Test for detecting thiamine deficiency

Abstract

The present invention concerns a diagnostic device and method for detecting thiamine and Vitamin A deficiencies using salivary biomarkers. The device utilizes a lateral flow assay to detect secretory Immunoglobulin A (sIgA) and amylase in saliva, providing a non-invasive, easy-to-use, and reliable method for assessing nutritional deficiencies. This invention is particularly useful for monitoring health status in populations with limited access to medical facilities.

Description

[0001] Test for Detecting Thiamine Deficiency

[0002] Technical Field

[0003] The present invention generally relates to the field of nutritional status, diagnostic tests, assays, in particular lateral flow assays, and to the determining or detection of deficiencies of vitamins in a subject, and methods and uses related to the above.

[0004] Background Art and Problems Solved by the Invention

[0005] Thiamine (Vitamin Bl) is essential for the proper functioning of the immune system, particularly in the programming of B cells to IgA-secreting plasma cells in Peyer's patches (Kunisawa et al., 2015; Sutherland et al., 2016). Thiamine deficiency is challenging to diagnose, especially in remote areas with limited medical infrastructure (World Health Organization, 2001). Additionally, Biesalski (2004) highlighted the role of Vitamin A in immune function, which can also affect mucosal immunity over prolonged deficiencies. Current diagnostic methods for nutritional deficiencies often involve invasive procedures, are limited to detecting single biomarkers, or require sophisticated laboratory equipment. These limitations make it difficult to perform widespread and timely assessments, particularly in resource-limited settings.

[0006] Thiamine deficiency can occur within three months due to the depletion of physiological stores (Lonsdale, 2006). In contrast, Vitamin A deficiency takes about three years to manifest due to its larger physiological stores (Biesalski, 2004).

[0007] It is an objective of the invention to detect acute thiamine deficiency.

[0008] It is a further objective to detect also detect chronic malnutrition such as Vitamin A deficiency.

[0009] It is an objective to provide a tool for assessing nutritional status, ideally in a comprehensive manner.

[0010] B8370WO; 07 / 07 / 2025 It is a further objective of the invention to provide a tool that can be produced at low costs, that can be used easily and rapidly, and that ideally allows for a rapid, visual read out.

[0011] It is an objective to provide a diagnostic test that does not require complex reading machines for determining the test outcome.

[0012] It is a further objective of the invention to provide a test or assay that can be used everywhere, for example at home, in the field, or, more generally, outside laboratories and / or health facilities such as hospitals and pharmacies, for example.

[0013] Further objectives and problems addressed by the present invention will become apparent from the description of the aspects and embodiments of the invention herein below.

[0014] Summary of the Invention

[0015] In an aspect, the invention provides an assay for detecting thiamine deficiency.

[0016] In another aspect, the invention provides an assay for detecting Vitamin A deficiency.

[0017] In a preferred embodiment, the invention provides an assay for detecting one or both selected from thiamine deficiency and Vitamin A deficiency.

[0018] In a preferred embodiment, the invention provides an assay for diagnosing thiamine deficiency and / or Vitamin A deficiency on the basis of a body liquid sample taken from an subject and / or individual, preferably a human individual. The liquid sample preferably comprises a sample of saliva.

[0019] In a preferred embodiment, the assay is an immunoassay, more preferably a lateral flow assay (LFA). The assay may be a sandwich-type or competitive immune assay.

[0020] In an embodiment, the assay comprises a binding molecule that is capable of specifically binding to secretory Immunoglobulin A (slgA).

[0021] B8370WO; 07 / 07 / 2025 The diagnosis or read out with respect to thiamine deficiency is preferably based on the detection of slgA in the sample.

[0022] The diagnosis or read out with respect to Vitamin A deficiency is preferably based on the detection of slgA in the sample, preferably over a period of time and / or by using the assay repeatedly.

[0023] In an embodiment, the assay comprises a binding molecule that is capable of specifically binding to amylase.

[0024] In other aspects, the invention provides a method for detecting and / or diagnosing thiamine and / or Vitamin A deficiency in an individual. In some embodiments, the method comprises loading a body liquid sample on a sample application area or port, and detecting and / or diagnosing thiamine and / or Vitamin A deficiency on the basis of the detection of slgA in said sample.

[0025] In an embodiment, the individual is an alcoholic and / or alcohol dependent.

[0026] In an embodiment, the individual is potentially malnourished and / or is at a risk of being malnourished.

[0027] In an aspect, the invention provides the use of a binding molecule binding specifically to slgA in a diagnostic test for detecting thiamine deficiency and / or Vitamin A deficiency.

[0028] In an aspect, the invention provides the use of a binding molecule binding specifically to amylase in the diagnostic test of the invention.

[0029] In an embodiment, the diagnostic assay and method for detecting thiamine and / or Vitamin A deficiencies uses one or more salivary biomarkers. In a preferred embodiment, the device utilizes a lateral flow assay to detect secretory Immunoglobulin A (slgA) and / or amylase in saliva, providing a non-invasive, easy-to-use, and reliable method for assessing nutritional deficiencies. This invention is particularly useful for monitoring health status in populations with limited access to medical facilities

[0030] B8370WO; 07 / 07 / 2025 A particularly novel embodiment of the invention is the combination of a defined saliva dilution step (for example approximately 1 : 10'000) with the simultaneous detection of secretory Immunoglobulin A (slgA) and amylase. This combination allows for precise quantification of slgA, which at lower dilution factors would otherwise exceed detection limits, while amylase acts as a robust internal control to verify both the functionality of the assay and the accuracy of the dilution step. This design uniquely ensures reliability of the diagnostic result even in settings with minimal operator training.

