WO2023141305A2

WO2023141305A2 - Assays, test kits and methods for detection of lacritin

Info

Publication number: WO2023141305A2
Application number: PCT/US2023/011291
Authority: WO
Inventors: Sergei Svarovsky; Alim SEIT-NEBI
Original assignee: Sapphire Biotech, Inc.
Priority date: 2022-01-20
Filing date: 2023-01-20
Publication date: 2023-07-27
Also published as: WO2023141305A3

Abstract

Assays, test kits and methods for detecting and measuring monomeric lacritin in a test sample are provided herein. A method includes the steps of obtaining the test specimen from a subject, transferring the test specimen to a sample receiving portion of an assay of a test kit, and reading the results from the assay. An exemplary test kit can comprise a first molecule having a first affinity to bind to a first portion of monomeric lacritin. In some contemplated test kits, a second molecule having a second affinity to bind to a second portion of monomeric lacritin is provided. In some contemplated test kits, a second molecule is provided, wherein the first molecule has a second affinity to bind to a portion of the second molecule.

Description

ASSAYS, TEST KITS AND METHODS FOR DETECTION OF LACRITIN

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/301,437, filed January 20, 2022. This and all other extrinsic materials discussed herein, including publications, patent applications, and patents, are incorporated by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of the term in the reference does not apply.

TECHNICAL FIELD

[0002] This disclosure relates to assays, test kits, and methods for the detection of monomeric Lacritin in a sample.

BACKGROUND

[0003] Lacritin is a tear protein, which in its monomeric form, autonomously promotes tearing and ocular surface survival. Reduced Lacritin levels occur in autoimmune Sjogren’s syndrome when ocular cells become stressed. Lacritin is the only identified growth-like factor decreased in tears from patients with ocular surface inflammation resulting from blepharitis, and it is downregulated in contact lens-related dry eye.

SUMMARY

[0004] Various assays, test kits and methods for the detection of monomeric lacritin are described herein, which can advantageously be used, for example, to diagnose blepharitis, Sjogren's syndrome, Dry Eye Disease and other inflammatory conditions, or as a companion diagnostic at point of care settings.

[0005] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first molecule having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a second molecule having a second affinity to bind to a second portion of monomeric lacritin. In some embodiments, at least one of the first and second portions comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, the first molecule lacks an affinity to bind to non-monomeric lacritin. In some embodiments, the second molecule lacks an affinity to bind to non-monomeric lacritin.

[0006] In some aspects of the disclosure, an antibody is provided that binds to a glutamine juncture (Q juncture) where one lacritin reacts with another forming a polymer. The antibody only recognizes this Q juncture in monomeric lacritin. When the Q juncture is occupied by another lacritin, the antibody cannot bind. In some aspects of the disclosure, an antibody is provided to a juncture present in polymeric lacritin but not in monomeric lacritin. Such antibody can be used to deplete any polymeric lacritin from a test sample before running an assay using any of the test kits described herein.

[0007] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first antibody having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a second antibody having a second affinity to bind to a second portion of monomeric lacritin. In some embodiments, at least one of the first and second portions comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, the first antibody lacks an affinity to bind to non-monomeric lacritin. In some embodiments, the second antibody lacks an affinity to bind to non-monomeric lacritin.

[0008] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first molecule having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first molecule is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a second molecule immobilized thereon, and wherein the first antibody has a second affinity to bind to a portion of the second molecule. In some embodiments, the second molecule comprises a lacritin derived peptide. In some embodiments, the first portion comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, it is contemplated that the second molecule comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, the first molecule lacks an affinity to bind to non-monomeric lacritin. [0009] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first antibody having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a lacritin derived peptide, and wherein the first antibody has a second affinity to bind to a portion of the lacritin derived peptide. In some embodiments, the first portion comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, it is contemplated that the lacritin derived peptide comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, the first antibody lacks an affinity to bind to non-monomeric lacritin.

[0010] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first molecule having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first molecule is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a second molecule immobilized thereon. In some embodiments, the first molecule comprises an anti-lacritin antibody, and the second molecule comprises Syndecan. Syndecan specifically recognizes the C-term lacritin domain and binds C-term of lacritin only in monomeric form. In some embodiments, the first portion comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, the first antibody lacks an affinity to bind to non-monomeric lacritin.

[0011] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first anti-lacritin antibody having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises Syndecan. In some embodiments, the first portion comprises a C-term epitope comprising SEQ ID NO. 3.

[0012] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first molecule and a second molecule, wherein the first molecule is coupled to at least one of a first label and a first tag, and wherein the second molecule is coupled to at least one of the first label and the first tag; and wherein the detection zone comprises a test location, and wherein the test location comprises a first anti-tag, wherein the first tag and the first anti-tag comprise a first tag/anti-tag pair. In some embodiments, the first molecule comprises Syndecan, and the second molecule comprises an anti-lacritin antibody. In some embodiments, the first molecule has an affinity to bind to a first portion of monomeric lacritin, wherein the first portion comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, the second molecule has an affinity to bind to a first portion of monomeric lacritin, wherein the first portion comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, the first molecule lacks an affinity to bind to non-monomeric lacritin. In some embodiments, the second molecule lacks an affinity to bind to non-monomeric lacritin.

[0013] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises Syndecan and a first anti-lacritin antibody, wherein Syndecan is coupled to at least one of a first label and a first tag, and wherein the first anti-lacritin antibody is coupled to at least one of the first label and the first tag; and wherein the detection zone comprises a test location, and wherein the test location comprises a first anti-tag, wherein the first tag and the first anti -tag comprise a first tag/anti-tag pair. In some embodiments, the first anti-lacritin antibody has an affinity to bind to a first portion of monomeric lacritin, and the first portion comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, the first anti-lacritin antibody lacks an affinity to bind to non-monomeric lacritin.

[0014] In an aspect of the disclosure, a method for detection of monomeric Lacritin in a test specimen is provided, comprising obtaining the test specimen from a subject, transferring the test specimen to a sample receiving portion of an assay of a test kit, and reading the results from the assay. The test kit can comprise any of the test kits described herein. In some contemplated methods, a step comprises depleting any polymeric (or non-monomeric) lacritin from the test specimen prior to transferring it to the sample receiving portion of the assay. In some contemplated methods, a step comprises depleting any non-monomeric lacritin (but maintaining all monomeric lacritin) from the test specimen prior to transferring it to the sample receiving portion of the assay.

[0015] In an aspect of the disclosure, an antibody having an affinity to bind to a portion of monomeric lacritin, the portion comprising SEQ ID NO. 3, is provided. In another aspect, a reagent for use in an immunoassay method for detecting and quantifying monomeric lacritin in a sample is provided, the reagent comprising an antibody having an affinity to bind to a portion of monomeric lacritin, the portion comprising SEQ ID NO. 3.

[0016] Other advantages and benefits of the disclosed proteins, and related assays, test kits and methods will be apparent to one of ordinary skill with a review of the following drawings and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0018] Fig. 1A illustrates a sandwich immunoassay for the detection of monomeric lacritin, according to an embodiment;

[0019] Fig. IB is a graph illustrating signal intensity relative to monomeric lacritin concentration in test samples using the assay of Fig. 1 A;

[0020] Fig. 2A illustrates a competitive assay for the detection of monomeric lacritin, according to an embodiment;

[0021] Fig. 2B is a graph illustrating signal intensity relative to monomeric lacritin concentration in test samples using the assay of Fig. 2A;

[0022] Fig. 3A illustrates a sandwich immunoassay for the detection of monomeric lacritin using a Syndecan/antibody pair, according to an embodiment;

[0023] Fig. 3B illustrates another sandwich immunoassay for the detection of monomeric lacritin using a Syndecan/antibody pair, according to another embodiment;

[0024] Fig. 4A illustrates a homogenous sandwich immunoassay for the detection of monomeric lacritin using a Syndecan/antibody pair, according to an embodiment; and

[0025] Fig. 4B illustrates another homogenous sandwich immunoassay for the detection of monomeric lacritin using a Syndecan/antibody pair, according to another embodiment. [0026] At least one drawing is executed in color.

