Detection of intestinal barrier dysfunction and / or liver cirrhosis

Abstract

The present invention relates to methods, kits, and test strips for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject. Additionally, methods of treating a subject with intestinal barrier dysfunction and / or liver cirrhosis are provided.

Description

[Technical Field]

[0001] The field of this document is generally the detection of intestinal barrier dysfunction and / or liver cirrhosis in subject.More specifically, the method, kit and test strip for identifying and monitoring intestinal barrier dysfunction and / or liver cirrhosis in subject.In addition, the method for treating the subject with intestinal barrier dysfunction and / or liver cirrhosis is provided. [Background technology]

[0002] Bibliographic details of the subject specification references are also provided at the end of the specification.

[0003] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[0004] Intestinal barrier dysfunction, also known as "increased intestinal permeability" or "leaky gut," refers to a state of impaired intestinal barrier function in patients, allowing the penetration of harmful substances, including viable microorganisms or their by-products (e.g., toxic lipopolysaccharide (LPS) and antigens that are normally excluded from the circulation). This then leads to chronic immune activation (either systemic, as in HIV infection, or local, as in inflammatory bowel disease), which is recognized as a major driver of pathogenesis in many conditions and is suspected to play a role in many others. Pathological conditions associated with intestinal barrier dysfunction can be broadly classified into three types: 1) intestinal barrier dysfunction observed in postoperative patients undergoing major surgery for a variety of reasons; 2) critically ill patients admitted to the ICU due to severe injury, burns, or sepsis. Increased intestinal permeability is associated with a systemic inflammatory response and the development of multiple organ dysfunction syndrome (MODS) in these patients; and 3) patients with chronic pathological conditions in which intestinal barrier dysfunction leads to chronic immune activation, which is associated with disease progression and / or the development of complications and comorbidities from other organs. This category of leaky gut patients includes those with cirrhosis, HIV infection, chronic viral hepatitis B or C, nonalcoholic steatohepatitis or nonalcoholic fatty liver disease, irritable bowel syndrome, obesity, and various autoimmune conditions. It is also notable that several studies have focused on intestinal barrier dysfunction in relation to neurological conditions, although findings across these studies currently appear inconsistent.

[0005] While intestinal biopsy and excellent pathology can provide fairly reliable indications of intestinal barrier dysfunction, this is invasive and impractical. Currently, there is no "gold standard" noninvasive test for intestinal barrier dysfunction. Noninvasive biomarkers such as serum LPS, bacterial 16S RNA, and soluble CD14 are generally accepted as indicative of dysfunction on a population basis. However, these have no prognostic or diagnostic value for individual patients due to the high degree of patient-to-patient variability. For example, a "glucose tolerance" test, which measures urinary excretion of lactulose and mannitol over 6–12 hours after an oral dose containing a mixture of these non-metabolizable sugars, requires patients to collect this urine for subsequent mass spectrometry analysis, making it impractical for widespread use (samples are sent to a central laboratory for analysis). While home glucose tolerance tests are available for home use, their results have not been well accepted clinically.

[0006] The lack of reliable biomarkers suitable for widespread use is a major obstacle to: 1) better understanding the role of intestinal barrier dysfunction in many diseases (i.e., research applications), 2) clinical development of pharmacological, dietary, and other interventions to reduce intestinal barrier dysfunction (research applications in defined clinical settings), and 3) diagnosis of intestinal barrier dysfunction in conditions commonly recognized as problematic in the clinic (e.g., cirrhosis, post-surgery, burns, sepsis, irritable bowel disease).

[0007] Not only is there a lack of markers for detecting intestinal barrier dysfunction, but there is also a lack of markers for detecting liver cirrhosis. Early detection of cirrhosis (Child-Pugh A) can allow for therapeutic or behavioral intervention to prevent and / or delay progression to more severe cirrhosis (Child-Pugh B and Child-Pugh C), which may require transplantation. This is particularly important in subjects with nonalcoholic fatty liver disease (NAFLD). NAFLD affects approximately 25% of the world's population but is typically not diagnosed until advanced disease (NASH and grade B cirrhosis). NAFLD, along with other metabolic diseases related to obesity, is on the rise, and as treatments become available, population-based screening is in high demand within the next few years.

[0008] Detection of more severe levels of cirrhosis can allow for more rapid intervention with therapy and surgery.

