WO2022169842A1

WO2022169842A1 - Rapid detection kits and methods for diagnosing infections of the respiratory tract

Info

Publication number: WO2022169842A1
Application number: PCT/US2022/014902
Authority: WO
Inventors: James M. ROLKE; Robin L. MARSDEN; Joseph M. NINOSKY; George F. TIDMARSH
Original assignee: Revelation Biosciences, Inc.
Priority date: 2021-02-02
Filing date: 2022-02-02
Publication date: 2022-08-11

Abstract

The present disclosure provides a lateral flow immunoassay, kits and methods for rapidly detecting interferon gamma inducible protein- 10 (IP- 10) in a sample from the mucosa of a subject, particularly the oral or nasal mucosa. The immunoassay may advantageously include a sample control for detecting the presence of IgA. The immunoassay described herein permits determination of whether an individual's mucosal sample contains IP-10, which in turn signals that the individual has been infected with a respiratory pathogen, such as a virus or bacteria that infects the respiratory tract of humans. The immunoassay further permits the identification of subjects who should be quarantined and/or subjected to further specific testing for respiratory pathogens and treatment.

Description

RAPID DETECTION KITS AND METHODS FOR DIAGNOSING

INFECTIONS OF THE RESPIRATORY TRACT

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/144,576, filed on February 2, 2021, and U.S. Provisional Patent Application No. 63/193,989, filed on May 27, 2021, each of which is hereby incorporated by reference in its entirety.

BACKGROUND

Coronavirus disease 2019 (COVID-19) causes serious respiratory illness including pneumonia and lung failure. As the COVID-19 pandemic swept the globe, gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. Moreover, early diagnosis that could lead to effective contact tracing and quarantine procedures was thwarted not only by the lack of available testing, but also by asymptomatic incubation and infection in a sizable percentage of infected individuals.

The World Health Organization recommends that respiratory tract specimens from all suspected cases of COVID-19 be shipped to authoritative laboratories for definitive diagnosis (Ahn et al. 2020). Detection of COVID-19 is currently based on molecular approaches, with polymerase chain reaction (PCR) being favored. This test is highly specific but can take hours to days for results and cannot be widely utilized in home settings or as a point-of-care diagnostic. In the United States, issues with testing availability were widely reported, even for patients exhibiting symptoms consistent with COVID-19. In addition, asymptomatic carriers of the virus are unlikely to seek testing, thereby potentially further exposing contacts and spreading infection.

There remains a need for rapid detection assays and methods for detecting SARS- CoV-2 as well as other respiratory pathogens.

SUMMARY OF THE INVENTION

The invention provides a lateral flow immunoassay, kit, and method for rapidly detecting interferon gamma inducible protein-10 (IP-10), also known as C-X-C motif chemokine ligand 10 (CXCL-10), in a sample from the mucosa of a subject. The invention permits determination of whether an individual’s mucosal sample contains IP- 10, which in turn indicates that the individual has been infected with a respiratory pathogen, such as a virus or bacteria that infects the respiratory tract of humans. According to the present disclosure, a lateral flow immunoassay for detecting IP- 10 in a mucosal sample from a subject, comprises: a. a sample application portion; b. a diagnostic antibody portion having dispersed thereon a first population of unfixed signal antibodies specific for IP- 10, wherein the antibodies of the first population comprise a label moiety, and c. a detection portion comprising a test zone;

The lateral flow assay may further comprise an assay control zone having fixed thereon capture antibodies that specifically bind to the first population of unfixed signal antibodies specific for IP- 10.

The antibody portion described above may further comprise a population of unfixed signal antibodies specific for IgA, wherein the antibodies specific for IgA comprise a second label moiety, and the detection portion further comprises a sample control zone having fixed thereon capture antibodies that specifically bind to the signal antibodies specific for IgA.

The assay of the present invention may further comprise a second population of unfixed signal antibodies specific for IP- 10, wherein the antibodies of the second population comprise a tag. Exemplary tags include Biotin, streptavidin, avidin, ruthenium, digoxin, azide, or desthiobiotin.

The test zone of the immunoassay may comprise streptavidin, biotin, avidin, ruthenium, digoxin, azide, or desthiobiotin fixed thereon.

Additionally, the immunoassay may further include: d. wicking portion; e. a membrane on which the diagnostic antibody portion, the detection portion, and the wicking portion are disposed; and f. a backing card supporting the membrane.

The lateral flow immunoassay may be configured such that i) the sample application portion is communicably arranged adjacent to or coextensive with the diagnostic antibody portion; ii) the diagnostic antibody portion is communicably arranged adjacent to the detection portion; and iii) the wicking portion is disposed on the membrane such that the sample flows from the sample application portion through the diagnostic antibody portion and subsequently through the detection portion.

The immunoassay may be configured such that the sole cytokine detected is IP-10, or it may be configured to detect one or more cytokines in addition to IP- 10. For example, the assay may be configured to also detect IL-la, IL-ip, IL-6, IL-8, IL-12, IL-18, IFN-a, IFN-P, IFN-Y, TNF-a, GM-CSF, MIP-la, MIP-lp, MCP-1, MCP-3, TRAIL, or any combination thereof.

The mucosal sample may be collected from the nasal or oral mucosa of the subject. The mucosal sample may include nasal lavage, nasal discharge, saliva, or sputum.

Mucosal samples may be collected by contacting a swab with an interior surface of the nasal or oral mucosa and placing the swab in a buffer to provide a liquid sample.

The first and second label moieties are advantageously visible in the test zone without the need for additional instrumentation. The first and second label moieties can independently be cellulose nanobeads, colloidal gold, gold nanoshells, covalent gold, covalent latex beads, fluorescent beads, silver particles, platinum particles, fluorescent cyanic dyes, magnetic beads, upconverting phosphors, or any combination thereof.

Signal antibodies may be derived from a mouse, goat, camel, chicken, llama, rat, rabbit, or dog host. Capture antibodies may be derived from a mouse, goat, camel, chicken, llama, rat, rabbit, or dog host.

The immunoassay described herein may further comprise a housing comprising a sample portal exposing at least a portion of the sample application portion and at least one reading portal exposing the test zone and the control zone.

Kits also are provided by the present invention. A kit may comprise a lateral flow immunoassay of claim and a sample collection swab. The sample collection swab may comprise a collection tip, wherein the tip comprises a flocked material, a sponge, cotton, polypropylene, or polystyrene. The kit may further include a sample collection vessel comprising a liquid carrier, phosphate buffered saline, Tween 20, bovine serum albumin, one or more protease inhibitors, one or more preservatives, and sodium deoxycholate. The kit may further comprise a sample applicator.

The housing may further include a cavity for holding the sample collection swab and a cavity for holding a sample collection vessel.

The present invention also provides a method of detecting IP- 10 in a sample comprising: a. collecting the sample from the mucosa of a subject with a sample collection swab; b. adding the sample to a liquid carrier to form a liquid sample; c. applying the liquid sample to a sample application portion of a lateral flow immunoassay as described herein; d. allowing sufficient time for at least a portion of the liquid sample to traverse the sample application portion, the diagnostic antibody portion, and the detection portion of the lateral flow immunoassay; e. observing the detection portion of the lateral flow immunoassay wherein: i. an observed signal in the test zone indicates IP-10 is present in the sample at a concentration of at least 80 pg/ml; and ii. an observed signal in the assay control zone indicates the lateral flow immunoassay is functional.

An observed signal in the sample control zone advantageously indicates that a sufficient amount of the mucosal sample was applied to the assay.

The aforementioned subject may be asymptomatic. Asymptomatic subjects should undergo a further diagnostic test to confirm the subject has been infected with a respiratory pathogen.

Further diagnostic tests may detect infection with SARS-CoV-2, influenza viruses, parainfluenza viruses, adenoviruses, rhinoviruses, enteroviruses, human metapneumoviruses, respiratory syncytial viruses, coronaviruses, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, or Moraxella catarrhalis.