DETAILED DESCRIPTION

[0027] After reading this description it will become apparent to one skilled in the art how to implement the disclosed devices, components and methods in various alternative embodiments and alternative applications. However, all the various embodiments of the present disclosure will not be described herein. It is understood that the embodiments presented here are presented by way of an example only, and not limitation. As such, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present disclosure as set forth below.

[0028] Various assays, test kits, and methods for detecting monomeric lacritin in a test sample are provided herein. In some aspects, detecting monomeric lacritin in a test sample comprises measuring monomeric lacritin in the test sample.

[0029] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first antibody having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a second antibody having a second affinity to bind to a second portion of monomeric lacritin.

[0030] In some embodiments, a “first zone” of an immunoassay can comprise a sample receiving portion and conjugate pad (also referred to as conjugation pad or conjugate release pad). The sample receiving portion can comprise at least one of a sample port, a sample pad, and a sample filter. In some embodiments, a “detection zone” can comprise a region of a nitrocellulose membrane strip having one or more test lines (which can be test regions that are not necessarily lines), and optionally one or more control lines (which can be control regions that are not necessarily lines). In some embodiments, an immunoassay can comprise an absorption pad on an end opposite the sample pad. The second antibody having a second affinity to bind to a second portion of monomeric lacritin can be immobilized on a test line of a detection zone. A control molecule (e.g., donkey anti-mouse pAb) can be immobilized on a first control line of a detection zone. The conjugate pad can comprise the detection reagents or conjugate (e.g., the first antibody coupled to/conjugated with a label). As a sample flows from the sample pad through the conjugate release pad, the conjugate is released into the sample and binds with the target analyte (monomeric lacritin, if present) and flows along the detection zone until captured by the immobilized second antibody on the test line. Any conjugate that does not bind with the target analyte can flow along the detection zone until captured by the immobilized control molecule (e.g., control protein) on the control line.

[0031] In some embodiments, the first label is selected from a nanoparticle (e.g., nanosphere, nanoshell), bead, latex bead, aptamer, oligonucleotide, a quantum dot, and a combination thereof. In some embodiments, the detection zone further comprises a control location, and wherein the control location comprises a control molecule (e.g., protein).

[0032] In some embodiments, the first portion is a N-terminal of monomeric lacritin. In some embodiments, the second portion is a C-terminal of monomeric lacritin. In some embodiments, the first portion is a C-terminal of monomeric lacritin. In some embodiments, the second portion is a N-terminal of monomeric lacritin. In some embodiments, the first antibody comprises an anti-N-term lacritin antibody and the second antibody comprises an anti-C-term lacritin antibody. In some embodiments, an anti-N-term antibody is a capture antibody, and an anti-C-term antibody is a detector antibody. In some embodiments, the first antibody comprises an anti-C-term lacritin antibody and the second antibody comprises an anti-N-term lacritin antibody. In some embodiments, the first antibody comprises a N-term lacritin antibody and the second antibody comprises a C-term lacritin antibody. In some embodiments, the first antibody comprises a C-term lacritin antibody and the second antibody comprises a N-term lacritin antibody. In some embodiments, at least one of the first and second portions comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, the first antibody lacks an affinity to bind to non-monomeric lacritin. In some embodiments, the second antibody lacks an affinity to bind to non-monomeric lacritin. In some embodiments, a signal density increases with an increase in monomeric lacritin concentration in the test specimen. In some embodiments, the detection zone further comprises a second test location. In some embodiments, the first zone comprises a sample receiving portion and a conjugate release pad, and wherein the conjugate release pad comprises the first antibody coupled to the first label. In some embodiments, the detection zone comprises a nitrocellulose membrane. In some embodiments, the immunoassay is a lateral flow assay.

[0033] Figure 1A illustrates a traditional sandwich assay in which an anti-lacritin antibody is sprayed onto the membrane. In the example shown, an antibody that binds to a first portion of lacritin (e.g., a N-terminal domain of lacritin) is labeled with a reporter moiety (a nanoparticle, enzyme or a dye). The conjugate pad holds the labeled antibody, which binds to the first portion (e.g., N-terminal peptide) of native lacritin in a sample (if present), flows along the membrane/detection zone and forms an immunosandwich with a second antibody sprayed onto the test line. The second antibody can have an affinity to bind to a second portion of native lacritin (e.g., C-terminal peptide of native lacritin) in a sample (if present). In this case, the more monomeric lacritin is present in the sample, the more intense the signal on the test line is, which is reflected in the shape of the standard curve of Figure IB. To use the assay of Figure 1A, a user can collect a sample (e.g., a tear sample, a saliva sample) from an individual, and add the sample to a sample pad of the assay. In some embodiments, all non-monomeric lacritin can optionally be depleted from the sample (e.g., using a molecule that binds to a juncture present in non-monomeric lacritin but not monomeric lacritin). When assay (chase) buffer is added to the sample well, the dried components on the strip interact with monomeric lacritin in the sample, if present. If the sample contains monomeric lacritin that binds to the labeled first antibody on the conjugate pad and to the second antibody bound to the test line, the test will show a visible test line - the intensity of which may vary depending on the amount of lacritin in the test sample. The antibody that binds to a first portion (e.g., N-terminal domain) of Lacritin, flows and forms an immunosandwich with the antibody sprayed onto the test line. In some embodiments, the first antibody, which can be sprayed onto the conjugate pad, binds to the N-terminal peptide of native lacritin, and the second antibody, which can be sprayed onto the test line, binds to the C-terminal peptide of native lacritin. In some embodiments, the first antibody, which can be sprayed onto the conjugate pad, binds to the C-terminal peptide of native lacritin, and the second antibody, which can be sprayed onto the test line, binds to the N-terminal peptide of native lacritin.

[0034] In an embodiment, a test uses immobilized second antibody (test line) and donkey anti-mouse pAb (control line) on a nitrocellulose strip. In some embodiments, a biological sample as the test-specimen or test sample is saliva and/or tears. In some embodiments, the saliva and/or tears is obtained from a subject either known or suspected of having an ocular disease or disorder. In some embodiments, a tear sample can be obtained directly from an eye of a subject. It is contemplated that the sample(s) can be obtained using any technique known in the art or later developed.

[0035] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first antibody having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a lacritin derived peptide, and wherein the first antibody has a second affinity to bind to a portion of the lacritin derived peptide. In some embodiments, the first portion comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, the first antibody lacks an affinity to bind to non-monomeric lacritin.

[0036] In some embodiments, the lacritin derived peptide comprises a segment of lacritin that includes the N-terminal portion. In some embodiments, the first portion of monomeric lacritin and the portion of the lacritin derived peptide each comprises a N-terminal portion. In some embodiments, the first portion of monomeric lacritin and the portion of the lacritin derived peptide each comprises a C-terminal portion. In some embodiments, a signal density decreases with an increase in monomeric lacritin concentration in the test specimen.