[0031] Further aspects and preferred embodiments of the invention are defined herein below and in the appended claims. Further features and advantages of the invention will become apparent to the skilled person from the description of the preferred embodiments given below.

[0032] Detailed Description of Preferred Embodiments

[0033] The present invention provides an assay and / or diagnostic test for detecting thiamine deficiency, Vitamin A deficiency, or both. Thiamine is also known as Vitamin Bl .

[0034] In some embodiments, the assay is provided in the form of and / or comprises a device. The assay and / or device is preferably portable and / or hand-held.

[0035] The assay of the invention is preferably non-invasive and / or easy-to-use.

[0036] The assay of the invention preferably provides a reliable method for assessing nutritional deficiencies.

[0037] In a preferred embodiment, the assay is an immunoassay.

[0038] In a preferred embodiment, the assay is a lateral flow assay (LFA), for example a sandwichtype or competitive type LFA.

[0039] In an embodiment, the assay comprises one or more selected from the group consisting of: a sample application area, a conjugate pad comprising preferably binding molecules specific for slgA and / or amylase, one or more detection regions with lines or symbols indicating the

[0040] B8370WO; 07 / 07 / 2025 presence of slgA and / or amylase. Preferably, the assay comprises a control line to confirm the proper function of the assay.

[0041] In the assay of the invention, thiamine status is preferably determined on the basis of the presence or absence slgA in a sample of saliva of an individual. The presence of slgA in saliva, or the presence of slgA above a minimum or detectable level is indicative for the absence of thiamine deficiency. On the other hand, absence of slgA in saliva, or the presence of slgA below a minimum or detectable level is indicative for thiamine deficiency.

[0042] In some embodiments, the sample is and / or comprises diluted saliva, for example saliva that is diluted to a predetermined dilution factor. An exemplary dilution factor is 1 : 10'000. More generally, the dilution factor is preferably in the range of 1 :100 to 1 : 1'000'000, more preferably in the range of 1 : 1'000 to 1 :100'000, for example 1 :5'000 to 1 :30'000.

[0043] For the purpose of the present specification, the expressions "sample of saliva", "saliva sample" or just "sample" encompass and / or mean a "sample comprising saliva", and in particular encompass diluted saliva as indicated above, for example.

[0044] Utilizing saliva as a diagnostic sample is a novel approach that ensures the method is non- invasive, easy to administer, and suitable for widespread use in various populations, including children and vulnerable groups.

[0045] In an embodiment, the assay of the invention comprises one or more detection regions, areas, and / or zones. The assay of the invention is preferably suitable to provide a readable signal at said detection zone, said readable signal allowing a user to determine the test outcome.

[0046] In an embodiment, the assay of the invention is suitable to produce a readable signal allowing determining if said sample comprises slgA, or if a level (e.g. amount, concentration) of slgA in said sample is present at detectable and / or minimum levels.

[0047] In an embodiment, positive detection of slgA in the sample indicates no thiamine deficiency.

[0048] In an embodiment, a readable signal indicating absence of slgA or the presence of slgA in said sample below said detectable and / or minimum levels indicates thiamine deficiency.

[0049] B8370WO; 07 / 07 / 2025 In an embodiment, the detectable threshold for slgA following dilution is approximately X ng / ml, wherein X preferably represents a value of a physiological range in a subject not suffering from thiamine deficiency. Similarly, amylase levels should be within the expected physiological range post-dilution, serving as a confirmation of both correct sampling and dilution.

[0050] SlgA in saliva can serve as a non-invasive biomarker for thiamine deficiency. Studies have shown that thiamine deficiency leads to the depletion of IgA plasma cells, potentially indicated by reduced slgA levels in saliva (Porter et al., 1966; Kunisawa et al., 2015).

[0051] In a preferred embodiment, the assay comprises a binding molecule that is capable of specifically binding to slgA. The binding molecule is or comprises preferably an antibody.

[0052] The term "antibody", for the purpose of the present specification, is intended to encompass immunoglobulins in general, antibodies and fragments thereof. Exemplary fragments are an Fc fragment (crystallizable fragment), an Fab fragment (antigen binding fragment), an Fv fragment (variable fragment), a scFv (single chain variable fragment), an Ab heavy chain, an Ab light chain, a VH domain (heavy chain variable region), a VL domain (light chain variable region), a CH (heavy chain constant region), a CL (light chain constant region). Exemplary Fab fragments encompass Fab, Fab', F(ab')2 fragments. In a preferred embodiment, the antibody comprises at least one variable region or domain of an antibody and / or at least a complementarity-determining region (CDR) of an antibody. The antibody may be independently selected from IgG, IgM, IgA, IgD and IgE, for example.