The subject may be symptomatic, for example with one or more symptoms selected from cough, difficulty breathing, fever, muscle aches, loss of taste, and loss of smell. Alternatively or additionally, the subject may be experiencing one or more symptoms selected from rhinorrhea, nasal congestion, sneezing, cough, fever, chills, fatigue, headache, pain during swallowing, wheezing, and mucus production. The subject may have experienced one or more symptoms for less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day. An observed signal in the test zone indicates that subjects should receive treatment for COVID-19, or other virus, and/or be quarantined.

The immunoassay may further be used to monitor the infectiousness of a subject infected with SARS-CoV-2, or other virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. l is a schematic drawing of an embodiment of a lateral flow immunoassay of the invention and several aspects of the immunoassay in operation.

FIG. 2 is a schematic drawing of an embodiment of a kit of the invention including a lateral flow immunoassay contained within a housing and a sample collection swab. FIG. 3 is a compilation of schematic drawings showing front, side, end, and rear views of an embodiment of a kit of the invention including a lateral flow immunoassay contained within a housing, where the housing further includes a concave portion adapted to store a sample collection swab.

FIG. 4 is a visual grade scale used for assessment of test line and control line intensity on lateral flow immunoassay test strips.

FIG. 5 is a photographic image showing test results on lateral flow immunoassay test strips specific for detecting IP-10, using antibody pair 555048/555046.

FIG. 6 is a photographic image showing test results on lateral flow immunoassay test strips specific for detecting IP-10, using antibody pair 555048/555046 and varying concentrations of sodium deoxycholate in the running buffer.

FIG. 7 is a is a photographic image showing test results on lateral flow immunoassay test strips specific for detecting IP- 10, showing improved assay performance (reduced aggregation in control line) upon addition of a sample pad.

FIG. 8 is a photographic image showing test results on a lateral flow immunoassay test strip specific for detecting IP- 10, showing the effects of increasing the concentrations of the cellulose nanobead-labelled antibody and the biotinylated antibody, respectively.

FIG. 9 is a photographic image showing test results on a lateral flow immunoassay for detecting IP- 10 showing a positive test line result, a positive sample control line, and a positive assay control line.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a portable, easy-to-use, rapid lateral flow immunoassay to detect IP- 10, alone or in combination with one or more additional cytokines in a mucosal sample. Such cytokines may include, e.g., interleukins IL- la, IL-ip, IL-6, IL-8, IL- 12, IL- 18, interferons IFN-a, IFN-P, and IFN-y, tumor necrosis factor alpha (TNF-a), granulocytemacrophage colony stimulating factor (GM-CSF), macrophage inflammatory proteins MIP- l or MIP-ip, or macrophage chemotactic proteins MCP-1, MCP-3, or TRAIL. In some embodiments, the invention provides a portable, easy-to-use, rapid lateral flow immunoassay for detecting only IP- 10 in a sample collected from a nasal cavity of a subject. Lateral flow immunoassays are generally based on well-understood immunoassay principles, which rely on an interaction between an antibody and its corresponding, specific analyte. These assays are advantageously highly selective and have low limits of detection. Other advantages of lateral flow immunoassays include ease of use and result interpretation, rapid results, prolonged shelf life at room temperature, and low-cost manufacture, among others.

As will be appreciated, the immunoassay of the invention will have many useful applications, including providing the ability to identify and quarantine infected individuals to prevent community spread of respiratory infections. Pathogens that cause respiratory illness are often highly contagious and capable of airborne transmission. Limiting exposure by quarantine of infected individuals is thus an effective public health measure. Such viruses include influenza viruses, parainfluenza viruses, adenoviruses, rhinoviruses, enteroviruses, human metapneumoviruses, respiratory syncytial viruses, and coronaviruses (e.g., including those that cause COVID-19). These pathogens are variously causally related to illnesses and syndromes in humans including the common cold, croup, influenza-like illness, bronchiolitis, and pneumonia, resulting in significant morbidity and mortality worldwide. Bacteria that cause respiratory illness are also highly contagious. Such bacteria may include Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis.

The invention is also useful in identifying asymptomatic carriers of respiratory infections. As used herein, “asymptomatic” carriers or subjects are those who may be infectious, but are not, or are not yet, exhibiting symptoms. This aspect of the invention will prove beneficial in that it can be administered, e.g., to visitors to extended care or health care facilities prior to permitting entry in order to protect vulnerable populations. In certain embodiments, the invention is directed to the detection of IP- 10 in a sample collected from a nasal cavity of a subject as a means of rapidly and easily identifying persons who are potentially infected with coronaviruses (e.g., including those that cause COVID-19).

Advantageously, the invention also provides a means by which infection with novel respiratory pathogens, for which diagnostic testing is not yet available or in short supply, can be detected. As will be appreciated, use of the immunoassay of the invention as a preliminary test will assist in prioritizing further diagnostic testing, for example for COVID-19, where specific diagnostic laboratory tests may be in short supply and must be sparely administered.

As cytokine responses to different respiratory pathogens are expected to be similar, infection with novel variants of respiratory pathogens would lead to elevated IP-10 levels in mucosal samples that can be detected by way of the present invention. In contrast to diagnostic strategies that are used to detect antigens or polynucleotides of specific pathogens, the present invention presents a diagnostic tool that is used to detect hallmarks of the host immune response to the pathogen. Some embodiments of the invention can be illustrated with reference to FIG. 1. A liquid sample 1 containing a cytokine or chemokine 2 (e.g. IP- 10), and a protein such as IgA 3 is applied to a sample application portion 4 of the immunoassay. The cytokine and IgA present in the sample travel by capillary action from the sample application portion 4 into the diagnostic antibody portion 5 having dispersed thereon signal antibodies 10 and 11, specific for IP-10 2 and IgA 3, respectively. Labels 10' and 11' are conjugated to antibodies 10 and 11. The cytokines bind specifically to the signal antibodies and the bound antibody complexes travel from the diagnostic antibody portion to the detection portion 6 with test zone 12 and sample control zone 13, each zone having fixed thereon capture antibodies 12' and 13' specific for cytokine 2 and IgA 3, and assay control zone 14 having fixed thereon capture antibodies 14' specific for antibodies 10. A wicking portion 7 draws the liquid through the immunoassay in the direction of flow designated by arrow 26. The sample application portion 4, diagnostic antibody portion 5, detection portion 6, and wicking portion 7 are disposed on a membrane support 8, and further supported by a rigid backing 9.

Although FIG. 1 illustrates an embodiment of the invention in which one cytokine (IP- 10) and IgA may be detected, some embodiments of the invention relate to detection of more than one cytokine.

Additionally, although FIG. 1 exemplifies a “sandwich” strategy, further embodiments of the invention include the use of a biotin-streptavidin amplification strategy. In these embodiments, the test zone in the detection portion has streptavidin fixed thereon. Unfixed biotin-tagged signal antibodies specific for the cytokine of interest (e.g., IP- 10) bind the streptavidin, and additional unfixed antibodies specific for the cytokine of interest (e.g., IP-10) bearing a label also will bind in the test zone to produce a visible signal.

FIG. 2 depicts an embodiment of a kit of the invention, showing a lateral flow immunoassay contained within a housing 15 and a sample collection swab 25. The housing containing the immunoassay includes a sample portal 21 for receiving the liquid sample from the sample collection swab. The housing further includes a reading portal 16 for test zone 17 and sample control zone 18, which correspond to test zone 12 and sample control zone 13 in FIG. 1, respectively. The housing also includes a reading portal 19 for assay control zone 20, which corresponds to assay control zone 14 in FIG. 1. The sample collection swab 25 includes reservoir 22 for carrier liquid, a hollow connecting tube 23 and a permeable collection tip 24. FIG. 3 depicts side (28, 29), top 30, bottom 31, end (32, 33), and perspective 34 views of an embodiment of the invention wherein a housing 35 contains test zones 36 and 37, control zone 38, and application zone 39. Housing 35 further includes a first cavity 40 adapted to hold and store a sample collection swab 41. The housing further includes a second cavity 43 for holding a sample collection container 44.