[0037] In some embodiments, a “first zone” of an immunoassay can comprise a sample receiving portion and conjugate pad. The sample receiving portion can comprise at least one of a sample port, a sample pad, and a sample filter. In some embodiments, a “detection zone” can comprise a region of a nitrocellulose membrane strip having one or more test lines, and optionally one or more control lines. In some embodiments, an immunoassay can comprise an absorption pad on an end opposite the sample pad. The lacritin derived peptide can be immobilized on a test line of a detection zone. The conjugate pad can comprise the detection reagents or conjugate (e.g., the first antibody coupled to/conjugated with a label). As a sample flows from the sample pad through the conjugate release pad, the conjugate is released into the sample. If the target analyte (monomeric lacritin) is present, the conjugate will bind to the target analyte. In some embodiments of a competitive assay, if the conjugate binds to the monomeric lacritin in a sample, it will not bind to the immobilized lacritin derived peptide on the test line.

[0038] Figure 2A illustrates a competitive assay for the detection of monomeric lacritin. In some embodiments, a lacritin-derived N-terminal peptide can be sprayed onto a membrane on a test line, and an antibody that binds to the N-terminal domain of lacritin is labeled with a reporter moiety (a nanoparticle, enzyme or a dye) and present on a conjugate pad of the assay. When the labeled antibody binds to N-terminal peptide of native lacritin in a sample, it is unable to bind to N-terminal peptide deposited on the test line. The more monomeric lacritin is present in the sample, the less intense the signal is, which is reflected in the shape of the standard curve of Figure 2B. [0039] In some embodiments, a lacritin-derived C-terminal peptide can be sprayed onto a membrane on a test line, and an antibody that binds to the C-terminal domain of lacritin is labeled with a reporter moiety (a nanoparticle, enzyme or a dye) and present on a conjugate pad of the assay. When the labeled antibody binds to C-terminal peptide of native lacritin, it is unable to bind to C-terminal peptide deposited on the test line.

[0040] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone (e.g., a conjugate pad comprising zone) comprises a first anti-lacritin antibody having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises Syndecan. In another aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises Syndecan coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a first anti-lacritin antibody having a first affinity to bind to a first portion of monomeric lacritin.

[0041] In some embodiments, the first portion comprises a C-terminal portion. In some embodiments, the first portion comprises a N-terminal portion. In some embodiments, the first portion comprises a C-term epitope comprising SEQ ID NO. 3. In some embodiments, the first antibody lacks an affinity to bind to non-monomeric lacritin. In some embodiments, the first label is selected from a nanoparticle, bead, latex bead, aptamer, oligonucleotide, a quantum dot, and a combination thereof.

[0042] In some embodiments, a “first zone” of an immunoassay can comprise a sample receiving portion and conjugate pad. The sample receiving portion can comprise at least one of a sample port, a sample pad, and a sample filter. In some embodiments, a “detection zone” can comprise a region of a nitrocellulose membrane strip having one or more test lines, and optionally one or more control lines. In some embodiments, an immunoassay can comprise an absorption pad on an end opposite the sample pad. The Syndecan or first anti-lacritin antibody can be immobilized on a test line of a detection zone. The conjugate pad can comprise the detection reagents or conjugate (e.g., the anti-lacritin antibody or Syndecan coupled to/conjugated with a label). As a sample flows from the sample pad through the conjugate release pad, the conjugate is released into the sample. If the target analyte (monomeric lacritin) is present, the conjugate will bind to a first portion of the target analyte and the antibody or Syndecan sprayed on the test line will bind to a second portion of the target analyte different from the first portion. In some embodiments, a signal density increases with an increase in monomeric lacritin concentration in the test specimen.

[0043] Figure 3A-3B illustrate example sandwich immunoassays for the detection of monomeric lacritin using a Syndecan/ Antibody pair, according to some embodiments. The Syndecan/ Antibody sandwich immunoassay of Figures 3A-3B is similar to Antibody/ Antibody sandwich immunoassay shown in Figure 1 A. Syndecan is known to specifically recognize the C-terminal domain of monomeric lacritin. In this case the Syndecan is used as a surrogate for anti-C-terminal domain antibody. Figures 3A-3B show alternative configurations where Syndecan is sprayed onto the assay membrane / test line (Figure 3A) or labeled (Figure 3B). Anti-lacritin antibody can be on the test line when Syndecan is labeled, and anti-lacritin antibody can be labeled when Syndecan is on a test line. The more monomeric lacritin is in the sample, the stronger the signal observed at the test line.

[0044] In an aspect of the disclosure, a test kit is provided for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises Syndecan and a first anti-lacritin antibody, wherein Syndecan is coupled to at least one of a first label and a first tag, and wherein the first anti-lacritin antibody is coupled to at least one of the first label and the first tag; and wherein the detection zone comprises a test location, and wherein the test location comprises a first anti-tag, wherein the first tag and the first anti-tag comprise a first tag/anti-tag pair.

[0045] In some embodiments, Syndecan is coupled to the first label, and wherein the first anti-lacritin antibody is coupled to the first tag. In some embodiments, Syndecan is coupled to the first tag, and wherein the first anti-lacritin antibody is coupled to the first label. In some embodiments, Syndecan has a first affinity to bind to a first portion of monomeric lacritin comprising an C-terminal domain portion, and wherein the first anti-lacritin antibody has a second affinity to bind to a second portion of monomeric lacritin different from the first portion. In some embodiments, the first anti-lacritin antibody comprises a anti-N-term lacritin antibody. In some embodiments, a signal density increases with an increase in monomeric lacritin concentration in the test specimen. In some embodiments, the detection zone further comprises a second test location. In some embodiments, the detection zone further comprises a control location. In some embodiments, the first zone comprises a sample receiving portion and a conjugate release pad, and wherein the conjugate release pad comprises the Syndecan and the first anti-lacritin antibody. In some embodiments, the detection zone comprises a nitrocellulose membrane and the first anti-tag is immobilized on a test line/region of the detection zone.

[0046] Figures 4A-4B illustrate homogenous sandwich immunoassays for detection of monomeric lacritin using a Syndecan/antibody pair. The Syndecan/antibody sandwich immunoassay in homogeneous format uses a labeled antibody or Syndecan and a tagged Syndecan or antibody. In this assay format either the antibody or Syndecan is labeled and the other of the pair is tagged. In Figure 4A, Syndecan is labeled and the anti-lacritin antibody is tagged. In Figure 4B, Syndecan is tagged and the anti-lacritin antibody is labeled. A moiety that binds the tag (anti-tag) is deposited onto the membrane on the test line. The reagents mixture (including (1) the labeled antibody or Syndecan and (2) the tagged Syndecan or antibody) is placed onto the conjugate pad. In presence of monomeric lacritin, the reagent pair forms an immunocomplex that is captured upstream by the anti-tag. The more monomeric lacritin is in the sample the stronger the signal observed at the test line. In some embodiments, each of the Syndecan and the antibody have an affinity to bind to monomeric lacritin but not polymeric lacritin.

[0047] The assays described herein can be used to, among other things, at least one of detect and measure monomeric Lacritin in a test specimen in order to diagnose one or more ocular diseases or disorders. As a few non-limiting examples, detection of low levels of monomeric lacritin (e.g., lower than a threshold concentration) can indicate one or more diseases associated with dry eyes and/or dry mouth (e.g., Sjogren’s syndrome).

[0048] In some embodiments, assay systems, test-cassette devices and methods of the present disclosure include an analytical membrane that is divided into one or more test regions and optionally one or more control regions. The detection region or regions can include a target analyte binding agent immobilized to a portion of the detection region such that it is not displaced when facilitating lateral flow across the analytical membrane. Lateral flow assay systems of the present invention can also include a sample receiving portion and/or conjugate pad within which target analyte binding agents are contained. In some embodiments, target analyte binding agents are contained within the conjugate pad but flows from the conjugate pad and across the analytical membrane (also referred to herein as detection zone) towards the detection and control regions when lateral flow occurs. Lateral flow assay systems of the present disclosure can also include a sample pad that is positioned at one distal end of the lateral flow assay system (e.g., opposite an absorbent pad).