[0053] Different antibodies mentioned in this specification may independently be selected from proteins comprising the exemplary antibodies and fragments recited herein.

[0054] The binding molecule is preferably specific to slgA, and preferably does not bind to, or to a much lesser extent, to other compounds or molecules that may be present in the sample.

[0055] In an embodiment, the assay comprises slgA or an slgA variant suitable to specifically bind to said binding molecule. Such an embodiment applies, for example, to a competitive LFA. The "variant", for the purpose of the invention, is a molecule comprising at least a fragment of the

[0056] B8370WO; 07 / 07 / 2025 original molecule (e.g. slgA) and / or is capable of binding to and / or forming a complex with the binding molecule that is specific to the original molecule.

[0057] In a competitive LFA, the slgA or a slgA variant may be immobilized at the detection region, and the assay detects binding of the binding molecule to said slgA or slgA variant.

[0058] In a competitive LFA, slgA in the sample of saliva binds to the binding molecule, generally in the conjugate pad and / or the transfer region, and transports the same by sample flow through the flow assay to the detection region, where the binding molecule binds to the immobilized molecule (e.g. immobilized slgA and / or slgA variant). Competitive assays are generally used when the analyte is relatively small and / or has only one epitope allowing specific binding.

[0059] In an embodiment of the assay, said binding molecule suitable to specifically bind to slgA, is immobilized at said one or more detection regions. The binding molecule may be present in mobile or free form in the transfer region and may be immobilized in the detection region.

[0060] In an embodiment of the assay, said slgA or a slgA variant suitable to specifically bind to said binding molecule, if present, is provided at said one or more detection regions, preferably immobilized at said one or more detection regions.

[0061] In an embodiment, said assay, preferably said one or more detection regions, is arranged for producing a readable signal serving as a positive control signal indicating that the assay is functional, is not defect and / or that the assay has been used correctly such that the read out is expected to be correct. A particular control signal, e.g. if absent, may indicate that the test outcome is invalid and / or not reliable.

[0062] The "positive control read out" or "positive control" is a read out allowing the conclusion of whether the outcome of the assay, as determined preferably at the one or more detection regions, is likely to be reliable and / or correct.

[0063] In an embodiment, said slgA is a first analyte and wherein said binding molecule is a first binding molecule. An "analyte", for the purpose of this specification, is a molecule in the sample that is to be detected by the assay. Exemplary analytes include slgA and amylase.

[0064] B8370WO; 07 / 07 / 2025 In an embodiment, said lateral flow test comprises a second binding molecule. The second binding molecule is preferably capable of specifically binding to at least a second analyte. Said second analyte is preferably selected from amylase, a mucin or mucins, lysozyme and lactoferrin, but preferably is amylase.

[0065] In an embodiment, said readable signal is a first readable signal, and wherein said assay, preferably said one or more detection regions, is arranged for producing at least a second readable signal allowing determining if said sample comprises said second analyte or if an amount of said second analyte in said sample is present at required levels, for example at detectable and / or minimum levels.

[0066] In an embodiment, the assay of the invention is suitable to detect amylase. Amylase detection may be used as a positive control or may be detected in addition to a separate positive control, for example.

[0067] While amylase is a preferred internal control for the LFA application due to its high relative abundance in saliva, alternatives such as mucins, lysozyme, and lactoferrin are also encompassed. Amylase, representing around 40-50% of the total protein content in saliva, is significantly abundant, making it a reliable marker (Humphrey & Williamson, 2001; Denny et al., 1997). Mucins (20-30%), lysozyme (5-10%), and lactoferrin (2-5%) are less abundant and more variable depending on health and environmental factors (Spielmann & Wong, 2011; Vitorino et al., 2004). This high abundance and stability make amylase a superior internal control for ensuring the reliability of the LFA.

[0068] In some embodiment, the invention provides the simultaneous detection of secretory Immunoglobulin A (slgA) and amylase in saliva. This preferably provides a comprehensive assessment of nutritional status, specifically detecting both thiamine and Vitamin A deficiencies. The dual biomarker approach of some embodiments is unique and offers significant advantages over traditional single-biomarker methods.

[0069] Alternatively or in addition, amylase is used as an internal control. Amylase, due to its high abundance and stability in saliva, as an internal control in the lateral flow assay, ensures the reliability and accuracy of the diagnostic results. This inventive choice enhances the assay's performance compared to other potential internal controls.

[0070] B8370WO; 07 / 07 / 2025 The control line based on amylase detection may also be used to assess if saliva has been correctly diluted.

[0071] In a preferred embodiment, said positive control signal is based on the presence of a second analyte in the sample. Preferably, the positive control signal is based on the binding of said second binding molecule to said second analyte, or on the binding of said second binding molecule to a molecule comprising said second analyte or a variant of said second analyte.

[0072] In a preferred embodiment, said second analyte is amylase.

[0073] In a preferred embodiment, said second binding molecule is capable of specifically binding to amylase.

[0074] In a preferred embodiment, said one or more detection regions is arranged for producing a readable signal allowing determining if said sample comprises amylase, or if a levels of amylase in said sample is present at required levels, for example detectable and / or minimum levels.

[0075] In an embodiment of the assay, positive detection of amylase indicates that the assay functions correctly and / or that the saliva has been correctly diluted.