In some embodiments, the sample is collected from the mucosa, such as nasal or oral mucosa (e.g., nasal cavity) of a subject. As used herein, the term “nasal cavity” includes the nostrils, the nasal fossae, nasal turbinates, and the nasopharynx. In some embodiments, the sample is collected from one or both nostrils of the subject. In some embodiments, the sample is collected by contacting a flocked swab tip with the epithelial surfaces of both nostrils and placing the swab tip in a liquid carrier to provide a liquid sample. In some embodiments, the sample includes nasal lavage, nasal discharge, and/or sputum. The lavage, discharge, and/or sputum may also be added to a liquid carrier.

The sample application portion functions to evenly distribute the sample and direct it to the diagnostic antibody portion of the immunoassay. The sample application portion may include filters of varying pore size to remove, e.g., solids from the sample. It may be impregnated with buffers, salts, proteins, surfactants, or other components to prepare the cytokines, chemokines, and/or protein for optimum interaction with the other test components. Buffers include sodium chloride-based or potassium chloride-based solutions such as phosphate buffered saline, potassium buffered saline, or borate buffered saline. Materials for manufacture of the sample application portion include cotton, cellulose, glass fiber, rayon, and filter materials. The sample application portion of the lateral flow immunoassay may advantageously exhibit consistent absorbency, thickness, and density to ensure assay reproducibility. The material should exhibit low protein binding to avoid loss of cytokines. One exemplary sample application material is a FUSION 5 sample pad, produced by Whatman. One exemplary sample application material is an Ahlstrom 1667 sample pad. In some embodiments, the sample application portion is treated with phosphate-buffered saline, casein and PLURONIC F127 (Poloxamer 188, 2-methyloxirane). The treatment may be allowed to cure for at least 7 days.

In some embodiments, the sample application portion is communicably arranged adjacent to the diagnostic antibody portion. In other embodiments, the sample application portion and the diagnostic antibody portion are coextensive. By “coextensive,” it is meant that the sample is applied directly to the diagnostic antibody portion. The diagnostic antibody portion may be impregnated with a buffer for maintaining the unfixed signal antibodies. Buffers include sodium chloride-based or potassium chloride-based solutions such as phosphate buffered saline, potassium buffered saline, or borate buffered saline. In some embodiments, the buffer may contain salts, detergents, or carbohydrates which preserve the antibodies upon drying and aid in resolubilizing the antibodies upon encounter with the liquid sample containing the cytokines. Carbohydrates include trehalose, sucrose, glucose, and mannose. In some embodiments, the diagnostic antibody portion is impregnated with phosphate-buffered saline, PLURONIC F127, casein and borate. In some embodiments, the buffer maintains the pH of the diagnostic antibody portion at pH 4.0-10.0, pH 5.0-9.0, or pH 6.0-8.0. In some embodiments, the pH of the diagnostic antibody portion is pH 6.5-7.5, or pH 7.3-7.5.

The diagnostic antibody portion has dispersed thereon unfixed signal antibodies, as further described herein below. In some embodiments, the unfixed signal antibodies are specific for IP- 10. At least one population of the signal antibodies specific for IP- 10 include a label that is visible to the naked eye, i.e., detectable without instrumentation, as further described herein. In certain embodiments, the label comprises cellulose nanobeads, colloidal gold, gold nanoshells, covalent gold, covalent latex beads, fluorescent beads, silver particles, platinum particles, fluorescent cyanic dyes, magnetic beads, upconverting phosphors, or any combination thereof.

In some embodiments, biotin and streptavidin are incorporated into the lateral flow immunoassay to amplify the signal in the test zone. As used herein, “streptavidin” refers to streptavidin homo-tetramers or polystreptavidin species that bind non-covalently to biotin molecules with high affinity. In some embodiments, the diagnostic antibody portion has dispersed thereon a first population of unfixed signal antibodies specific for IP- 10, wherein the antibodies of the first population include a label moiety; and a second population of unfixed signal antibodies specific for IP- 10, wherein the antibodies of the second population include a biotin tag.

As used herein, a “population of antibodies” refers to antibodies having identical variable regions such that each antibody in the population binds to the same epitope of an analyte of interest and, if present, has the same label or tag. For example, one population can bind to an epitope of IP- 10 and include a label moiety and another population can bind to an epitope of IP- 10 and include a streptavidin, biotin, avidin, ruthenium, digoxin, azide, or desthiobiotin tag. A third population can bind to an epitope of TRAIL and include a label moiety and a fourth population can bind to an epitope of TRAIL and include a streptavidin, biotin, avidin, ruthenium, digoxin, azide, or desthiobiotin tag. In some embodiments, the diagnostic antibody portion has dispersed thereon 3, 4, 5, 6, 7, 8, 9, or 10 populations of antibodies. In some embodiments, the diagnostic antibody portion has dispersed thereon 1 population of antibodies. In some embodiments, the diagnostic antibody portion has dispersed thereon 2 populations of antibodies. The unfixed signal antibodies are specific for epitopes of the cytokines, chemokines, and/or proteins of interest, specifically epitopes derived from human cytokines that are secreted in nasal passages, as well as adjacent structures in the upper respiratory tract, in response to infection by respiratory pathogens, such as certain viruses and bacterial species. In some embodiments, the unfixed signal antibodies are specific for epitopes of IP- 10. In some embodiments, the signal antibodies may be monoclonal antibodies produced in non-human species such as, e.g., mouse, rabbit, goat, donkey, or rat species. In some embodiments, the signal antibodies are derived from a mouse host.

In some embodiments, the detection portion includes one or more control zones. In some embodiments, the control zone includes an assay control zone, wherein a positive signal indicates that the assay is functioning properly. In some embodiments, the detection portion has fixed thereon capture antibodies that specifically bind to the signal antibodies specific for the cytokines.

In other embodiments, the control zone further includes a sample control zone wherein a positive signal indicates that sufficient test material was present in the appropriate sample to permit detection of a biological sample. With respect to these embodiments, the detection portion has fixed thereon capture antibodies that specifically bind to IgA. As will be understood, the fixed capture antibodies in the control zones can be monoclonal antibodies raised in non-human species to specifically bind the signal antibodies specific for the biological control, or monoclonal antibodies raised in non-human species to specifically bind human IgA. In some embodiments, the fixed capture antibodies are specific for human IgA. In some embodiments, the signal antibodies specifically bind to one or more of interleukins IL-la, IL-ip, IL-6, IL-8, IL-12, IL-18, interferons IFN-a, IFN-P, and IFN-y, tumor necrosis factor alpha (TNF-a), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage inflammatory proteins MIP-la or MIP-ip, or macrophage chemotactic proteins MCP-1, MCP-3, or TRAIL. In some embodiments, the signal antibodies specifically bind to IP- 10. These cytokines are produced in the epithelium of the upper airway during respiratory infection and can be detected by way of the present invention. In some embodiments, the immunoassay of the invention includes at least two different populations of signal antibodies designed to detect the concurrent presence of at least two cytokines in a sample. In some embodiments, cytokines for detection by the present invention include IP- 10, IL-8, IL-6, IFN- a, TNF-a and IFN-y, TRAIL, and combinations thereof. In some embodiments, the immunoassay of the invention includes at least one population of signal antibodies designed to detect IP-10 in a sample. In some embodiments, the lateral flow immunoassay is designed to detect IP-10 only, i.e., is not designed to detect further cytokines.

Cytokines secreted by the nasal epithelium during respiratory infection are often produced in accordance with a specific time course, with some appearing early in infection and others appearing later, during resolution of infection. Therefore, some embodiments of the invention include a population of signal antibodies specific for a cytokine produced early in infection (e.g., IL-6, INF-a, IL-10, TRAIL) and a population of signal antibodies specific for a cytokine produced later in infection (e.g., TNF-a, IL-8, MIP-10). As will be appreciated, an immunoassay of the invention can include populations of signal antibodies for any number of different cytokines, advantageously covering those produced throughout the course of infection. In some embodiments, the invention includes a population of signal antibodies specific for IP- 10, which is produced throughout the time course of infection. In other embodiments, the invention includes a population of signal antibodies specific for IP- 10, a population of signal antibodies specific for a cytokine produced early in infection, and a population of signal antibodies specific for a cytokine produced later in infection.

In some embodiments, the immunoassay of the invention is configured to detect only IP- 10. In some embodiments, the assay is configured to detect one or more cytokines in addition to IP- 10.