[0049] A sample that contains (or may contain) a target analyte (e.g., monomeric lacritin) is applied to the sample pad. In some embodiments, a lateral flow assay system also comprises a wicking pad at an end of the device distal to the sample pad. The wicking pad generates capillary flow of the sample from the sample pad through the conjugate pad, analytical membrane, detection region, and control region.

[0050] In accordance with some embodiments, upon addition of a test-specimen to the sample pad, the facilitation of flow (e.g., lateral flow) causes a target-analytes within the sample, if any, to contact a target analyte binding agent within the conjugate pad; subsequently, flow (e.g., lateral flow) causes the target analytes and the target analyte binding agents to contact a second binding agent immobilized to a detection region of the analytical membrane. The presence and/or quantity of the target analytes is then determined based on detection of the analytes in the detection region(s) also referred to herein as a “test line” or “test region” and/or in comparison to the control.

[0051] As used herein the term “tag/anti-tag pair” or vice versa (anti-tag/tag pair) refers to 2 moieties that are known to bind (e.g., non-covalently) to each other. For example, tag/anti- tag pairs can be ligand/receptor pairs; where the anti-tag is the binding partner to the tag.

[0052] The tag/anti-tag interaction can be a noncovalent interaction, such as a proteinligand interaction. In an embodiment, the noncovalent protein-ligand interaction is an interaction between biotin and avidin or streptavidin. Biotin (or other tag) is conjugated to Syndecan or anti- lacritin antibody, and avidin or streptavidin (or other anti-tag) is conjugated to the nitrocellulose membrane. For example, with biotin-Syndecan / biotin-antibody and streptavidin-conjugated nitrocellulose membrane, the high-affinity interaction between biotin and avidin or streptavidin tethers the biotin-Syndecan / biotin-antibody conjugate to the streptavidin-conjugated nitrocellulose membrane. In an embodiment, where monomeric lacritin is present in the test sample, the biotin-Syndecan or biotin-antibody binds with a first portion of monomeric lacritin, a labeled Syndecan or labeled-antibody binds with a second portion of monomeric lacritin different from the first portion, and the biotin tagged molecule binds to the streptavidin immobilized on the test line. Streptavidin is a tetramer and each subunit binds biotin with equal affinity; thus, wild-type streptavidin binds four biotin molecules. For some applications it is useful to generate a strong 1 : 1 complex of two molecules, i.e., monovalent binding. Monovalent streptavidin is an engineered recombinant form of streptavidin which is still a tetramer but only one of the four binding sites is functional. A streptavidin with exactly two biotin binding sites per tetramer (divalent streptavidin) can be produced by mixing subunits with and without a functional biotin binding site. A streptavidin with exactly three biotin binding sites per tetramer (trivalent streptavidin) can also be produced using the same principle as to produce divalent streptavidins. The streptavidin used in the inventive assay can be wild-type (binding four biotins), or it may be monovalent, divalent, or trivalent. Methods of conjugating biotin and streptavidin to proteins and substrates are known to those of skill in the art and any such methods can be used to conjugate biotin or streptavidin to a binding molecule, and to conjugate biotin or streptavidin to the substrate.

[0053] In another embodiment, the noncovalent protein-ligand interaction is a Halo interaction. Halo-Tag is a 33 kDa mutagenized haloalkane dehalogenase that forms covalent attachments to substituted chloroalkane linker derivatives (Halo-Ligand). Similarly to the streptavidin-biotin connection, the chloroalkane linker extends 1.4 nm into the hydrophobic core of Halo-Tag. Commercially available Halo-ligand derivatives include the invariant chloroalkane moiety followed by 4 ethylene glycol repeats, and a reactive sulfahydryl, succinimidyl ester, amine, or iodoacetamide group, among many other options. Methods of conjugating biotin and streptavidin to proteins and substrates are known to those of skill in the art and any such methods can be used to conjugate Halo-Tag or Halo-Ligand to a molecule (e.g., a binding molecule), and to Halo-Tag or Halo-Ligand to the nitrocellulose membrane.

[0054] In another embodiment, the noncovalent protein-ligand interaction is a His-tag interaction. The His-tag (also called 6xHis-tag) contain six or more consecutive histidine residues. These residues readily coordinate with transition metal ions such as Ni2+ or Co2+ immobilized on beads or a resin. The His-tag is added to a molecule (e.g., a binding molecule) used in the assay, with the beads or resin with immobilized Ni2+ or Co2+ conjugated or otherwise bound to the nitrocellulose membrane. Methods of adding His-tags to proteins and beads or resin with immobilized Ni2+ or Co2+ to substrates are known to those of skill in the art and any such methods can be used to add a His-tag to a molecule (e.g., a binding molecule), and beads or resin with immobilized Ni2+ or Co2+ to the nitrocellulose membrane. In other embodiments, the noncovalent interaction utilizes a ligand tag that is calmodulin-binding peptide, glutathione, amylose, a c-my tag, or a Flag tag. The ligand tag is attached to the molecule (e.g., a binding molecule), and the respective ligand-binding molecule is attached to the nitrocellulose membrane using methods known to those of skill in the art. The ligand tag can also be single-strand DNA (ssDNA) attached to the molecule (e.g., binding molecule), where the complementary ssDNA is immobilized on the nitrocellulose membrane.

[0055] In some embodiments, the conjugate pad can comprise a mixture of (a) a first molecule (e.g., Syndecan, an antibody) coupled to a first label, and (b) a second molecule (e.g., Syndecan, an antibody) bound to a tag. In some embodiments, the conjugate pad can comprise a first molecule (e.g., an antibody, Syndecan) coupled to a first label. In some embodiments, the conjugate pad can comprise a first molecule (e.g., an antibody, Syndecan) coupled to a first tag. In some embodiments, at least one of the first and second molecules comprises an antibody having an affinity to bind to a portion of monomeric lacritin, the portion comprising SEQ ID NO. 3.

[0056] In some embodiments, the label(s) is/are selected from a nanoparticle, bead, latex bead, aptamer, and/ or a quantum dot. As used herein, the term “label” refers to a moiety, the presence of which can be detected using methods well-known in the art for label-detection. In an embodiment, a first molecule is coupled to a label such that it can be detected when bound to a second molecule immobilized on the nitrocellulose membrane (or otherwise bound to the nitrocellulose membrane such that it remains affixed when a flow medium flows through the nitrocellulose membrane), thus demonstrating a presence of a target analyte in the sample. In an embodiment, a first molecule is coupled to a label such that it can be detected when bound to a second molecule immobilized on the nitrocellulose membrane (or otherwise bound to the nitrocellulose membrane such that it remains affixed when a flow medium flows through the nitrocellulose membrane), thus demonstrating an absence of a target analyte in the sample. In an embodiment, the control protein (for example, an anti-IgG monoclonal antibody) is coupled to a label such that it can be detected when bound to its target on the nitrocellulose membrane (for example, IgG on the Control Line), thus demonstrating that the test is functional and has been performed properly. In an embodiment, the label is detectable by the naked eye. In another embodiment, the label is detectable by fluorescence. In another embodiment, the label is detectable by chemiluminescence. Methods for coupling the labels to proteins are known to those of skill in the art.