[0009] Thus, there is a need for improved methods for identifying, screening, and monitoring intestinal barrier dysfunction, liver disease, and / or cirrhosis in subjects, including methods and kits for identifying individuals at any of the different stages of cirrhosis (Child-Pugh A, Child-Pugh B, and Child-Pugh C).

[0010] Those skilled in the art will appreciate that many devices can be programmed and automated to detect deviations from threshold values ​​as described herein. Summary of the Invention

[0011] (Disclosure Summary) The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as providing explicit support for both meanings or either meaning. As used herein, the term "about," unless otherwise specified, refers to + / - 10%, more preferably + / - 5%, and even more preferably + / - 1% of the specified value.

[0012] It will be understood that throughout this specification the term "comprise" or variations such as "comprises" or "comprising" imply the inclusion of a specified element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. As used herein, the singular forms "a," "an," and "the" include singular and plural references unless the context dictates otherwise. Each embodiment herein should apply mutatis mutandis to every other embodiment unless expressly stated otherwise.

[0013] In one aspect, the present invention provides a method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining dIgA and mIgA levels and a ratio thereof in a biological sample from the subject, and comparing the ratio to a threshold value.

[0014] In one aspect, the present invention provides a method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining dIgA1 or dIgA2 levels and mIgA1 or mIgA2 levels and their ratio in a biological sample from the subject, and comparing the ratio to a threshold value.

[0015] In certain embodiments, the present invention provides methods for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, comprising the methods described herein.

[0016] In one aspect, a difference in the level or ratio of dimeric and monomeric IgA forms described herein from the threshold value indicates intestinal barrier dysfunction and / or cirrhosis in the subject, or a change in the level or severity of intestinal barrier dysfunction and / or cirrhosis in the subject. In one embodiment, the subject is retested by the subject method, and the threshold value is determined from the subject's previous test results.

[0017] In one aspect, the present invention provides a method for detecting intestinal barrier dysfunction and / or cirrhosis in a subject, the method comprising determining the level of IgA2 in a biological sample from the subject, wherein an elevated level of IgA2 compared to a threshold value indicates intestinal barrier dysfunction and / or cirrhosis in the subject.

[0018] In one aspect, the present invention provides a method for treating intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining dIgA and mIgA levels and a ratio thereof in a biological sample from the subject, and comparing the ratio to a threshold value; and if the subject has a ratio different from the threshold value, administering a treatment for intestinal barrier dysfunction and / or liver cirrhosis to the subject.

[0019] In one embodiment, the present invention provides a method of treating intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining the level of IgA2 in a biological sample from the subject, and if the control has an elevated level of IgA2 compared to a threshold, administering to the subject a treatment for intestinal barrier dysfunction and / or liver cirrhosis.

[0020] In one embodiment, the present invention provides a kit for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, comprising: (i) an agent that binds to dIgA and forms a detectable dIgA complex; (ii) an agent that binds to mIgA and forms a detectable mIgA complex; (i) specifically binds to dIgA, and / or (ii) specifically binds to mIgA.

[0021] In one embodiment, the present invention provides a test strip for a lateral flow device comprising at least one sample loading area, a) the strip comprises a capture portion comprising an agent that binds to dIgA; b) the strip comprises a capture portion comprising an agent that binds to mIgA; a) is closer to the sample loading area than b) so that the sample contacts a) before b).

[0022] In one aspect, the present invention provides a method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining a dIgA to mIgA ratio or an mIgA to dIgA ratio in a biological sample from the subject, wherein an elevated dIgA to mIgA ratio compared to a threshold value or a decreased mIgA to dIgA ratio compared to a threshold value indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject.

[0023] In one aspect, the present invention provides a method of treating intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining a dIgA to mIgA ratio or an mIgA to dIgA ratio in a biological sample from the subject, and if the subject has an elevated dIgA to mIgA ratio compared to a threshold value, or if the subject has a decreased mIgA to dIgA ratio compared to a threshold value, administering a treatment for intestinal barrier dysfunction and / or liver cirrhosis to the subject.

[0024] In one embodiment, the present invention provides a method of treating intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining the level of IgA2 in a biological sample from the subject, and if the control has an elevated level of IgA2 compared to a threshold, administering to the subject a treatment for intestinal barrier dysfunction and / or liver cirrhosis.

