comprising the step of:

(5) determining that anti-SARS-CoV-2 antibodies present in the sample were generated in response to infection based on the result at the capture site containing the target antigen that comprises at least a portion of a SARS-CoV- 2 protein other than the spike protein.

14. The method of claim 13, further comprising the step of:

(6) determining that anti-SARS-CoV-2 antibodies present in the sample were generated in response to vaccination if a result is observed at the capture site containing the target antigen that comprises at least a portion of a SARS-CoV- 2 spike protein but not observed at the capture site containing the target antigen that comprises a SARS-CoV-2 protein other than the spike protein.

15. The method of claim 12, wherein the sample is a biological sample selected from the group consisting of whole blood or any portion thereof, urine, saliva, sputum, cerebrospinal fluid, skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder wash, semen, fecal, pleural fluid, nasopharyngeal fluid, and combinations thereof.