[0057] Labels detectable by the naked eye include metal nanoparticles and nanoshells (e.g., green gold nanoshells; red, orange, or blue gold nanoparticles; copper oxide nanoparticles; silver nanoparticles), gold colloid, platinum colloid, polystyrene latex or natural rubber latex colored with respective pigments such as red and blue. Labels detectable by the naked eye include textile dyes, such as for example, a Direct dye, a Disperse dye , a Dischargeable acid dye, a Kenanthol dye, a Kenamide dye, a Dyacid dye, a Kemtex reactive dye, a Kemtex acid dye, a Kemtex Easidye acid dye, a Remazol dye, a Kemazol dye, a Caledon dye, a Cassulfon dye, an Isolan dye, a Sirius dye, an Imperon dye, a phtalogen dye, a naphtol dye, a Levaftx dye, a Procion dye, and an isothiocyanate dye. Examples of textile dyes that can be used to label proteins include, for example, Remazol brilliant blue, Uniblue A, malachite green isothiocyanate, and Orange 16 (Remazol orange). Any label known to those of skill in the art to both be fluorescent and used to label proteins can be used.

[0058] Fluorescent labels include any of the Alexa fluor dyes, any of the BODIPY dyes, any of the eFluor dyes, any of the Super Bright dyes, fluorescein or a derivative thereof, eosin or a derivative thereof, tetramethylrhodamine, rhodamine or a derivative thereof, Texas red or a derivative thereof, pyridyl oxazole or a derivative thereof, NBD chloride, NBD fluoride, ABD- F, lucifer yellow or a derivative thereof, 8-anilino-l-naphthalenesulfonic acid (8-ANS) or a derivative thereof, Oregon green or a derivative thereof, Pacific blue or a derivative thereof, Pacific green or a derivative thereof, Pacific orange or a derivative thereof Cy3, Cy5, Cyanine7, Cyanine5.5, or coumarin or a derivative thereof. Fluorescent labels include any fluorescent protein, such as green fluorescent protein (GFP), red fluorescent protein (e.g., dsRed), cyan fluorescent protein, blue fluorescent protein, yellow fluorescent protein, enhanced green fluorescent protein (EGFP), or any derivative of such fluorescent proteins thereof. Any label known to those of skill to both be fluorescent and be used to label proteins can be used.

[0059] Chemiluminescent labels include enzyme labels that catalyze formation of ATP which is then assayed by the firefly reaction or that catalyze formation of peroxide which is determined by luminol, isoluminol, or peroxyoxalate CL. Such enzyme labels include luciferase and horseradish peroxidase. Any label known to those of skill in the art to both be chemiluminescent and used to label proteins can be used.

[0060] In some embodiments, reading the results from the test-cassette/assay further comprises determining the intensity of a test region (e.g., test line) in the assay compared with a reference standard. In a particular embodiment, the reference standard is a scorecard. As used herein, the phrase “reference standard” refers to a control set of values, either obtained simultaneously with the assay results or obtained from a previous control experiment such that they are indicative of the presence and/or level of a target analyte (e.g., monomeric lacritin) in the test-specimen. In a particular embodiment, the reference standard is a scorecard. [0061] In certain embodiments, the level of a target analyte (e.g., monomeric lacritin) in the test-specimen is reported as falling within a range of pre-determined values. In some embodiments, the range of pre-determined values corresponds to High (H), Moderate-High (MH), Moderate to Moderate-High (M-MH), Moderate (M), Moderate to Not Detectable (M- ND) and Not Detectable (ND). As used herein, the phrase “reported as falling within a range of pre-determined values” refers to the manner in which the level of the target analyte are analyzed relative to the reference standard or set of control values. The range of pre-determined values can be as few as two levels of monomeric lacritin values (or concentrations) up to about 10 or more distinct concentration (or quantity) levels of monomeric lacritin values. In one embodiment corresponding to 2 levels of values, for example, falling either above or below a predetermined set value may indicate the presence of an insufficient amounts of monomeric lacritin in a test-specimen, such that there is a greater likelihood of a disease or disorder associated with dry eyes.

[0062] In some aspects, detecting or measuring monomeric lacritin in a sample can comprise using an electronic device.

[0063] In one embodiment, the following reagent configuration is employed. A conjugate pad is infused with a mixture of the labeled first molecule and a tagged second molecule. In one embodiment, the following reagent configuration is employed. A conjugate pad is infused with a labeled first molecule, wherein the first molecule (e.g., an antibody, Syndecan) has an affinity to bind to a first portion of monomeric lacritin. In one embodiment, the following reagent configuration is employed. A conjugate pad is infused with a mixture of the labeled first molecule, a tagged second molecule, and a control antibody coupled to control label as a constant assay control. The purpose of the control bead is to provide reassurances regarding sample addition, reconstitution, and flow. In some embodiments, if a control line cannot be visualized with the human eye, the test is considered invalid. In one embodiment, the following reagent configuration is employed. A conjugate pad is infused with a labeled first molecule and a control antibody coupled to control label as a constant assay control, wherein the first molecule (e.g., an antibody, Syndecan) has an affinity to bind to a first portion of monomeric lacritin.

[0064] When assay (chase) buffer is added to the sample well, the dried components on the strip interact with the target analyte (monomeric lacritin) in the test sample, if present. As a test sample flows through the conjugate release pad, the labeled and/or tagged conjugate(s) are released into the sample. The labeled and/or tagged conjugate(s) bind to a first portion of monomeric lacritin, if such target analytes are present. In some embodiments, the labeled and/or tagged conjugate(s) bound to the target analyte can flow through the nitrocellulose membrane of the detection region and be captured by a molecule bound to a test line and having an affinity to bind to a second portion of the monomeric lacritin. The presence of the target analyte can be detectable using methods well-known in the art for lab el -detection (e.g., Figures 1A, 3A, 3B, 4A, 4B). In some embodiments, the labeled and/or tagged conjugate(s) can bind to the target analyte, which can prevent the labeled and/or tagged conjugate(s) from binding to a molecule immobilized on the test line, (e.g., Figure 2A).

[0065] Non-Limiting Embodiments.

[0066] Embodiment 1. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first antibody having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a second antibody having a second affinity to bind to a second portion of monomeric lacritin.

[0067] Embodiment 2. The test kit of embodiment 1, wherein the first label is selected from a nanoparticle, bead, latex bead, aptamer, oligonucleotide, a quantum dot, and a combination thereof.

[0068] Embodiment 3. The test kit of any of embodiments 1-2, wherein the detection zone further comprises a control location, and wherein the control location comprises a control antibody.

[0069] Embodiment 4. The test kit of any of embodiments 1-3, wherein the first portion is a N-terminal of monomeric lacritin.

[0070] Embodiment 5. The test kit of any of embodiments 1-4, wherein the second portion is a C-terminal of monomeric lacritin.

[0071] Embodiment 6. The test kit of any of embodiments 1-5, wherein a signal density increases with an increase in monomeric lacritin concentration in the test specimen.

[0072] Embodiment 7. The test kit of any of embodiments 1-6, wherein the detection zone further comprises a second test location.

[0073] Embodiment 8. The test kit of any of embodiments 1-7, wherein the first zone comprises a sample receiving portion and a conjugate release pad, and wherein the conjugate release pad comprises the first antibody coupled to the first label. [0074] Embodiment 9. The test kit of any of embodiments 1-8, wherein the detection zone comprises a nitrocellulose membrane.

[0075] Embodiment 10. The test kit of any of embodiments 1-9, wherein the immunoassay is a lateral flow assay.

[0076] Embodiment 11. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first antibody having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a lacritin derived peptide, and wherein the first antibody has a second affinity to bind to a portion of the lacritin derived peptide. [0077] Embodiment 12. A test kit of embodiment 11, wherein the lacritin derived peptide comprises a segment of lacritin that includes the N-terminal portion.

[0078] Embodiment 13. A test kit of any of embodiments 11-12, wherein the first portion of monomeric lacritin and the portion of the lacritin derived peptide each comprises a N- terminal portion.