[0025] In one embodiment, the present invention provides a kit for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising: (i) obtaining a biological sample containing an antibody; (ii) determining the level of dimeric and polymeric IgA (dIgA) in the sample; and (iii) determining the level of monomeric IgA (mIgA) in the sample; (iv) optionally determining the level of IgA2 in the sample; (v) determining the ratio of dIgA to mIgA in the samples from (ii) and (iii); An elevated dIgA to mIgA ratio compared to a control and / or elevated IgA2 levels compared to a control indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject. [Brief explanation of the drawings]

[0026] [Figure 1] 1A) Different IgA forms in blood and their percentages. B) An embodiment of the method of the present invention is shown. Protein L can be used to form complexes containing monomeric IgA. Mouse anti-human IgA2 can be used to form complexes containing IgA2. Recombinant chimeric secretory component can be used to form complexes containing dimeric IgA. Complexes can be visualized by adding anti-human IgA colloidal gold and measuring absorbance with an Axxin AX-2X reader (run time: 30 minutes or less). In some embodiments of the present invention, only dIgA and mIgA are evaluated. In some embodiments of the present invention, dIgA, IgA2, and mIgA are evaluated. In some embodiments of the present invention, dIgA complexes are formed before mIgA complexes. In some embodiments of the present invention, IgA2 complexes are formed before mIgA complexes. [Figure 2] 1 shows a diagrammatic representation, including dimensions, of an example of a lateral flow test used in Examples 2 and 3. [Figure 3] 1 shows a diagrammatic representation, including dimensions, of an example of a lateral flow test used in Examples 2 and 3. [Figure 4]The steps for one embodiment of the POC test for dIgA, IgA2, and mIgA are illustrated below: Plasma (5 μl) is added to well A of the cassette. Running buffer (1 drop) is added to well A and incubated for 10 minutes. Next, 4 drops of running buffer are added to well B. The test is run for 20-30 minutes and read on an Axxin AX-2X reader. [Figure 5-1] 5A) An example readout of the POC test in Figure 2 is shown. B) The results of running the test strip for 20 minutes (30 minutes total assay time) are shown. The test strip should show three visible lines representing (starting from the sample well): dimeric IgA, IgA2, and monomeric IgA. Absence of the third line (monomeric IgA) or all three lines indicates either assay failure or that the patient is IgA-deficient, making the test unusable in these rare individuals. The intensity of each test line is proportional to the amount of the respective analyte and is interpreted visually or, more preferably, using an automated reader such as the Axxin AX-2X reader (Axxin Ltd, Melbourne). The AX-2X is used to obtain a numerical readout for each test line. The workflow for using the Axxin AX-2X is shown in Figure 5A, and an example readout is provided in Figure 5B. [Figure 5-2] 5A) An example readout of the POC test in Figure 2 is shown. B) The results of running the test strip for 20 minutes (30 minutes total assay time) are shown. The test strip should show three visible lines representing (starting from the sample well): dimeric IgA, IgA2, and monomeric IgA. Absence of the third line (monomeric IgA) or all three lines indicates either assay failure or that the patient is IgA-deficient, making the test unusable in these rare individuals. The intensity of each test line is proportional to the amount of the respective analyte and is interpreted visually or, more preferably, using an automated reader such as the Axxin AX-2X reader (Axxin Ltd, Melbourne). The AX-2X is used to obtain a numerical readout for each test line. The workflow for using the Axxin AX-2X is shown in Figure 5A, and an example readout is provided in Figure 5B. [Figure 6] Representative sample strips for dIgA, IgA2, and mIgA POC tests are shown. dIgA, IgA2, and mIgA POC tests were performed on 121 samples from 16 healthy controls, 77 patients with hepatitis B virus infection, and 121 patients with cirrhosis. Cutoffs were established for IgA2 levels (healthy control mean + 2 standard deviations (≥ 3500)) and dIgA / mIgA ratios (healthy control mean + 1 SD (≥ 0.65)). [Figure 7] Evaluation of dIgA, IgA2, and mIgA in healthy individuals (n=16) compared with individuals with hepatitis B (n=77) or cirrhosis (n=121) is shown. Regarding individual IgA species, mIgA was elevated in chronic hepatitis B but not in cirrhotic patients, while IgA2 was elevated in both hepatitis B and cirrhotic patients (some patients showed highly elevated levels), but dIgA levels showed the greatest difference, being highly elevated in cirrhotic and some hepatitis B patients (only 16 / 77 HBV patients had cirrhosis). [Figure 8-1] Figure 1 shows the IgA2 / dIgA and dIgA / mIgA ratios in healthy individuals, individuals with hepatitis B, or individuals with cirrhosis. While the IgA2 / dIgA or IgA2 / mIgA ratios were not significant, the dIgA / mIgA ratio showed highly significant differences between cirrhosis and healthy controls, and in 10 / 16 hepatitis B patients