[0076] In a preferred embodiment, a readable signal indicating the absence of slgA, or the presence of slgA in said sample below said detectable and / or minimum levels indicates Vitamin A deficiency, preferably chronic Vitamin A deficiency. This diagnose applies in particular if slgA is found low, for example absent, over a period in which the assay is used repeatedly.

[0077] In a preferred embodiment, the assay comprises a sample application area or port adapted to receiving said sample.

[0078] For example, in an LFA, the assay comprises a sample pad. The sample pad is preferably suitable to distribute the sample and / or to direct it to a conjugate pad / transfer region. The sample pad may comprise one or more selected from buffer salts, proteins, surfactants, liquids, and sugars. The sample pad may be impregnated with one or more of these

[0079] B8370WO; 07 / 07 / 2025 components. The sample pad is preferably porous and comprises pores determining the flow rate of the sample through the sample pad and / or towards the conjugate pad.

[0080] The assay of the invention may or may not comprise a housing, for example made of plastic. If the assay comprises a housing, e.g. made of plastic., the housing may comprise an opening to said sample pad and forming a sample port.

[0081] In a preferred embodiment, the assay comprises one or more detection regions. The detection region preferably comprises a membrane comprising reagents immobilized in on one or more lines, symbols, and / or patterns. The membrane may be a nitrocellulose membrane, but other materials are encompassed.

[0082] The one or more detection region may comprise one or more test lines, symbols, and / or patterns.

[0083] Preferably, one or more detection region may comprise at least one control line, symbol and / or pattern.

[0084] For the purpose of this specification, and for reasons of convenience, the expressions "test line" and "control line" encompass forms, contours, patterns, symbols, etc. other than lines.

[0085] At the test and / or control lines, the assay preferably comprises immobilized reagents, such as antibodies, antigens, proteins and / or fragments thereof. The type of immobilized agent generally depends on the type of assay, e.g. sandwich-type or competitive LFA. In case of a sandwich-type assay, antibodies specifically binding to the one or more analytes, in particular the first and second analytes, to be detected are immobilized on a test line. In case of a competitive assay, an artificial, for example synthetic or recombinant version of the analyte, in particular of the first and second analyte, or a variants thereof, are immobilized at the test line. These versions of the analyte are generally referred to as "variants" in this specification.

[0086] A binding event at the test line may be considered to indicate a positive test outcome. E.g. binding of slgA at the test line results in a positive test signal, which, in the test of the invention, can be used to determine that the individual whom the sample has been obtained

[0087] B8370WO; 07 / 07 / 2025 from does not suffer from thiamine deficiency and / or does not suffer from Vitamin A deficiency.

[0088] It is noted in this context that the terms "positive" and "negative", in the context of the outcome of the assay, is to some extent arbitrary in that they can be inverted by definition. For example, the detection of a binding event at the test line could be considered as negative test outcome in that thiamine deficiency and / or Vitamin A deficiency is not found and is thus absent. In the example section hereinafter, the symbol [+] is used for indicating a binding event.

[0089] In an embodiment, the assay of the invention comprises one test line and one control line. In an embodiment, the test line is suitable to detect if slgA is present in the sample, and the control line is suitable to determine if the test is functional, if sample dilution has been correctly conducted and / or if amylase is present in the sample. Preferably, the assay comprises at least one binding molecule, which specifically binds to slgA. The control line may be suitable to detect if amylase is present in the sample. The assay preferably comprises a second binding molecule that specifically binds to amylase. Mobile and immobilized forms of the second binding molecule may be present, for example in the conjugate pad and detection regions, respectively.

[0090] In an embodiment, the assay of the invention comprises two test lines and one control line. In an embodiment, a first test line is suitable to detect if slgA is present in the sample and a second test line is suitable to detect if amylase is present in the sample. The assay preferably comprises a first binding molecule, that specifically binds to slgA, and a second binding molecule, that specifically binds to amylase. The control line may detect another analyte, may be unrelated to amylase and / or slgA, or may be a binding molecule specific to slgA or amylase but present in excess.

[0091] If the assay comprises a housing, e.g. made of plastic, the housing may comprise an opening to said membrane, the housing delimiting a read-out window, for example, at said detection region.

[0092] The assay may comprise one, two or more test lines, for example for detecting if one, two or more different analytes are present in the sample.

[0093] B8370WO; 07 / 07 / 2025 In a preferred embodiment, the assay comprises a transfer region and / or a conjugate pad, allowing said sample and / or analytes comprised in said sample to move from said sample application area or port to said one or more detection regions.

[0094] One or more binding molecules, for example first and second binding molecules, are preferably provided mobile in said conjugate pad.

[0095] In an embodiment, the assay comprises at least one tag and / or label. The tag or label is the part of the test that allows the read out, e.g. the detection of a binding reaction at the test and / or control lines.

[0096] The one or more mobile binding molecules may be conjugated to the tag and / or label, which tag and / or label is detectable at said detection region, for example by visual inspection of the detection region, or, in a less preferred embodiment, using of an appropriate reading equipment. Alternatively, the tag and / or label may be conjugated to one or more separate, preferably also mobile, binding molecules, which may be specific to the first and / or second mobile binding molecules.