[0079] Embodiment 14. A test kit of any of embodiments 11-13, wherein the first portion of monomeric lacritin and the portion of the lacritin derived peptide each comprises a C- terminal portion.

[0080] Embodiment 15. A test kit of any of embodiments 11-14, wherein a signal density decreases with an increase in monomeric lacritin concentration in the test specimen.

[0081] Embodiment 16. A test kit of any of embodiments 11-15, wherein the immunoassay is a lateral flow assay.

[0082] Embodiment 17. A test kit of any of embodiments 11-16, wherein the detection zone comprises a nitrocellulose membrane.

[0083] Embodiment 18. A test kit of any of embodiments 11-17, wherein the first zone comprises a sample receiving portion and a conjugate release pad, and wherein the conjugate release pad comprises the first antibody coupled to the first label.

[0084] Embodiment 19. A test kit of any of embodiments 11-18, wherein the detection zone further comprises a second test location.

[0085] Embodiment 20. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first anti-lacritin antibody having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises Syndecan. [0086] Embodiment 21. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises Syndecan coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a first anti-lacritin antibody having a first affinity to bind to a first portion of monomeric lacritin.

[0087] Embodiment 22. The test kit of any of embodiments 20-21, wherein the first portion comprises a C-terminal portion.

[0088] Embodiment 23. The test kit of any of embodiments 20-22, wherein a signal density increases with an increase in monomeric lacritin concentration in the test specimen.

[0089] Embodiment 24. The test kit of any of embodiments 20-23, wherein the first label is selected from a nanoparticle, bead, latex bead, aptamer, oligonucleotide, a quantum dot, and a combination thereof.

[0090] Embodiment 25. The test kit of any of embodiments 20-24, wherein the immunoassay is a lateral flow assay

[0091] Embodiment 26. The test kit of any of embodiments 20-25, wherein the detection zone comprises a nitrocellulose membrane

[0092] Embodiment 27. The test kit of any of embodiments 20-26, wherein the first zone comprises a sample receiving portion and a conjugate release pad, and wherein the conjugate release pad comprises the first anti-lacritin antibody coupled to the first label.

[0093] Embodiment 28. The test kit of any of embodiments 20-27, wherein the detection zone further comprises a second test location.

[0094] Embodiment 29. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises Syndecan and a first anti-lacritin antibody, wherein Syndecan is coupled to at least one of a first label and a first tag, and wherein the first anti-lacritin antibody is coupled to at least one of the first label and the first tag; and wherein the detection zone comprises a test location, and wherein the test location comprises a first anti-tag, wherein the first tag and the first anti-tag comprise a first tag/anti-tag pair.

[0095] Embodiment 30. The test kit of embodiment 29, wherein Syndecan is coupled to the first label, and wherein the first anti-lacritin antibody is coupled to the first tag.

[0096] Embodiment 31. The test kit of any of embodiments 29-30, wherein Syndecan is coupled to the first tag, and wherein the first anti-lacritin antibody is coupled to the first label. [0097] Embodiment 32. The test kit of any of embodiments 29-31, wherein Syndecan has a first affinity to bind to a first portion of monomeric lacritin comprising an N-terminal domain portion, and wherein the first anti-lacritin antibody has a second affinity to bind to a second portion of monomeric lacritin different from the first portion.

[0098] Embodiment 33. The test kit of any of embodiments 29-32, wherein a signal density increases with an increase in monomeric lacritin concentration in the test specimen.

[0099] Embodiment 34. The test kit of any of embodiments 29-33, wherein the detection zone further comprises a second test location.

[0100] Embodiment 35. The test kit of any of embodiments 29-34, wherein the detection zone further comprises a control location.

[0101] Embodiment 36. The test kit of any of embodiments 29-35, wherein the first zone comprises a sample receiving portion and a conjugate release pad, and wherein the conjugate release pad comprises the Syndecan and the first anti-lacritin antibody.

[0102] Embodiment 37. The test kit of any of embodiments 29-36, wherein the detection zone comprises a nitrocellulose membrane.

[0103] Embodiment 38. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first molecule having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first molecule is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a second molecule having a second affinity to bind to a second portion of monomeric lacritin.

[0104] Embodiment 39. The test kit of embodiment 38, wherein the first molecule comprises a first antibody having the first affinity to bind to the first portion of monomeric lacritin, and wherein the second molecule comprises a second antibody having the second affinity to bind to the second portion of monomeric lacritin.

[0105] Embodiment 40. The test kit of any of embodiments 38-39, wherein the first label is selected from a nanoparticle, bead, latex bead, aptamer, oligonucleotide, a quantum dot, and a combination thereof.

[0106] Embodiment 41. The test kit of any of embodiments 38-40, wherein the detection zone further comprises a control location, and wherein the control location comprises a control antibody.

[0107] Embodiment 42. The test kit of any of embodiments 38-41, wherein the first portion is a N-terminal of monomeric lacritin.

[0108] Embodiment 43. The test kit of any of embodiments 38-42, wherein the second portion is a C-terminal of monomeric lacritin. [0109] Embodiment 44. The test kit of any of embodiments 38-43, wherein a signal density increases with an increase in monomeric lacritin concentration in the test specimen.

[0110] Embodiment 45. The test kit of any of embodiments 38-44, wherein the detection zone further comprises a second test location.

[0111] Embodiment 46. The test kit of any of embodiments 38-45, wherein the first zone comprises a sample receiving portion and a conjugate release pad, and wherein the conjugate release pad comprises the first antibody coupled to the first label.

[0112] Embodiment 47. The test kit of any of embodiments 38-46, wherein the detection zone comprises a nitrocellulose membrane, and wherein the immunoassay is a lateral flow assay.

[0113] Embodiment 48. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first molecule having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first molecule is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a second molecule, and wherein the first molecule has a second affinity to bind to a portion of the second molecule.

[0114] Embodiment 49. The test kit of embodiment 48, wherein the first molecule is a first antibody having the first affinity and the second affinity, wherein the second molecule comprises a lacritin derived peptide, and wherein a signal density decreases with an increase in monomeric lacritin concentration in the test specimen.

[0115] Embodiment 50. The test kit of any of embodiments 48-49, wherein the lacritin derived peptide comprises a segment of lacritin that includes the N-terminal portion.

[0116] Embodiment 51. The test kit of any of embodiments 48-50, wherein the first portion of monomeric lacritin and the portion of the lacritin derived peptide each comprises a N- terminal portion.

[0117] Embodiment 52. The test kit of any of embodiments 48-51, wherein the first portion of monomeric lacritin and the portion of the lacritin derived peptide each comprises a C- terminal portion.

[0118] Embodiment 53. Atestkit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first anti-lacritin antibody having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a first molecule having a second affinity to bind to a second portion of monomeric lacritin. [0119] Embodiment 54. Atestkit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the detection zone comprises a test location, and wherein the test location comprises a first anti- lacritin antibody having a first affinity to bind to a first portion of monomeric lacritin; and wherein the first zone comprises a first molecule having a second affinity to bind to a second portion of monomeric lacritin, the first molecule coupled to a first label.

[0120] Embodiment 55. The test kit of any of embodiments 53-54, wherein the first molecule comprises Syndecan, wherein the second portion comprises a N-terminal portion, and wherein the first portion comprises a C-terminal portion.

[0121] Embodiment 56. The test kit of any of embodiments 53-55, wherein a signal density increases with an increase in monomeric lacritin concentration in the test specimen.

[0122] Embodiment 57. The test kit of any of embodiments 53-56, wherein the first label is selected from a nanoparticle, bead, latex bead, aptamer, oligonucleotide, a quantum dot, and a combination thereof.