with cirrhosis, with an overall sensitivity of 81.8% and a specificity of 88.3%. [Figure 8-2] Figure 1 shows the IgA2 / dIgA and dIgA / mIgA ratios in healthy individuals, individuals with hepatitis B, or individuals with cirrhosis. While the IgA2 / dIgA or IgA2 / mIgA ratios were not significant, the dIgA / mIgA ratio showed highly significant differences between cirrhosis and healthy controls, and in 10 / 16 hepatitis B patients with cirrhosis, with an overall sensitivity of 81.8% and a specificity of 88.3%. [Figure 9-1]Analysis of the dIgA / mIgA ratio by Child-Pugh cirrhosis classification is shown. Patients with cirrhosis (after excluding individual patients with multiple time points) were further examined according to the severity of their cirrhosis according to the Child-Pugh classification, with CP A being the least severe and exhibiting a good 1-year survival rate, CP B being more severe and exhibiting a moderate 1-year survival rate, and CP C being the most severe and exhibiting a poor 1-year survival rate. The dIgA / mIgA ratio was most sensitive in CP C and progressively less sensitive in CP B and CP A. However, including elevated levels of IgA2 progressively identified additional cirrhotic patients within each CP class, improving sensitivity overall from 83.7% to 90.8%. [Figure 9-2] Analysis of the dIgA / mIgA ratio by Child-Pugh cirrhosis classification is shown. Patients with cirrhosis (after excluding individual patients with multiple time points) were further examined according to the severity of their cirrhosis according to the Child-Pugh classification, with CP A being the least severe and exhibiting a good 1-year survival rate, CP B being more severe and exhibiting a moderate 1-year survival rate, and CP C being the most severe and exhibiting a poor 1-year survival rate. The dIgA / mIgA ratio was most sensitive in CP C and progressively less sensitive in CP B and CP A. However, including elevated levels of IgA2 progressively identified additional cirrhotic patients within each CP class, improving sensitivity overall from 83.7% to 90.8%. [Figure 9-3]Analysis of the dIgA / mIgA ratio by Child-Pugh cirrhosis classification is shown. Patients with cirrhosis (after excluding individual patients with multiple time points) were further examined according to the severity of their cirrhosis according to the Child-Pugh classification, with CP A being the least severe and exhibiting a good 1-year survival rate, CP B being more severe and exhibiting a moderate 1-year survival rate, and CP C being the most severe and exhibiting a poor 1-year survival rate. The dIgA / mIgA ratio was most sensitive in CP C and progressively less sensitive in CP B and CP A. However, including elevated levels of IgA2 progressively identified additional cirrhotic patients within each CP class, improving sensitivity overall from 83.7% to 90.8%. [Figure 10] This indicates that most patients with mild cirrhosis (CP A) who tested negative for the dIgA / mIgA ratio were those whose primary cause of cirrhosis was HBV infection. Detection rates were high for all other causes of cirrhosis (alcoholic, unknown cause, HCV, NASH, HIV, primary biliary cirrhosis, and primary sclerosing cholangitis), but the numbers were low for some causes, reflecting their low incidence in the region. The IgA test (dIgA / mIgA ratio) had good sensitivity for cirrhosis whose primary cause was alcoholic, unknown cause, HCV, NASH, primary biliary cirrhosis, and primary sclerosing cholangitis. [Figure 11-1] When the same cohort shown in Figures 7-9 was tested for alanine aminotransferase 1 (ALT-1) using the BioPoint ALT1 Rapid Test, the results show that only a small percentage (6.6%) of cirrhotic patients had evidence of liver disease when tested using ALT, the most commonly used biomarker of liver disease. Of the cirrhotic samples (8 / 121), 8 had ALT-1 levels above 40 U / L. dIgA / mIgA testing complements biochemical or immunochemical tests of liver function, such as the BioPoint ALT1 Test, in detecting more severe liver disease. [Figure 11-2]When the same cohort shown in Figures 7-9 was tested for alanine aminotransferase 1 (ALT-1) using the BioPoint ALT1 Rapid Test, the results show that only a small percentage (6.6%) of cirrhotic patients had evidence of liver disease when tested using ALT, the most commonly used biomarker of liver disease. Of the cirrhotic samples (8 / 121), 8 had ALT-1 levels above 40 U / L. dIgA / mIgA testing complements biochemical or immunochemical tests of liver function, such as the BioPoint ALT1 Test, in detecting more severe liver disease. [Figure 12-1] Separate analyses of A) dIgA1, B) dIgA2, and C) their ratios are shown in healthy subjects (n=17) versus cirrhotic subjects (n=121). D) A schematic representation of the assay for the detection of dIgA1 and dIgA2 is provided. Briefly, dIgA1 and dIgA2 bind to chimeric secretory components (CSCs) treated with 5 μL of mouse anti-human IgA1 or mouse anti-human IgA2 bound to a solid support and detected by the addition of anti-mouse IgG gold. The graph shows that levels of both dIgA subclasses (dIgA1 and dIgA2) can be measured, and both subclasses are elevated in cirrhotic livers. The dIgA2 / dIgA1 ratios