[0097] To make an example, the tag may be conjugated to an antibody that is specific to an antibody that specifically binds slgA and / or to an antibody that binds amylase, or to both antibodies, the latter being said first and second binding molecules. For example, the tag may be conjugated to an antibody that specifically binds to the constant region or to the framework region of the first and / or second binding molecules. In this manner, it is possible to use the same tag-antibody conjugate for detecting both, the binding reaction of slgA with the first binding molecule and the binding reaction of amylase to the second binding molecule.

[0098] In some embodiments, the assay is provided for qualitative use, and suitable to detect and / or diagnose one or both selected from thiamine deficiency and Vitamin A deficiency.

[0099] In other embodiments, the assay detects and / or diagnoses, independently, one or both selected from thiamine deficiency and Vitamin A deficiency semi-quantitively and / or quantitatively. Different types of semi-quantitative and / or quantitative LFAs have been reported. Semi- quantitative and / or quantitative outcomes of the assay may be determined on the strength of

[0100] B8370WO; 07 / 07 / 2025 the signal that the assay produces as a test result. For example, the intensity of color at a test line may be assessed approximately, for example by visual inspection of the read out, or more accurately, for example using equipment suitable to determine the strength of the signal produced.

[0101] A test outcome in which the test line related to slgA is visible but not as strong as would be expected from an individual having no thiamine and / or Vitamin A deficiency, respectively, may be interpreted semi-quantitatively to indicate an increased risk of thiamine and / or Vitamin A deficiency occurring in the future if no measures are taken, partial deficiency, and / or sub-optimal thiamine and / or Vitamin A status, for example.

[0102] It is noted that the invention encompasses that one analyte is detected qualitatively and the other semi-quantitatively, one semi-quantitatively and the other quantitatively, one qualitatively and the other quantitatively, for example. In some embodiments, slgA is determined qualitatively and amylase semi-quantitatively or quantitatively. In another embodiment, amylase is determined qualitatively and slgA semi-quantitatively or quantitatively. In yet another embodiment, only one of the two analytes is detected.

[0103] In some aspects and embodiments, the invention provides a method for detecting and / or diagnosing thiamine and / or Vitamin A deficiency in a subject and / or individual. The method comprises applying a saliva sample to an assay, preferably to an assay suitable to detect, respectively, slgA and / or amylase in the sample.

[0104] Preferably, the method comprises diluting the sample of saliva, for example with water, before applying the sample to a loading area and / or to a sample pad. Dilution factors are disclosed elsewhere in this description. A preferred dilution factor is approximately 10'000. Dilution is a preferred or critical factor for accurate differentiation of slgA concentrations within the assay's dynamic range. The amylase detection in the assay serves to verify correct dilution and assay validity.

[0105] The method preferably comprises reading the test result at the detection region. In preferred embodiments, the test result is read by visual inspection, for example by the individual and / or of a trained healthcare specialist, such as a nurse, a doctor, a pharmacist, and the like. In some embodiments, the test is easy to read and can be read by untrained personnel.

[0106] B8370WO; 07 / 07 / 2025 In other embodiments, the invention encompasses reading the test outcome with the use of an equipment, such as a particular reading apparatus, the equipment being adapted to read and interpret the readable signal. In such cases, the readable signal may be read by the equipment only. Equipment may be used, for example, for semi-quantitative and / or even quantitative assessment of the test outcome.

[0107] In an embodiment, the method comprises finding and / or diagnosing thiamine deficiency in the individual from which the sample was taken if the assay does not detect slgA in the sample or if the thiamine levels of in the sample are below detectable and / or predefined minimum values.

[0108] In an embodiment, the method comprises finding and / or diagnosing Vitamin A deficiency in the individual from which the sample was taken if the assay does not detect slgA in the sample or if the slgA levels of samples taken over a period of time are regularly and / or consistently below detectable and / or predefined minimum values. The period of time may be at least 2-4 weeks, 1-6 months, 7-12 months, and / or 1-3 years, for example. The method of the invention using the assay may be conducted every two weeks, every month, every 1-6 months, or every year, for example. Preferably, the assay is conducted regularly, at approximately regular intervals.

[0109] Vitamin A deficiency can be suspected if slgA remains low over time in repeated tests, and if it persists after thiamine treatment. A single test showing absence of slgA is generally not interpreted as potential Vitamin A deficiency. The longitudinal decline in slgA, unresponsive to thiamine supplementation, is a strong indicator of chronic Vitamin A deficiency.

[0110] In some embodiments, the method comprises taking a saliva sample from an individual. In other embodiments, the saliva sample has been previously taken from the individual or subject. The individual is preferably a human subject.

[0111] In some embodiments, the sample is diluted prior to applying the sample to a loading area and / or sample pad.

[0112] B8370WO; 07 / 07 / 2025 In some embodiments, the invention encompasses one or more of the following diagnosis on the basis of the reading of the test outcome:

[0113] - Positive detection of both amylase and slgA indicates no thiamine or Vitamin A deficiency.

[0114] - Positive detection of amylase but negative detection of slgA indicates thiamine deficiency.

[0115] Absence of both slgA and amylase, or presence of slgA and absence of amylase, indicates that the test is invalid.