[0123] Embodiment 58. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first molecule and a first anti-lacritin antibody, wherein the first molecule is coupled to at least one of a first label and a first tag, and wherein the first anti- lacritin antibody is coupled to at least one of the first label and the first tag; and wherein the detection zone comprises a test location, and wherein the test location comprises a first anti-tag, wherein the first tag and the first anti-tag comprise a first tag/anti-tag pair.

[0124] Embodiment 59. The test kit of embodiment 58, wherein the first molecule comprises Syndecan, wherein Syndecan is coupled to the first label, and wherein the first anti- lacritin antibody is coupled to the first tag.

[0125] Embodiment 60. The test kit of any of embodiments 58-59, wherein the first molecule comprises Syndecan, wherein Syndecan is coupled to the first tag, and wherein the first anti-lacritin antibody is coupled to the first label.

[0126] Embodiment 61. The test kit of any of embodiments 58-60, wherein the first molecule has a first affinity to bind to a first portion of monomeric lacritin comprising an N- terminal domain portion, and wherein the first anti-lacritin antibody has a second affinity to bind to a second portion of monomeric lacritin different from the first portion.

[0127] Embodiment 62. The test kit of any of embodiments 58-61, wherein a signal density increases with an increase in monomeric lacritin concentration in the test specimen. [0128] Embodiment 63. The test kit of any of embodiments 58-62, wherein the detection zone further comprises a second test location.

[0129] Embodiment 64. The test kit of any of embodiments 58-63, wherein the detection zone further comprises a control location.

[0130] Embodiment 65. The test kit of any of embodiments 58-64, wherein the first zone comprises a sample receiving portion and a conjugate release pad, and wherein the conjugate release pad comprises the Syndecan and the first anti-lacritin antibody.

[0131] Embodiment 66. The test kit of any of embodiments 58-65, wherein the detection zone comprises a nitrocellulose membrane.

[0132] Embodiment 67. The test kit of any of embodiments 1-10, wherein the first antibody lacks an affinity to bind to non-monomeric lacritin.

[0133] Embodiment 68. The test kit of any of embodiments 1-10 or 67, wherein the second antibody lacks an affinity to bind to non-monomeric lacritin.

[0134] Embodiment 69. The test kit of any of embodiments 11-19, wherein the first antibody lacks an affinity to bind to non-monomeric lacritin.

[0135] Embodiment 70. The test kit of any of embodiments 11-19 or 69, wherein the lacritin derived peptide lacks an affinity to bind to non-monomeric lacritin.

[0136] Embodiment 71. The test kit of any of embodiments 20-28, wherein the first anti-lacritin antibody lacks an affinity to bind to non-monomeric lacritin.

[0137] Embodiment 72. The test kit of any of embodiments 29-37, wherein the first antibody lacks an affinity to bind to non-monomeric lacritin.

[0138] Embodiment 73. The test kit of any of embodiments 38-47, wherein the first molecule lacks an affinity to bind to non-monomeric lacritin.

[0139] Embodiment 74. The test kit of any of embodiments 38-47 or 69, wherein the second molecule lacks an affinity to bind to non-monomeric lacritin.

[0140] Embodiment 75. The test kit of any of embodiments 48-52, wherein the first molecule lacks an affinity to bind to non-monomeric lacritin.

[0141] Embodiment 76. The test kit of any of embodiments 48-52 or 76, wherein the second molecule lacks an affinity to bind to non-monomeric lacritin.

[0142] Embodiment 77. The test kit of any of embodiments 53-57, wherein the first anti-lacritin antibody lacks an affinity to bind to non-monomeric lacritin.

[0143] Embodiment 78. The test kit of any of embodiments 53-57 or 77, wherein the first molecule lacks an affinity to bind to non-monomeric lacritin. [0144] Embodiment 79. The test kit of any of embodiments 58-66, wherein the first anti-lacritin antibody lacks an affinity to bind to non-monomeric lacritin.

[0145] Embodiment 80. The test kit of any of embodiments 58-66 or 79, wherein the first molecule lacks an affinity to bind to non-monomeric lacritin.

[0146] Embodiment 81. The test kit of any of embodiments 1-80, wherein the first portion comprises a C-term epitope comprising SEQ ID NO. 3.

[0147] Embodiment 82. The test kit of any of embodiments 1-81, wherein the immunoassay comprises an enzyme-linked immunosorbent assay (ELISA).

[0148] Embodiment 83. The test kit of any of embodiments 1-82, wherein at least one of the first and second portions consists of a C-term epitope consisting of SEQ ID NO. 3.

[0149] Embodiment 84. The test kit of any of embodiments 1-10, wherein the first antibody is hindered from binding to non-monomeric lacritin.

[0150] Embodiment 85. The test kit of any of embodiments 1-10 or 67, wherein the second antibody is hindered from binding to non-monomeric lacritin.

[0151] Embodiment 86. The test kit of any of embodiments 11-19, wherein the first antibody is hindered from binding to non-monomeric lacritin.

[0152] Embodiment 87. The test kit of any of embodiments 11-19 or 69, wherein the lacritin derived peptide is hindered from binding to bind to non-monomeric lacritin.

[0153] Embodiment 88. The test kit of any of embodiments 20-28, wherein the first anti-lacritin antibody is hindered from binding to non-monomeric lacritin.

[0154] Embodiment 89. The test kit of any of embodiments 29-37, wherein the first antibody is hindered from binding to non-monomeric lacritin.

[0155] Embodiment 90. The test kit of any of embodiments 38-47, wherein the first molecule is hindered from binding to non-monomeric lacritin.

[0156] Embodiment 91. The test kit of any of embodiments 38-47 or 69, wherein the second molecule is hindered from binding to non-monomeric lacritin.

[0157] Embodiment 92. The test kit of any of embodiments 48-52, wherein the first molecule is hindered from binding to non-monomeric lacritin.

[0158] Embodiment 93. The test kit of any of embodiments 48-52 or 76, wherein the second molecule is hindered from binding to non-monomeric lacritin.

[0159] Embodiment 94. The test kit of any of embodiments 53-57, wherein the first anti-lacritin antibody is hindered from binding to non-monomeric lacritin.

[0160] Embodiment 95. The test kit of any of embodiments 53-57 or 77, wherein the first molecule is hindered from binding to non-monomeric lacritin. [0161] Embodiment 96. The test kit of any of embodiments 58-66, wherein the first anti-lacritin antibody is hindered from binding to non-monomeric lacritin.

[0162] Embodiment 97. The test kit of any of embodiments 58-66 or 79, wherein the first molecule is hindered from binding to non-monomeric lacritin.

[0163] Embodiment 98. The test kit of any of embodiments 1-80, wherein the second portion comprises a C-term epitope comprising SEQ ID NO. 3.

[0164] Thus, specific examples of assays, test kits and methods for detecting and measuring monomeric lacritin have been described. The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles described herein can be applied to other embodiments without departing from the spirit or scope of the invention. Thus, it is to be understood that the description and drawings presented herein represent a presently preferred embodiment of the invention and are therefore representative of the subject matter which is broadly contemplated by the present invention. It is further understood that the scope of the present invention fully encompasses other embodiments that may become obvious to those skilled in the art and that the scope of the present invention is accordingly not limited.

[0165] Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

[0166] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0167] Also, as used herein, and unless the context dictates otherwise, the term “coupled to” is intended to include both direct coupling (in which two elements that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two elements).

[0168] Reference throughout this specification to “an embodiment” or “an implementation” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment or implementation. Thus, appearances of the phrases “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment or a single exclusive embodiment. Furthermore, the particular features, structures, or characteristics described herein may be combined in any suitable manner in one or more embodiments or one or more implementations.

[0169] The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any aspect described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects. Unless specifically stated otherwise, the term “some” refers to one or more.