The signal antibodies used in the diagnostic antibody portion are conjugated to a label that is visible to the naked eye when sufficient signal antibodies are in close proximity. In some embodiments, the label moiety is visible in the test zone without instrumentation. In some embodiments, the labels are conjugated to the signal antibodies using standard bioconjugation techniques (see, e.g., Hermanson, 2013). Particularly useful methods include conjugation to a free amine via alkylation or acylation, conjugation to a free carboxyl group via carbodiimide activation, conjugation to a free sulfhydryl group via alkylation, redox or Michael addition reaction. In some embodiments, labels for use with the invention: (1) permit simple conjugation chemistry without loss of biological integrity and activity; have no (or low) nonspecific binding under the salt, buffer, and detergent conditions of the immunoassay; (2) have high stability at different pH values and temperatures; are easily and inexpensively manufactured; and (3) have high color contrast against the detection portion of the immunoassay. In some embodiments, labels include colloidal gold nanoparticles or nanoshells, latex particles or microbeads, cellulose particles or microbeads, carbon particles or selenium particles. In some embodiments, the first and second label moieties can independently be cellulose nanobeads, colloidal gold, gold nanoshells, covalent gold, covalent latex beads, fluorescent beads, silver particles, platinum particles, fluorescent cyanic dyes, magnetic beads, upconverting phosphors, or any combination thereof.

The labels may be colored. In some embodiments, the label conjugated to a signal antibody is unique to the cytokine, chemokine, or protein that the signal antibody specifically binds. Detection and identification of multiple cytokines, chemokines, and/or proteins in a sample thereby corresponds to a unique visible color for each cytokine, chemokine, or protein. For example, a label visible as a blue color in a test zone may correspond to the presence of a first cytokine in the sample and a pink color in a test zone may correspond to the presence of a second cytokine in the sample. In alternate embodiments, different populations of signal antibodies may be conjugated to the same label, such that a positive signal in any of the test zones will indicate that at least one cytokine was detected in the sample.

Several different embodiments of the detection portion are within the scope of the invention. In some embodiments, the detection portion is made of a polymeric material such as nitrocellulose, nylon or polyvinyl fluoride, or combinations thereof. In some embodiments, the detection portion has at least two test zones, each test zone having fixed thereon capture antibodies that are specific for the same cytokines as (and thus correspond to) one of the populations of signal antibodies. In some embodiments, the detection portion comprises a test zone having fixed thereon capture antibodies that specifically bind to signal antibodies specific for IP- 10. In other embodiments, the detection portion comprises a test zone comprising streptavidin fixed thereon. In some embodiments, streptavidin at a concentration of 0.5 mg/mL is applied to the test zone at a dispensing rate of 1 pL/cm.

As will be understood, the test zones can be arranged on the detection portion in any one- or two-dimensional shape, as long as the aggregated fixed capture antibodies are present in sufficient amount and at sufficient density such that a positive signal may be visible upon binding of the cytokine-signal antibody complexes to the fixed capture antibodies. In some embodiments, streptavidin, biotin, avidin, ruthenium, digoxin, azide, or desthiobiotin is fixed in the test zone in sufficient amount and at sufficient density such that a positive signal may be visible upon binding of 1) the biotinylated signal antibodies and 2) the labeled signal antibodies. In some embodiments, the test zones are arranged as single lines. In other embodiments, the test zones are arranged in circles or squares. As will be appreciated, any shape may be used, limited only by the unidirectional flow of the immunoassay. In an alternative arrangement of the lateral flow immunoassay, separate immunoassay strips are used to detect each cytokine or control of interest. The separate immunoassay strips can be conveniently arranged side-by side in a housing to provide a simple, easy-to-use readout format.

The detection portion further comprises a control zone having fixed thereon capture antibodies that specifically bind to the signal antibodies, wherein a positive signal indicates that the appropriate sample has been added, and that the assay is functioning properly. In some embodiments, the detection portion comprises a control zone having fixed thereon capture antibodies that specifically bind to the signal antibodies specific for IP-10. In some embodiments, the non-human species in which the control zone capture antibodies are raised may be a mouse, rabbit, goat, donkey, or rat. In some embodiments, the capture antibodies are derived from a goat host. As will be understood, the control zones can be arranged on the detection portion in any one- or two-dimensional shape, as long as the aggregated fixed capture antibodies are present in sufficient amount and at sufficient density such that a positive signal (caused by binding of the signal antibodies to the fixed capture antibodies) may be visible upon binding of the signal antibodies. In some embodiments, the control zones are arranged as single lines. In other embodiments, the control zones are arranged in circles or squares. As will be appreciated, any shape may be used, limited only by the unidirectional flow of the immunoassay.

The wicking portion of the immunoassay pulls the liquid from the liquid sample sequentially through the sample application portion, the diagnostic antibody portion, the detection portion and into the wicking portion. In some embodiments, each of these portions are communicably arranged adjacent to the previous and subsequent portions. In some embodiments, at least one of these portions (e.g., the sample application portion) is communicably arranged adjacent to or coextensive with a subsequent portion (e.g., the diagnostic antibody portion). As used herein, “communicably arranged” means that the portions are in sufficient contact, either through a lateral edge of each portion or by partially overlapping the portions, such that capillary action draws liquid through one portion to the portion in contact with it. Specifically, in some embodiments, the sample application portion is communicably arranged adjacent to or coextensive with the diagnostic antibody portion, the diagnostic antibody portion is communicably arranged adjacent to the detection portion, and the wicking portion is arranged so that liquid flows sequentially through these portions. In some embodiments, the diagnostic antibody portion, the detection portion and the wicking portion are disposed on a membrane.

In some embodiments, the wicking portion is made of cotton or high-density cellulose. In some embodiments, the wicking portion serves as a sink for excess liquid and prevents liquid from traveling in the opposite direction, which could result in false positives. In some embodiments, the wicking portion comprises cotton fibers.

The membrane support of the immunoassay is generally made of an inert polymeric material that will not interfere with the test conditions. Several possible materials include nitrocellulose, nylon, poly ethersulfone, polyethylene or fused silica, or combinations thereof. In some embodiments, the membrane comprises nitrocellulose.

In some embodiments, a rigid backing is provided if the membrane support is not sufficiently rigid to support the test strip of the lateral flow immunoassay. In some embodiments, a backing card supports the membrane. In certain embodiments, the backing may be made of polystyrene, polyvinyl chloride or polyester, or combinations thereof. In some embodiments, the backing comprises a vinyl polymer. In some embodiments, the wicking portion comprises cotton fibers, the membrane comprises nitrocellulose, and the backing comprises a vinyl polymer.

In some embodiments, the immunoassay is placed inside and protected by a housing to preserve the assay from environmental conditions and contaminants. In some embodiments, the housing is made from a plastic material such as polypropylene, polystyrene, or copolymers thereof. In some embodiments, the housing includes a sample application portal for application of the liquid sample to the sample application portion of the immunoassay. In some embodiments, the housing includes portals for viewing the test zones and control zones. As will be understood, the housing may have individual portals for each test zone and control zone. Alternatively, the housing may have a single portal that shows all test zones and control zones. A further embodiment provides a single portal that shows all test zones and a single portal for the control zone or zones. As will be understood, any configuration of portals is within the scope of the invention, on the condition that all test zones and control zones are visible to the user. In some embodiments, the housing comprises a sample portal exposing at least a portion of the sample application portion and at least one reading portal exposing the test zone and the control zone. In some embodiments of the invention, the sample collection swab is stored within a concave region of the housing. In some embodiments, the housing further comprises a cavity for holding a sample collection vessel.

In some embodiments, the lateral flow immunoassay (which may be contained within a housing) is included in a kit with a sample collection swab having a collection tip. The sample collection swab is in some embodiments made of a flexible plastic material such as, e.g., polypropylene, polystyrene, or copolymers thereof. In some embodiments, the tip may be manufactured from a flocked material, including but not limited to, cotton, polyester, paper, polyvinyl chloride, nylon and rayon. In some embodiments, the tip may be manufactured from flocked polyester.