[0116] The present invention encompasses and covers various settings, such as clinical, field, and home testing and various target groups, such as, children, adults, and different geographical regions. In some embodiments, the individual is a person suffering from alcohol dependence, or a person having previously suffered from alcohol dependence.

[0117] The assay and method of the invention may be advantageously used for detecting acute and chronic malnutrition. Thiamine deficiency can occur within three months due to the depletion of physiological stores (Lonsdale, 2006). In contrast, Vitamin A deficiency takes about three years to manifest due to its larger physiological stores (Biesalski, 2004). The assay of the invention can detect both acute thiamine deficiency and chronic malnutrition such as Vitamin A deficiency, providing a comprehensive tool for assessing nutritional status.

[0118] Furthermore, the assay and method of the invention may be advantageously used for monitoring the health of alcoholics. Chronic alcohol consumption is known to cause thiamine deficiency (Mancinelli et al., 2003). The assay of the invention can be particularly useful in monitoring the health status of alcoholics, allowing for early intervention and treatment.

[0119] The assay and method of the invention has utility in various populations. The assay is particularly useful for NGOs working with malnourished populations, detainees, and regions with poor infrastructure (Tomkins, 2003). It offers a non-invasive, easy-to-administer, and reliable method for detecting nutritional deficiencies.

[0120] Examples

[0121] B8370WO; 07 / 07 / 2025 Example 1: Lateral Flow Assay 1 (LSA1) for detecting thiamine deficiency

[0122] LSA1 is designed as a sandwich-type lateral flow immunoassay allowing for visual reading of the test result not requiring read out equipment, with two lines at the detection region: a test line and a control line.

[0123] LSA1 comprises a sample pad, on which a sample of 1 : 10'000 diluted saliva is to be loaded, a conjugate pad, comprising mobile anti-sIgA antibodies and mobile anti-amylase antibodies both conjugated to gold particles, and a nitrocellulose membrane at the read-out region. At the test line, anti-sIgA antibodies are immobilized and at the control line anti-amylase antibodies are immobilized, wherein the immobilized antibodies do not bind to the same epitopes of slgA and amylase, respectively, as the mobile antibodies. LSA1 comprises an adsorbent pad in contact with the membrane, for driving the capillary flow of saliva from the sample pad through the conjugate pad and to the membrane containing the test and control lines.

[0124] Upon loading a saliva sample on the sample application port of LSA1, the saliva migrates through the conjugate pad, in which slgA and amylase analytes, in as far as present, bind to the mobile conjugates, and migrate further with the bound conjugate to the detection zone. slgA and amylase are detected due to binding to the immobilized antibodies on the test and control line, respectively.

[0125] Secretory (s)IgA is found to be present if the test line appears red due to the gold particles [+] and is found to be absent if the test line remains white [-]. The test is considered to be functional if the control line turns red due to the gold particles conjugated to the anti-amylase antibody.

[0126] The following read outs are theoretically possible and will be interpreted as indicated:

[0127] Test line Control line

[0128] [+] [+] test valid, no thiamine deficiency

[0129] [-] [+] test valid, thiamine deficiency is diagnosed

[0130] [+] test invalid, needs to be repeated test invalid, needs to be repeated

[0131] B8370WO; 07 / 07 / 2025 Example 2: Lateral Flow Test 2 (LSA2) for detecting thiamine deficiency and / or Vitamin A deficiency

[0132] LSA2 is a sandwich-type lateral flow immunoassay as LSA1, with the difference that it has three lines at the detection region: two test lines and a control line.

[0133] Test line 1 detects binding to slgA, as described above, and test line 2 detects binding to amylase as described for the control line in LSA1. The control line in LSA2 comprises an immobilized control protein (cP), and the conjugate pad comprises mobile conjugates of an anti-cP-antibody bound to gold particles.

[0134] As with LSA1, a red color [+] at the read out area indicates that an analyte is present in the sample (or that the assay is functional in the case of the control line), whereas absence of the red color [-] indicates absence of binding at the corresponding line.

[0135] In the case of the second test line (amylase detection), the reading may also include semi- quantitative interpretation in cases where the intensity of the color is low, indicating low levels of amylase ([+ / -]).

[0136] The read outs shown in Table 1 below are theoretically possible with LSA2 and will be interpreted as indicated:

[0137] Table 1: Test outcome interpretation based on LSA2

[0138] B8370WO; 07 / 07 / 2025

[0139] Prophetic Examples

[0140] Example 1: Diagnostic Test for Malnourished Children

[0141] A group of children in a malnutrition-affected area are tested for thiamine deficiency using the LFA1. Saliva samples are collected, diluted at a 1 : 10'000 ratio, and applied to the sample port. The test shows the presence of amylase but the absence of slgA, indicating that the children are suffering from thiamine deficiency. This allows for timely nutritional intervention to prevent severe complications. Expected outcome: Improved health and nutritional status of the children through timely intervention.