[0170] Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints and open-ended ranges should be interpreted to include only commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0171] Certain numerical values and ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating un-recited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0172] Combinations, described herein, such as “at least one of A, B, or C,” “one or more of A, B, or C,” “at least one of A, B, and C,” “one or more of A, B, and C,” and “A, B, C, or any combination thereof’ include any combination of A, B, and/or C, and may include multiples of A, multiples of B, or multiples of C. Specifically, combinations such as “at least one of A, B, or C,” “one or more of A, B, or C,” “at least one of A, B, and C,” “one or more of

A, B, and C,” and “A, B, C, or any combination thereof’ may be A only, B only, C only, A and

B, A and C, B and C, or A and B and C, and any such combination may contain one or more members of its constituents A, B, and/or C. For example, a combination of A and B may comprise one A and multiple B’s, multiple A’s and one B, or multiple A’s and multiple B’s.

[0173] All structural and functional equivalents to the components of the various aspects described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

SEQUENCE LISTING

SEQ ID NO 1 : An exemplary amino acid sequence of a monomeric lacritin. An exemplary amino acid sequence of an epitope for an exemplary antibody against N-term lacritin is indicated in bold, and an exemplary amino sequence of an epitope for an exemplary antibody against C-term lacritin is indicated in underlined in italic.

MKFTTLLFLA AVAGALVYAE DASSDSTGAD PAQEAGTSKP NEEISGPAEP

ASPPETTTTA QETSAAAVQG TAKVTSSRQE LNPLKSIVEK SILL TEQ ALA

KAGKGMHGGV PGGKQFIENG SEFAQKLLKK FSLLKPWA

SEQ ID NO 2: An exemplary amino acid sequence of an epitope for an exemplary antibody against N-term lacritin:

E DASSDSTGAD PAQEAGT

SEQ ID NO 3 : An exemplary amino acid sequence of an epitope for an exemplary antibody against C-term lacritin:

SEFAQKLLKK

Claims

1. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first molecule having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first molecule is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the first test location comprises a second molecule having a second affinity to bind to a second portion of monomeric lacritin.

2. The test kit of claim 1, wherein the first molecule comprises a first antibody having the first affinity to bind to the first portion of monomeric lacritin, and wherein the second molecule comprises a second antibody having the second affinity to bind to the second portion of monomeric lacritin.

3. The test kit of claim 2, wherein the first label is selected from a nanoparticle, bead, latex bead, aptamer, oligonucleotide, a quantum dot, and a combination thereof.

4. The test kit of claim 2, wherein the detection zone further comprises a control location, and wherein the control location comprises a control antibody.

5. The test kit of claim 2, wherein the first portion is a N-terminal of monomeric lacritin.

6. The test kit of claim 2, wherein the second portion is a C-terminal of monomeric lacritin.

7. The test kit of claim 2, wherein a signal density increases with an increase in monomeric lacritin concentration in the test specimen.

8. The test kit of claim 2, wherein the first molecule is hindered from binding to non-monomeric lacritin.

9. The test kit of claim 2, wherein the first zone comprises a sample receiving portion and a conjugate release pad, and wherein the conjugate release pad comprises the first antibody coupled to the first label.

10. The test kit of claim 1, wherein the second molecule is hindered from binding to non-monomeric lacritin.

11. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first molecule having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first molecule is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a second molecule, and wherein the first molecule has a second affinity to bind to a portion of the second molecule.

12. The test kit of claim 1, wherein the first molecule is a first antibody having the first affinity and the second affinity, wherein the second molecule comprises a lacritin derived peptide, and wherein a signal density decreases with an increase in monomeric lacritin concentration in the test specimen.

13. The test kit of claim 12, wherein the lacritin derived peptide comprises a segment of lacritin that includes the N-terminal portion.

14. The test kit of claim 12, wherein the first portion of monomeric lacritin and the portion of the lacritin derived peptide each comprises a N-terminal portion.

15. The test kit of claim 12, wherein the first molecule is hindered from binding to non-monomeric lacritin.

16. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first anti-lacritin antibody having a first affinity to bind to a first portion of monomeric lacritin, and wherein the first antibody is coupled to a first label; and wherein the detection zone comprises a test location, and wherein the test location comprises a first molecule having a second affinity to bind to a second portion of monomeric lacritin.

17. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the detection zone comprises a test location, and wherein the test location comprises a first anti-lacritin antibody having a first affinity to bind to a first portion of monomeric lacritin; and wherein the first zone comprises a first molecule having a second affinity to bind to a second portion of monomeric lacritin, the first molecule coupled to a first label; and

18. The test kit of any of claims 16-17, wherein the first molecule comprises Syndecan, wherein the second portion comprises a N-terminal portion, and wherein the first portion comprises a C-terminal portion.

19. The test kit of any of claim 16-17, wherein a signal density increases with an increase in monomeric lacritin concentration in the test specimen, and wherein the first anti- lacritin antibody is hindered from binding to non-monomeric lacritin.

20. The test kit of any of claims 16-17, wherein the first label is selected from a nanoparticle, bead, latex bead, aptamer, oligonucleotide, a quantum dot, and a combination thereof.

21. A test kit for detection of monomeric lacritin in a test specimen, comprising: an immunoassay having a first zone upstream from a detection zone; wherein the first zone comprises a first molecule and a first anti-lacritin antibody, wherein the first molecule is coupled to at least one of a first label and a first tag, and wherein the first anti-lacritin antibody is coupled to at least one of the first label and the first tag; and wherein the detection zone comprises a test location, and wherein the test location comprises a first anti-tag, wherein the first tag and the first anti-tag comprise a first tag/anti-tag pair.

22. The test kit of claim 21, wherein the first molecule comprises Syndecan, wherein Syndecan is coupled to the first label, and wherein the first anti-lacritin antibody is coupled to the first tag.

23. The test kit of claim 21, wherein the first molecule comprises Syndecan, wherein Syndecan is coupled to the first tag, and wherein the first anti-lacritin antibody is coupled to the first label.

24. The test kit of claim 21, wherein the first molecule has a first affinity to bind to a first portion of monomeric lacritin comprising a C-terminal domain portion, and wherein the first anti-lacritin antibody has a second affinity to bind to a second portion of monomeric lacritin different from the first portion.

25. The test kit of claim 21, wherein a signal density increases with an increase in monomeric lacritin concentration in the test specimen.

26. The test kit of claim 21, wherein each of the first molecule and the first anti- lacritin antibody is hindered from binding to non-monomeric lacritin.

27. The test kit of claim 21, wherein the detection zone further comprises a control location.

28. The test kit of claim 21, wherein the first zone comprises a sample receiving portion and a conjugate release pad, and wherein the conjugate release pad comprises the Syndecan and the first anti-lacritin antibody.

29. The test kit of claim 21, wherein the detection zone comprises a nitrocellulose membrane.

30. A reagent for use in an immunoassay method for detecting and quantifying monomeric lacritin in a sample, the reagent comprising an antibody having an affinity to bind to a portion of monomeric lacritin, the portion comprising SEQ ID NO. 3.

31. The test kit of claim 2, wherein and the first portion or the second portion comprises a C-term epitope comprising SEQ ID NO. 3.

32. The test kit of claim 12, wherein the first portion comprises a C-term epitope comprising SEQ ID NO. 3.

33. The test kit of claim 16, wherein and the first portion or the second portion comprises a C-term epitope comprising SEQ ID NO. 3.

34. The test kit of claim 17, wherein the first portion or the second portion comprises a C-term epitope comprising SEQ ID NO. 3.

35. The test kit of claim 24, wherein the first portion or the second portion comprises a C-term epitope comprising SEQ ID NO. 3.