The kit further may further include an inert carrier liquid that is compatible with the sample and which does not interfere with operation of the immunoassay. The liquid carrier may be an aqueous liquid carrier, such as buffered saline. Proteins may be added to the liquid carrier to stabilize the sample. Such proteins may include albumin, casein, and agar. Surfactants may be added to improve solubilization of the cytokines to be detected. Surfactants may include Tween compounds, polysorbates, and Triton-X. In some embodiments, the liquid carrier solubilizes the cytokines in the sample and facilitates binding by the signal antibodies. In some embodiments, the buffer is phosphate buffered saline. Other liquid carrier components may include Tween 20, bovine serum albumin, one or more protease inhibitors, and one or more preservatives. In particular embodiments, the liquid carrier comprises phosphate buffered saline, sodium deoxycholate, Tween 20, bovine serum albumin, one or more protease inhibitors, and/or one or more preservatives. In some embodiments, sodium deoxycholate is present in the liquid carrier at a concentration of 0.125% wt/vol.

In some embodiments, the liquid carrier is included in the kit in a sample collection vessel. The sample collection vessel can be manufactured from a rigid or semi-flexible material, such as, e.g., plastics or glass. The vessel may contain between 10 microliters and 1 milliliter of liquid carrier. The vessel may also contain between 50 microliters and 500 microliters, between 100 microliters and 500 microliters, between 200 microliters and 500 microliters, between 200 microliters and 400 microliters, or between 200 microliters and 300 microliters.

In alternative embodiments, the sample collection swab includes a reservoir for the liquid carrier, a hollow connecting tube, and a permeable collection tip. In some embodiments, the sample collection swab is a unitary article, including a reservoir for a carrier liquid, a hollow connecting tube and a permeable collection tip. In some embodiments, the permeable collection tip of the sample collection swab is of a different material from the reservoir and hollow connecting tube. The reservoir of the collection swab in some embodiments contains between 10 microliters and 1 milliliter. The reservoir may also contain between 50 microliters and 500 microliters, between 100 microliters and 500 microliters, between 200 microliters and 500 microliters, between 200 microliters and 400 microliters, or between 200 microliters and 300 microliters. Upon squeezing or depressing the reservoir, the liquid carrier is forced through the hollow connecting tube and the sample present on the permeable collection tip is wetted. In some embodiments, a single depression of the reservoir forces the liquid through the hollow connecting tube, wets the sample, and applies the sample to the sample application portion of the immunoassay by dripping or pressing the collection tip to the surface of the sample application portion of the lateral flow immunoassay. In other embodiments, a first depression is used to wet the permeable collection tip and a second depression applies the sample to the immunoassay. In some embodiments, the permeable collection tip is pre-wetted and the sample collection swab is placed in a container or package to maintain the moisture of the collection tip.

In some embodiments, the kit further comprises a sample applicator. The sample applicator may be used to apply the liquid sample (i.e., the sample collected from the nasal passages that has been added to the inert liquid carrier) to the sample application portion of a lateral flow immunoassay. Examples of sample applicators suitable for use with the invention include plastic pipettes, serological pipettes, micropipettes, and droppers.

In some embodiments, the kit may further include instructions for use of the kit. The instructions may inform the user regarding how to collect the sample, how to apply the sample to the immunoassay, and how to read and interpret the results.

The invention further includes methods of detecting one or more cytokines and/or chemokines in a sample. Cytokines and chemokines that may be detected by the methods include interleukins IL-1, IL-ip, IL-6, IL-8, IL- 12, IL- 18, interferons IFN-a and IFN-y, tumor necrosis factor alpha (TNF-a), granulocyte-macrophage colony stimulating factor (GM-CSF), IP- 10, and/or macrophage inflammatory protein (MIP-ip), monocyte chemoattractant protein 1 (MCP-1) or MCP-3, or TRAIL. In certain embodiments, the methods of the invention detect IP- 10 present in the liquid sample. IP- 10 may be the only cytokine detected by the methods, or additional cytokines may be detected with IP- 10. In other embodiments, two or more cytokines and/or chemokines are detected by the methods. In certain such embodiments, 3, 4, 5, 6, 7, 8, 9, or 10 cytokines are detected by the methods.

The methods of the invention include a step of collecting a sample from the nasal cavity of a subject and adding the sample to a liquid carrier, such as a buffer, to form a liquid sample. In some embodiments, the sample is collected by placing a collection tip of a sample collection swab inside the nasal passages (e.g., inside the nostrils) of the subject. In some embodiments, the sample collection swab contacts the nasal mucosa inside one or both nostrils. In other embodiments, the subject collects nasal discharge or nasal lavage and adds the discharge or lavage to a liquid carrier to form a liquid sample.

In some embodiments, the liquid sample is added to a sample application portion of a lateral flow immunoassay by using a sample applicator. In other embodiments, the liquid sample is added to the lateral flow immunoassay by depressing the reservoir of a sample collection swab and applying the liquid sample to the sample application portion of the immunoassay.

After application of the liquid sample to the lateral flow immunoassay, sufficient time is allowed for the liquid sample to travel from the sample application portion, through the diagnostic antibody portion and into the detection portion of the immunoassay. The liquid travels sequentially through the portions of the immunoassay by way of wicking, or capillary action. If the cytokine (e.g., IP-10) is present in the sample, a signal will be observed in the test zones of the detection portion of the immunoassay. As will be appreciated, the length of time required for development of signal in the test zones will vary depending on the materials used to construct the immunoassay, the liquid carrier, the concentration of cytokines present in the sample and other factors. Generally, a signal representing a positive result can be detected in one or more test zones and the control zone of the lateral flow immunoassay of the invention about 5-30 minutes after application of the sample. In some embodiments, signals in the test and control zones develop within 15 minutes of addition of the liquid sample to the immunoassay. As will be appreciated, observation of the sample control line signal in the sample control zone indicates that the appropriate sample has been added, and observation of the assay control line signal in the assay control zone indicates that the immunoassay is functional. As used herein, “functional” means that the immunoassay has operated according to its validated use.

In particular embodiments, the invention is directed to a method of detecting IP- 10 in a sample. The method includes a step of collecting the sample from the nasal cavity of a subject. In some embodiments, the sample is collected by contacting the interior surfaces of one or both nostrils with the sample collection swab. The collected sample on the swab is added to a liquid carrier to form a liquid sample.

A further step includes applying the liquid sample to a sample application portion of a lateral flow immunoassay constructed to detect IP- 10 in the liquid sample. As will be appreciated, the lateral flow immunoassay used in the methods of the invention may be any of the embodiments of lateral flow immunoassays described herein, expressly including those included in kits described herein.

A further step of the method requires allowing sufficient time for at least a portion of the liquid sample to traverse the sample application portion, a diagnostic antibody portion and a detection portion of the lateral flow immunoassay. In some embodiments, the “sufficient time” is 5-30 minutes, or 15 minutes or less.

After the liquid sample has traversed the lateral flow immunoassay, the detection portion of the lateral flow immunoassay is observed. If IP-10 is present in the sample, a visible signal will be generated in the test zone of the immunoassay. A visible sample control line signal in the control zone indicates the appropriate sample was added to the assay. A visible assay control line signal in the control zone indicates the immunoassay is functional. In some embodiments signals in the test and control zones are visible without the use of instrumentation. As used herein “visible without the use of instrumentation” means that the signal can be observed with the naked eye. An observed signal in the test zone indicates IP- 10 is present in the sample at a concentration of at least about 50-200 pg/mL. In some embodiments, the observed signal indicates that IP- 10 is present in the sample at a concentration of at least about 100-200 pg/mL. In some embodiments, the observed signal indicates that IP- 10 is present in the sample at a concentration of at least about 80 pg/mL, or at least 150 pg/mL.