for healthy and cirrhotic samples were all below 1. [Figure 12-2] Separate analyses of A) dIgA1, B) dIgA2, and C) their ratios are shown in healthy subjects (n=17) versus cirrhotic subjects (n=121). D) A schematic representation of the assay for the detection of dIgA1 and dIgA2 is provided. Briefly, dIgA1 and dIgA2 bind to chimeric secretory components (CSCs) treated with 5 μL of mouse anti-human IgA1 or mouse anti-human IgA2 bound to a solid support and detected by the addition of anti-mouse IgG gold. The graph shows that levels of both dIgA subclasses (dIgA1 and dIgA2) can be measured, and both subclasses are elevated in cirrhotic livers. The dIgA2 / dIgA1 ratios for healthy and cirrhotic samples were all below 1. [Figure 13]FIG. 1B provides a schematic representation of two capture methods for detecting mIgA in biological samples, including the detection of dIgA, IgA2, and mIgA. A) For detection using Protein L, Protein L is bound to a solid support, and mIgA in plasma binds to Protein L and is visualized by adding anti-human IgA gold and measuring absorbance on an Axxin AX-2X reader. B) For detection using anti-human IgA (an antibody that detects total IgA), anti-human IgA is bound to a solid support, and mIgA in plasma is bound by anti-human IgA, and the complex is visualized by adding anti-human IgA gold and measuring absorbance on an Axxin AX-2X reader. [Figure 14] Figure 1B provides a comparison of Protein L versus anti-IgA capture of mIgA strains in methods involving detection of dIgA, IgA2, and mIgA. A) In healthy controls (n=8), we show that using Protein L or anti-IgA to capture mIgA yields similar results for the majority of patients (6 / 8). B) In HIV patients (n=8), we show that much higher amounts of mIgA are detected using anti-IgA capture, likely due to competition for Protein L binding by the increased overall immunoglobulin concentrations commonly seen during HIV infection due to systemic immune activation. [Figure 15] A comparison of Protein L versus anti-IgA, which captures mIgA strains, in a method involving the detection of dIgA, IgA2, and mIgA in subjects with cirrhosis is provided in Figure 1B. The results show that for patients with cirrhosis, 17 / 24 had similar values ​​for mIgA using either Protein L or anti-IgA, suggesting that anti-IgA can be used to detect mIgA in the assay format described in Figure 1B. [Figure 16-1]The use of anti-IgA rather than Protein L in the dIgA / mIgA ratio test was shown to provide higher assay specificity in selected healthy controls and HIV patients, with no healthy or HIV patients having ratios above the cutoff. In this experiment, the cutoff used for patients and controls was 1. However, sensitivity was reduced in selected cirrhotic patients, with 15 / 24 being positive (ratio > 1.0) when anti-IgA was used to capture mIgA, compared with 19 / 24 being positive when Protein L was used to capture mIgA. [Figure 16-2] The use of anti-IgA rather than Protein L in the dIgA / mIgA ratio test was shown to provide higher assay specificity in selected healthy controls and HIV patients, with no healthy or HIV patients having ratios above the cutoff. In this experiment, the cutoff used for patients and controls was 1. However, sensitivity was reduced in selected cirrhotic patients, with 15 / 24 being positive (ratio > 1.0) when anti-IgA was used to capture mIgA, compared with 19 / 24 being positive when Protein L was used to capture mIgA. [Figure 17-1] Using a threshold of ≥0.5 for assessing cirrhosis in a cohort of hepatitis B patients demonstrates increased sensitivity. The left panel shows that assessing hepatitis B controls using a threshold of ≥0.65 (based on the mean + SD of healthy controls) achieves 62.5% sensitivity and 88.5% specificity. Lowering the threshold to ≥0.50 increases sensitivity to 75.0%, while specificity remains at 73.8% (samples between 0.5 and 0.65 are highlighted in blue). With regard to assessing cirrhosis in a hepatitis B patient population, sensitivity is more important than specificity, as the prevalence of cirrhosis is 20.8% in this hepatitis B cohort and is much higher in many populations, such as those in Africa and Asia. Therefore, the test can be tailored for use in populations where the infection status of conditions such as hepatitis B or HIV is known. [Figure 17-2]Using a threshold of ≥0.5 for assessing cirrhosis in a cohort of hepatitis B patients demonstrates increased sensitivity. The left panel shows that assessing hepatitis B controls using a threshold of ≥0.65 (based on the mean + SD of healthy controls) achieves 62.5% sensitivity and 88.5% specificity. Lowering the threshold to ≥0.50 increases sensitivity to 75.0%, while specificity remains at 73.8% (samples between 0.5 and 0.65 are highlighted in blue). With regard to assessing cirrhosis in a hepatitis B patient population, sensitivity is more important than specificity, as the prevalence of cirrhosis is 20.8% in this hepatitis B cohort and is much higher in many populations, such as those in Africa and Asia. Therefore, the test can be tailored for use in populations where the infection status of conditions such as hepatitis B or HIV is known. DETAILED DESCRIPTION OF THE INVENTION