[0142] Example 2: Monitoring Thiamine Levels in Chronic Alcoholics

[0143] A clinic monitors the health status of chronic alcoholics using the LFA1. Saliva samples from the patients are tested monthly. One patient's sample shows a significant reduction in slgA levels while amylase remains present, signaling the onset of thiamine deficiency. The clinic administers thiamine supplements, and subsequent tests show recovery in slgA levels. Expected outcome: Improved management of thiamine levels and overall health in chronic alcoholics.

[0144] Example 3: Field Use by NGOs

[0145] NGOs working in remote regions without access to advanced medical facilities use the LFA to screen for thiamine deficiency among the local population. The ease of sample collection and rapid results from the LFA enable the NGOs to identify individuals with thiamine

[0146] B8370WO; 07 / 07 / 2025 deficiency and provide appropriate nutritional support. Expected outcome: Enhanced ability of NGOs to address nutritional deficiencies in remote areas.

[0147] Example 4: Vitamin A Deficiency Detection

[0148] In a study to assess long-term vitamin deficiencies, individuals are periodically tested for slgA levels over three years. LSA1 or LSA2 can be used in the frame of this study.

[0149] Individuals showing a gradual decline in slgA levels, while amylase remains stable, are identified as potentially suffering from chronic Vitamin A deficiency. This early detection allows for dietary adjustments to prevent immune system impairments. Expected outcome: Prevention of chronic Vitamin A deficiency and related immune system impairments.

[0150] Example 5: Study in Incarceration Centers

[0151] A study is conducted involving detainees showing symptoms of thiamine deficiency at the MAC Abidjan incarceration center in collaboration with the Ministry of Justice and National Nutrition Program of Ivory Coast. Saliva and blood samples are collected and analyzed for thiamine levels, slgA, and amylase. This study can be conducted with LSA1.

[0152] Thiamine supplements are provided for six weeks, followed by a re-evaluation of thiamine and slgA levels to validate the correlation between thiamine deficiency and slgA levels. A control group without thiamine deficiency symptoms undergoes the same study to confirm the absence of slgA deficiency in this group. Expected outcome: Validation of the diagnostic device's effectiveness in detecting thiamine deficiency and monitoring supplementation outcomes.

[0153] Example 6: Use in Emergency Situations

[0154] In disaster-stricken areas where malnutrition is prevalent, the device can be used by emergency response teams to quickly assess the nutritional status of affected populations, allowing for rapid and targeted nutritional interventions. Expected outcome: Rapid identification and intervention for malnutrition in emergency situations. LSA1 and LSA2 can be used in such a case, with LSA2 providing more reliable readings.

[0155] Example 7: School Health Programs

[0156] B8370WO; 07 / 07 / 2025 Schools in regions with high rates of malnutrition can use the device as part of routine health check-ups to monitor the nutritional status of students, ensuring early detection and intervention. Expected outcome: Improved nutritional health and academic performance of students through regular monitoring and intervention.

[0157] References:

[0158] 1. Sutherland, D. B., Suzuki, K., & Fagarasan, S. (2016). "Fostering of advanced mutualism with gut microbiota by Immunoglobulin A." Immunological Reviews, 270(1), 20- 31. doi: 10.1111 / imr.12384.

[0159] 2. Porter, R. J., Arnold, L. L., & Berggren, G. G. (1966). "Thiamine deficiency in malnourished infants: A clinical and biochemical study." American Journal of Clinical Nutrition, 18(5), 317-329.

[0160] 3. Lonsdale, D. (2006). "A review of the biochemistry, metabolism and clinical benefits of thiamine and its derivatives." Evidence-Based Complementary and Alternative Medicine, 3(1), 49-59.

[0161] 4. Biesalski, H. K. (2004). "Vitamin A in health and disease: The role of retinoids and carotenoids in immunity." Nutrition, 20(7-8), 702-708.

[0162] 5. World Health Organization. (2001). "Thiamine deficiency and its prevention and control in major emergencies." Geneva: WHO.

[0163] 6. Tomkins, A. (2003). "Malnutrition, morbidity, and mortality in developing countries." Proceedings of the Nutrition Society, 62(1), 75-79.

[0164] 7. Mancinelli, R., Ceccanti, M., Guiducci, M. S., Sasso, G. F., Sebastiani, G., Attilia, M. L., & Rotondo, C. (2003). "Simultaneous liquid chromatographic assay of thiamine and its phosphate esters in human erythrocytes and plasma." Journal of Chromatography B, 789(2), 355-363.

[0165] 8. Kunisawa, J., Sugiura, Y., Wake, T., Nagatake, T., Suzuki, H., Nagasawa, R., Shikata,

[0166] S., Honda, K., Hashimoto, E., Suzuki, Y ., Setou, M., Suematsu, M., & Kiyono, H. (2015). "Mode of Bioenergetic Metabolism during B Cell Differentiation in the Intestine Determines the Distinct Requirement for Vitamin Bl." Cell Reports, 13(1), 122-131. doi: 10.1016 / j .celrep.2015.08.063.

[0167] 9. Humphrey, S. P., & Williamson, R. T. (2001). "A review of saliva: Normal composition, flow, and function." Journal of Prosthetic Dentistry, 85(2), 162-169.

[0168] B8370WO; 07 / 07 / 2025 10. Denny, P. C., Denny, P. A., & Tabak, L. A. (1997). "Saliva and the Salivary Glands." In The Biochemistry of Glycoproteins and Proteoglycans.