In some embodiments, the lateral flow immunoassay used in the methods is constructed to detect only IP- 10. In other embodiments, the lateral flow immunoassay is constructed to detect one or more additional cytokines. In some embodiments, an observed signal in the test zone indicates a further diagnostic test should be conducted to determine whether the subject has been infected with a respiratory pathogen. In some embodiments, the respiratory pathogen is SARS-CoV-2. In some embodiments, the respiratory pathogen is a viral pathogen, e.g., selected from influenza viruses, parainfluenza viruses, adenoviruses, rhinoviruses, enteroviruses, flavivirus, human metapneumoviruses, respiratory syncytial viruses, and coronaviruses. In some embodiments, the respiratory pathogen is a bacterial pathogen selected from Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, or Moraxella catarrhalis, for example. The further diagnostic test can be any diagnostic test known in the art to detect infection by specific pathogens. Such tests may include, but are not limited to, polymerase chain reaction (PCR), enzyme-linked immunosorbent assays (ELISA), electrochemiluminescence (ECL) immunoassays, multiplex assays such as LUMINEX, western blotting, flow cytometry, and bacterial culture, and identification. Such tests may also include respiratory pathogen panels and/or other high throughput screening tests.

In some embodiments, the subject is asymptomatic. In other embodiments, the subject is experiencing one or more symptoms selected from cough, difficulty breathing, fever, loss of taste and loss of smell. In some embodiments, the subject is experiencing one or more symptoms selected from rhinorrhea, nasal congestion, sneezing, cough, fever, fatigue, headache, pain during swallowing, wheezing, and mucus production. In some embodiments, the subject has experienced the one or more symptoms for less than 8 days. In some embodiments, the subject has experienced one or more symptoms for less than 7 days, less than 6 days, less than 5 days less than 4 days, less than 3 days, less than 2 days, or less than 1 day.

In some embodiments, an observed signal in the test zone indicates the subject should receive treatment for a respiratory virus infection. In some further embodiments, the respiratory virus is SARS-CoV-2. In some embodiments, an observed signal in the test zone indicates the subject should receive treatment for COVID-19. In some embodiments, an observed signal in the test zone indicates the subject should be quarantined. As used herein, “quarantine” has its customary meaning as used by those of skill in the art of public health. In some embodiments, the subject may be quarantined until symptoms have subsided, and/or the subject is no longer infectious. In some embodiments, the subject may choose to selfquarantine for a period of time to ensure an absence of infection (e.g., in the case of negative results), or until symptoms have subsided (e.g., in the case of positive results), and/or the subject is no longer infectious (e.g., in the case of serial testing).

The invention further includes methods for determining whether a subject suspected of infection with a respiratory pathogen should be quarantined. The steps of the method include: collecting a sample from the nasal cavity of the subject with a sample collection swab; adding sample to a liquid carrier to form a liquid sample; applying the liquid sample to a sample application portion of a lateral flow immunoassay constructed to detect IP- 10 in the liquid sample; allowing sufficient time for at least a portion of the liquid sample to traverse the sample application portion, a diagnostic antibody portion and a detection portion of the lateral flow immunoassay; and observing the detection portion of the lateral flow immunoassay wherein an observed signal in a test zone indicates the subject should be quarantined, an observed sample control line signal in the sample control zone indicates the appropriate sample has been added, and an observed assay control line signal in the assay control zone indicates the lateral flow immunoassay is functional. As will be understood, the lateral flow immunoassay used in the method may be any embodiment described herein, including those present in the kits of the invention.

In some embodiments, the subject suspected of infection with a respiratory pathogen was in contact with a person known or suspected to be infected with a respiratory pathogen within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days or within 1 day prior to collecting the sample. As will be appreciated, “known or suspected to be infected” includes persons who are experiencing symptoms consistent with respiratory infection, as well as persons who have received a diagnosis of infection with a known or unknown respiratory pathogen (whether experiencing symptoms or not). “Contact,” as used herein, has its customary meaning as used by those of skill in the art of public health. In some embodiments, the subject is asymptomatic. In some embodiments, the subject is experiencing one or more symptoms selected from cough, difficulty breathing, fever, loss of taste and loss of smell. In some embodiments, the subject is suspected of infection with SARS-CoV-2.

The invention further includes methods for monitoring the infectiousness of a subject infected with SARS-CoV-2. The method includes steps of collecting the sample from the nasal cavity of a subject with a sample collection swab; adding the sample to a liquid carrier form a liquid sample, applying the liquid sample to a sample application portion of a lateral flow immunoassay constructed to detect IP- 10, allowing sufficient time for at least a portion of the liquid sample to travel from the sample application portion through a diagnostic antibody portion of the lateral flow immunoassay, and subsequently through a detection portion of the lateral flow immunoassay, and observing the detection portion of the lateral flow immunoassay. The immunoassay may be any embodiment described herein, including those provided in kits. The steps of the method may also include any embodiment of the method of detecting IP-10 in a sample described herein. An observed signal in a test zone indicates IP-10 is present in the sample, an observed sample control line signal in a control zone indicates the appropriate sample has been added, and an observed assay control line signal in a control zone indicates the lateral flow immunoassay is functional. The steps of the method are repeated at regular intervals to support serial testing, such as testing daily, every other day, every third day, every fourth day, or weekly, until there is an absence of signal in the test zone. As will be appreciated, such absence indicates the subject is not infectious.

The invention further includes use of the currently described invention in tandem with the use of a read out from an alternate testing kit which is specific for detection of the pathogen of interest, such as an antigen testing kit or PCR test method. The method includes use of the kit prior to, in conjunction with, or subsequent to analysis with the paired alternate test method. The invention further supports the consolidation of the currently described method into a single test in conjunction with an alternate test method (e.g., to provide additional information in the case a patient is negative on a SARS-CoV-2 test).

EXAMPLES

The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

Example 1. Screening of Antibodies and Identification of Antibody Pair for Detection of IP- 10

A series of antibodies were screened to find a unique pair that produced the strongest test line signal, while maintaining low non-specific binding. Antibodies were screened in both a traditional sandwich assay and biotin system. The biotin system was shown to exhibit superior performance. In this system, the following antibody pairs (identified by catalogue number) resulted in the best performance:

BD= BD Biosciences; R&D= R&D Systems;

BD555046 - clone 4D5/A7/C5, purified mouse anti-human IP-10;

BD555048 - clone 6D4/D6/G2, Biotin mouse anti-human IP-10;

R&D MAB2661 - recombinant monoclonal mouse IgGl, anti-human IP-10 Clone # 33008R;

R&D AF266NA — polyclonal goat IgG anti -human IP- 10

The antibody pair (BD 555048/BD 555046) produced a clear test line (3.5 on a 0-7 visual scale, see FIG. 4) at 0.2 ng/mL recombinant IP- 10 diluted in running buffer, with no non-specific binding observed. See FIG. 5.

Example 2. Reduction of non-specific binding in prototype LFA strips

To further reduce non-specific binding, sodium deoxy cholate was added to the running buffer at varied concentrations (0.125%, 0.25%, 0.5%). The addition of 0.125% sodium deoxycholate improved the performance of the test kit by reducing non-specific binding. See FIG. 6.

Example 3. Reduction of aggregation due to sample viscosity

Inconsistencies observed in the control line were believed to be due to physical hindrance caused by sample viscosity. In order to address the observed aggregation, a Fusion 5 sample pad (treated with PBS, 0.25% casein and 1% Pluronic F127) was added to the test strip. The addition of the Fusion 5 sample pad for these samples significantly improved performance, demonstrating the cellulose nanobead-labeled antibody /IP- 10 conjugates were travelling the full length of the test strip. See FIG. 7.

Example 4. Titration of antibody concentrations

In order to compensate for reduction in the positive signals with addition of sample pad, as described in Example 3, the concentration of red cellulose nanobead (CNB)-labelled antibody and biotinylated antibody were respectively varied. Increasing either the biotinylated antibody or the cellulose nanobead-labelled antibody improved the test line signals by 1 visual grade (FIG. 4). However, when both the CNB conjugate and biotinylated antibody concentrations were increased, non-specific binding also increased. The best differentiation was seen with the CNB concentration was increased to 0.01% while the biotinylated antibody concentration remained at 0.01 pg/strip. See FIG. 8. An exemplary a lateral flow immunoassay for detecting IP- 10 is depicted in Fig. 9, showing a positive test line result, a positive sample control line, and a positive assay control line.

Example 5. Prototype LFA Materials and buffer compositions

Example 6. IP-10 is correlated with SARS-CoV-2 infection in cohort of COVID-19 clinic patients

Subjects presenting at a COVID-19 clinic seeking PCR diagnostic testing agreed to provide mucosal samples for detection of IP-10 by LFA. Swabs from each nostril were taken from each of 68 patients, exhibiting varying degrees of COVID-associated symptoms from 0 to 9 days prior to sample collection. For LFA testing, swabs were added to a sample buffer containing lx phosphate buffered saline (PBS), 0.1% Tween 20, 1% bovine serum albumin, lx Protease Inhibitor Cocktail Set I (Sigma-Aldrich cat. no. 539131-1 VL, containing AEBSF hydrochloride, aprotinin, E-64 protease inhibitor, EDTA and Leupeptin) and 0.05% Proclin 300 (Sigma-Aldrich cat no. 48912-U containing a 3: 1 mixture of 5-Chloro-2-methyl-4- isothiazolin-3-one and 2-methyl-2H-isothiazol-3-one). The sample was then diluted into running buffer (at a ratio of 2: 1 sample to running buffer) containing lx PBS, 0.1% Tween 20 and 0.125% sodium deoxycholate. 100 pl of the mixtures were then added to LFA test strips capable of detecting 150 pg/mL IP- 10. The LFA results were compared to PCR results as follows:

Additional subjects (including asymptomatic subjects) presenting at a COVID-19 clinic seeking PCR diagnostic testing agreed to provide mucosal samples for detection of IP- 10 by LFA. Swabs from each nostril were taken from each of 122 patients, exhibiting varying degrees of COVID-associated symptoms from 0 to 10 days prior to sample collection. For LFA testing, swabs were added to a sample buffer containing lx phosphate buffered saline (PBS), 0.1% Tween 20. The sample was then diluted into running buffer (at a ratio of 2: 1 sample to running buffer) containing lx PBS, 0.1% Tween 20 and 0.125% sodium deoxy cholate. 100 pl of the mixtures were then added to LFA test strips capable of detecting 150 pg/mL IP- 10. In addition to the samples noted above, the LFA results were compared to PCR results as follows:

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. REFERENCES

All references cited throughout the specification, including the following references, are herein specifically incorporated by reference:

Ahn D, et al., Current Status of Epidemiology, Diagnosis, Therapeutics, and Vaccines for Novel Coronavirus Disease 2019 (COVID-19). J. Microbiol. Biotechnol. 2020;

30(3):313-24.

Alper CM et al., Nasal Secretion Concentrations of IL-5, IL-6 and IL-10 in Children With and Without Upper Respiratory Tract Viruses. Arch Otolaryngol Head Neck Surg. 2010; 136(3):281-86.

Bobosha K et al., Field-Evaluation of a New Lateral Flow Assay for Detection of Cellular and Humoral Immunity against Mycobacterium leprae, PLoS Negl Trop Dis. 2014; 8(5):e2845, doi: 10.1371/journal.pntd.0002845.

Bunker et al., IgA Responses to Microbiota. Immunity. 2018; 49(2): 211-224.

Cheermala, N., et.al. Dynamic Innate Immune Response Determines Susceptibility to SARS-CoV-2 Infection and Early Replication Kinetics, J Exp Med. 2021; 218(8): 1 - 18.

Chen Y et al., IP-10 and MCP-1 as Biomarkers Associated with Disease Severity of COVID-19, Mol. Med. 2020; 26:97, 1-12.

Chi Y et al., Serum Cytokine and Chemokine Profile in Relation to the Severity of Coronavirus Disease 2019 in China, J Infectious Diseases (JID) 2020; 222:746-54.

Doyle WJ et al., Nasal Cytokines as Mediators of Illness During the Common Cold. Current Allergy and Asthma Reports 2005; 5: 173-81.

Fritz RS, Hayden FG, Calfee DP, Cass LM, Peng AW, Alvord, WG, Strober W, and Strauss SE. Nasal Cytokine and Chemokine Responses in Experimental Influenza A Virus Infection: Results of a Placebo Controlled Trial of Intravenous Zanamivir Treatment. J Inf Disease 1999; 180:586-93.

Gogate N et al., COVID-19 Biomarkers in Research: Extension of the OncoMX Cancer Biomarker Data Model to Capture Biomarker Data from Other Diseases, Preprint, bioRxiv. 2020; 2020.09.09.196220, doi: 10.1101/2020.09.09.196220.

Hayden FG, Fritz RS, Lobo MC, Alvord WG, Strober W, Straus SE. Local and systemic cytokine responses during experimental human influenza A virus infection. J Clin Invest 1998; 101 :643-9. Hayney MS et al., Serum IFN-y-Induced Protein 10 (IP-10) as a Biomarker for Severity of Acute Respiratory Infection in Healthy Adults, J Clinical Virology 2017; 90:32- 37.

Heggestad JT et al., Multiplexed, Quantitative Serological Profiling of COVID-19 from a Drop of Blood by a Point-of-Care Test, Preprint, medRxiv. 2020, 2020. 11.05.2022665'4, doi: 10.1101/2020.11.05.20226654.

Hermanson, Bioconjugate Techniques. 3^rd Ed., Academic Press 2013.

Hue S et al., Uncontrolled Innate and Impaired Adaptive Immune Responses in Patients with COVID-19 Acute Respiratory Distress Syndrome, Am J Respir Crit Care Med 2020; 202(11):1509-1519.

Kim YK et al., A Novel Diagnostic Approach to Detecting Porcine Epidemic Diarrhea Virus: The Lateral Immunochromatography Assay. J Virol. Methods 2015; 225:4-8.

Koczula KM and Gallotta A, Lateral Flow Assays. Essays in Biochemistry 2016; 60: 111-20.

Laham FR et al., Differential Production of Inflammatory Cytokines in Primary Infection with Other Common Respiratory Viruses of Infancy, J Infectious Diseases 2004; 189:47-56.

Lewis TC et al., Nasal Cytokine Responses to Natural Colds in Asthmatic Children, Clinical & Experimental Allergy 2012; 42: 1734-1744.

Lillard JW, Anti-CXCL9, Anti-CXCLIO, Anti-CXCLl l, Anti-CXCL13, Anti- CXCR3 and Anti-CXCR5 Agents for Inflammatory Disorders, U.S. Patent No. 8,563,476.

Oved K et al., Signatures and Determinants for Diagnosing Infections and Methods of Use Thereof, U.S. Patent No. 9,726,668.

Ozsurekci Y et al., Predictive Value of Cytokine/Chemokine Responses for the Disease Severity and Management in Children and Adult Cases with COVID-19, J Med Virol. 2020; 1-10.

Posthuma-Trumpie GA et al., Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal. Bioanal. Chem. 2009; 393:569-82.

Sajid M et al., Designs, formats, and applications of lateral flow assay: A literature review. J. Saudi Chem. Soc. 2015; 19:689-705.

Sutherland JS et al., Use of Lateral Flow Assays to Determine IP-10 and CCL4 Levels in Pleural Effusions and Whole Blood for TB Diagnosis, Tuberculosis 2016; 96:31-36. Turner RB et al., Association Between Interleukin-8 Concentration in Nasal Secretions and Severity of Symptoms of Experimental Rhinovirus Colds. Clin. Infectious Diseases 1997; 26:840-6.

Use of International Standard ISO 10993-1, "Biological evaluation of medical devices - Part 1 : Evaluation and testing within a risk management process" 2016. Guidance for Industry and Food and Drug Administration Staff.

Yetisen AK et al., Paper-based microfluidic point-of-care diagnostic devices. Lab Chip 2013; 13:2210-51.

Claims

CLAIMS We claim:

1. A lateral flow immunoassay for detecting IP- 10 in a mucosal sample from a subject, comprising: a. a sample application portion; b. a diagnostic antibody portion having dispersed thereon a first population of unfixed signal antibodies specific for IP- 10, wherein the antibodies of the first population comprise a label moiety, and c. a detection portion comprising a test zone; wherein at least one cytokine comprises IP- 10.

2. The immunoassay of claim 1, wherein the detection zone further comprises an assay control zone having fixed thereon capture antibodies that specifically bind to the first population of unfixed signal antibodies specific for IP- 10.