[0169] 11. Spielmann, N., & Wong, D. T. (2011). "Saliva: Diagnostics and Therapeutics." Wiley Interdisciplinary Reviews: Systems Biology and Medicine, 3(4), 429-445. 12. Vitorino, R., Lobo, M. J., Ferrer-Correia, A. J., Dubin, J. R., Tomer, K. B., & Amado,

[0170] F. M. (2004). "Identification and characterization of human whole saliva glycoproteins using a proteomic approach." Journal of Proteome Research, 3(5), 621-631.

[0171] B8370WO; 07 / 07 / 2025

Claims

Claims1. Lateral flow assay for detecting thiamine deficiency based on a sample of diluted saliva, the assay comprising a binding molecule that is capable of specifically binding to secretory Immunoglobulin A (slgA), wherein said assay is provided for producing a readable signal allowing determining if said sample comprises slgA, or if an amount of slgA in said sample is present at detectable and / or minimum levels, and wherein a readable signal indicating absence of slgA or the presence of slgA in said sample below said detectable and / or minimum levels indicates thiamine deficiency.

2. The assay of claim 1, which is provided for producing a readable signal allowing determining if said sample comprises amylase, or if an amount of amylase in said sample is present at required levels, for example at detectable and / or minimum levels.

3. The assay of claim 2, wherein a readable signal indicating absence of amylase or the presence of amylase in said sample below said detectable and / or minimum levels indicates that a sample dilution has not been correctly conducted and / or that said assay outcome is not valid.

4. The assay of any one of the preceding claims, wherein said sample of diluted saliva is saliva diluted with a dilution factor of approximately 1 : 10'000.

5. The assay of any one of the preceding claims, wherein said lateral flow assay comprises:- a sample application area or port adapted to receiving said sample,- one or more detection regions,- a transfer region, allowing said sample and / or analytes comprised in said sample to move from said sample application area or port to said one or more detection regions,- one or more tags and / or labels.

6. The lateral flow assay of any one of the preceding claims, wherein said assay comprises slgA or an slgA variant suitable to specifically bind to said binding molecule.B8370WO; 07 / 07 / 20257. The lateral flow assay of any one f the preceding claims, wherein:- said binding molecule suitable to specifically bind to slgA, and / or, if applicable,- said slgA or a slgA variant suitable to specifically bind to said binding molecule, is provided at said one or more detection regions, preferably immobilized at said one or more detection regions.

8. The lateral flow assay of any one of the preceding claims, wherein said slgA is a first analyte and wherein said binding molecule is a first binding molecule, and wherein said lateral flow test comprises a second binding molecule that is capable of specifically binding to at least a second analyte, wherein said second analyte is amylase.

9. The assay of any one of the preceding claims, wherein said readable signal is a first readable signal, and wherein said assay, preferably said one or more detection regions, is arranged for producing a second readable signal allowing determining if said sample comprises said second analyte or if an amount of said second analyte in said sample is present at required levels, for example at detectable and / or minimum levels.

10. The lateral flow assay of any one of the preceding claims, wherein said assay, preferably said one or more detection regions, is arranged for producing a readable signal serving as a control signal indicating that the assay is functional and / or that the assay has been used correctly such that the read out is expected to be correct.

11. The assay of claim 10, wherein said control signal is based on the binding of said second binding molecule to said second analyte, or on the binding of said second binding molecule to a molecule comprising said second analyte or a variant of said second analyte.

12. The assay of any one of the preceding claims, wherein a readable signal indicating the absence of slgA, or the presence of slgA in said sample below said detectable and / or minimum levels indicates Vitamin A deficiency, preferably chronic Vitamin A deficiency.

13. The assay of any one of the preceding claims, wherein:- said readable signal comprises the absence or presence of visible light,- said one or more tags and / or labels emit and / or reflect visible light, and / or- a read out, preferably at said one or more detection regions, is based on visible light,B8370WO; 07 / 07 / 2025such that the read out can be performed by visual inspection of the one or more detection regions.

14. A method of determining if a subject suffers from thiamine deficiency, the method comprising:- loading a sample of saliva of said subject on a lateral flow assay, wherein said assay is suitable to detect slgA in said sample, and,- determining that subject suffers from thiamine deficiency if one or more detection regions of the assay indicate the absence of slgA in said sample.

15. The method of claim 14, comprising, prior to loading the sample on the assay, diluting saliva taken from a subject to provide said sample of saliva, wherein a dilution factor is approximately 1 : 10'000.

16. The method of claim 14 or claim 15, which is further suitable to determine if said subject suffers from Vitamin A deficiency.

17. The method of claim 16, wherein said method comprises repeating the method of claims 15-17 several times and / or over a period of time, and wherein repeated finding of low levels of slgA is an indication that said subject suffers from Vitamin A deficiency.

18. The method of any one of claims 14-17, wherein said assay is suitable to detect amylase in said sample, the method comprising determining that amylase is absent and / or levels of amylase in said sample are different from the required levels, for example below detectable and / or minimum levels, indicates that a sample dilution has not been correctly conducted and / or that said assay outcome is not valid.B8370WO; 07 / 07 / 2025

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