3. The immunoassay of claim 1 or 2, wherein the antibody portion further comprises a population of unfixed signal antibodies specific for IgA, wherein the antibodies specific for IgA comprise a second label moiety, and the detection portion further comprises a sample control zone having fixed thereon capture antibodies that specifically bind to the signal antibodies specific for IgA.

4. The immunoassay of any preceding claim, further comprising a second population of unfixed signal antibodies specific for IP- 10, wherein the antibodies of the second population comprise a tag.

5. The immunoassay of claim 4, wherein the tag comprises biotin, streptavidin, avidin, ruthenium, digoxin, azide, or desthiobiotin.

6. The immunoassay of any preceding claim, wherein the test zone comprises streptavidin, biotin, avidin, ruthenium, digoxin, azide, or desthiobiotin fixed thereon.

7. The immunoassay of any preceding claim, further comprising

29 d. a wicking portion; e. a membrane on which the diagnostic antibody portion, the detection portion, and the wicking portion are disposed; and f. a backing card supporting the membrane,

8. The immunoassay of claim 7, wherein: i. the sample application portion is communicably arranged adjacent to or coextensive with the diagnostic antibody portion; ii. the diagnostic antibody portion is communicably arranged adjacent to the detection portion; and iii. the wicking portion is disposed on the membrane such that the sample flows from the sample application portion through the diagnostic antibody portion and subsequently through the detection portion.

9. The immunoassay of claim 7, wherein: i. two assay strips are arranged in parallel within one kit housing to accommodate an additional cytokine or chemokine or control; and ii. each sample application portion is communicably arranged adjacent to or coextensive with the diagnostic antibody portion; iii. each diagnostic antibody portion is communicably arranged adjacent to the detection portion; and iv. each wicking portion is disposed on the membrane such that the sample flows from the sample application portion through the diagnostic antibody portion and subsequently through the detection portion.

10. The immunoassay of any preceding claim, wherein the assay is configured to detect only IP- 10.

11. The immunoassay of any one of claims 1 to 8, wherein the assay is configured to detect one or more cytokines in addition to IP- 10 selected from IL- la, IL-ip, IL-6, IL-8, IL-

12. IL-18, IFN-a, IFN-p, IFN-y, TNF-a, GM-CSF, MIP-la, MIP-lp, MCP-1, MCP-3, TRAIL, and any combination thereof.

30

12. The immunoassay of claim 11, wherein the one or more cytokines in addition to IP-10 is selected from IL-8, IL-6, IFN-a, TNF-a, IFN-y, TRAIL, and any combination thereof.

13. The immunoassay of any preceding claim, wherein the mucosal sample is collected from the nasal or oral mucosa of the subject.

14. The immunoassay of any preceding claim, wherein the mucosal sample comprises nasal lavage, nasal discharge, saliva, or sputum

15. The immunoassay of any preceding claim, wherein the sample is collected by contacting a swab with interior surfaces of the nasal or oral mucosa and placing the swab in a buffer to provide a liquid sample.

16. The immunoassay of any preceding claim, wherein the first and second label moi eties are visible in the test zone without instrumentation.

17. The immunoassay of any preceding claim, wherein the first and second label moi eties independently comprise cellulose nanobeads, colloidal gold, gold nanoshells, covalent gold, covalent latex beads, fluorescent beads, silver particles, platinum particles, fluorescent cyanic dyes, magnetic beads, upconverting phosphors, or any combination thereof.

18. The immunoassay of any preceding claim, wherein the signal antibodies are derived from a mouse, goat, camel, chicken, llama, rat, rabbit, or dog host.

19. The immunoassay of claim 18, wherein the signal antibodies are derived from a mouse host.

20. The immunoassay of claim 18, wherein the capture antibodies are derived from a goat host.

21. The immunoassay of any preceding claim, wherein streptavidin at a concentration of 0.5 mg/mL is applied to the test zone at a dispensing rate of 1 pl/cm.

22. The immunoassay of any one of claims 7 to 21, wherein the wicking portion comprises cotton fibers, the membrane comprises nitrocellulose, and the backing comprises a vinyl polymer.

23. The immunoassay of any preceding claim, further comprising a housing comprising a sample portal exposing at least a portion of the sample application portion and at least one reading portal exposing the test zone and the control zone.

24. A kit comprising the lateral flow immunoassay of claim 22 and a sample collection swab.

25. The kit of claim 24, wherein the sample collection swab comprises a collection tip, wherein the tip comprises a flocked material, a sponge, cotton, polypropylene, or polystyrene.

26. The kit of claim 24 or 25, further comprising a sample collection vessel comprising a liquid carrier.

27. The kit of claim 26, wherein the liquid carrier comprises phosphate buffered saline, Tween 20, bovine serum albumin, one or more protease inhibitors, one or more preservatives, and sodium deoxycholate.

28. The kit of claim 27, wherein the sodium deoxycholate is present in the liquid carrier at a concentration of 0.125% wt/vol.

29. The kit of any one of claims 24 to 28, further comprising a sample applicator.

30. The kit of any one of claims 24 to 29, wherein the housing comprises a cavity for holding the sample collection swab.

31. The kit of any one of claims 24 to 30, wherein the housing comprises a cavity for holding a sample collection vessel.

32. A method of detecting IP- 10 in a sample comprising: a. collecting the sample from the mucosa of a subject with a sample collection swab; b. adding the sample to a liquid carrier to form a liquid sample; c. applying the liquid sample to a sample application portion of a lateral flow immunoassay of any one of claims 2 to 22; d. allowing sufficient time for at least a portion of the liquid sample to traverse the sample application portion, the diagnostic antibody portion and the detection portion of the lateral flow immunoassay; e. observing the detection portion of the lateral flow immunoassay wherein: i. an observed signal in the test zone indicates IP-10 is present in the sample at a concentration of at least 80 pg/ml; and ii. an observed signal in the assay control zone indicates the lateral flow immunoassay is functional.

33. The method of claim 32, wherein an observed signal in the sample control zone indicates that a sufficient amount of the mucosal sample was applied to the assay.

34. The method of claim 32 or 33, wherein the sufficient time of step d is 15 minutes or less.

35. The method of any one of claims 32 to 34, wherein the subject is asymptomatic.

36. The method of any one of claims 32 to 35, wherein an observed signal in the test zone indicates a further diagnostic test should be conducted to confirm the subject has been infected with a respiratory pathogen.

37. The method of claim 36, wherein the further diagnostic test detects infection with SARS-CoV-2.

38. The method of claim 37, wherein the further diagnostic test detects infection with a pathogen selected from influenza viruses, parainfluenza viruses, adenoviruses, rhinoviruses, enteroviruses, human metapneumoviruses, flavivirus, respiratory syncytial viruses,

33 coronaviruses, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis.

39. The method of any one of claims 37, wherein the subject is experiencing one or more symptoms selected from cough, difficulty breathing, fever, muscle aches, loss of taste and loss of smell.

40. The method of claim 38, wherein the subject is experiencing one or more symptoms selected from rhinorrhea, nasal congestion, sneezing, cough, fever, chills, fatigue, headache, pain during swallowing, wheezing, and mucus production.

41. The method of claim 39 or 40, wherein the subject has experienced the one or more symptoms for less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day.

42. The method of claim 39, wherein an observed signal in the test zone indicates the subject should receive treatment for COVID-19.

43. The method of claim 39, wherein an observed signal in the test zone indicates the subject should be quarantined.

44. A method for monitoring the infectiousness of a subject infected with SARS-CoV-2 comprising: a. collecting a sample from the mucosa of the subject; b. applying the sample to a sample application portion of the lateral flow immunoassay of any one of claims 4 to 23; d. allowing sufficient time for at least a portion of the sample to traverse the sample application portion, the diagnostic antibody portion and the detection portion of the lateral flow immunoassay; e. observing the detection portion of the lateral flow immunoassay wherein: i. an observed signal in the test zone indicates the subject is infectious; ii. an absence of signal in the test zone indicates the subject is not infectious; and

34 ii. an observed signal in the sample control zone indicates an appropriate sample has been applied to the lateral flow immunoassay; and iii. an observed signal in the assay control zone indicates the lateral flow immunoassay is functional, wherein steps a-e are repeated at regular intervals until there is an absence of signal in the test zone.

35