[0027] [Consideration of the embodiment] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Any materials and methods similar or equivalent to those described herein can be used to carry out or test this disclosure.Practitioners are particularly directed to Ausubel et al., Current Protocols in Molecular Biology, Supplement 47, John Wiley & Sons, New York, 1999; Colowick and Kaplan, eds., Methods in Enzymology, Academic Press, Inc.; Weir and Blackwell, eds., Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications, 1986; definitions and terms in the art and other methods known to those skilled in the art.

[0028] The detection of specific antibody (immunoglobulin (Ig)) classes is recognized as a critical step in the diagnosis and research of human and animal diseases. For example, the detection of antigen-specific IgM class antibodies is widely used as a diagnostic test for infections by viruses such as hepatitis A virus, hepatitis E virus, West Nile virus, dengue virus, measles virus, rubella virus, and bacterial infections such as syphilis (Treponema pallidum), because IgM class antibodies are typically produced in infected hosts during the acute phase of infection and remain detectable for only a few months.

[0029] Conversely, IgG class antibodies typically persist lifelong and can indicate either current or past infection with a particular pathogen. For chronic infections such as human immunodeficiency virus (HIV), where patients do not spontaneously clear the virus, detection of IgG class antibodies is diagnostic for infection, but for others, such as hepatitis C virus (HCV), where some patients clear the virus either spontaneously or after treatment, detection of antigen-specific IgG is not diagnostic for current or ongoing infection. IgG class antibodies are also primarily involved in antibody-mediated immunity within the body's plasma compartment.

[0030] IgA class antibodies are used to aid in the diagnosis of infectious diseases, including hepatitis E virus, hepatitis A virus, and dengue virus, as well as in the study of vaccines and immunity to infectious diseases. IgA is attractive for diagnostic purposes because it is primarily produced during the acute phase of infection, and high levels of antigen-specific IgA, with or without co-detection of IgM, can provide a marker of current infection. In addition, because IgA is the predominant antibody class secreted at mucosal epithelial surfaces, its presence is considered a marker of mucosal immunity. The role of different IgA structural forms, specifically dIgA, as biomarkers of infection is not fully understood.

[0031] As used herein, the terms "IgA" or "total IgA" collectively refer to both the IgA subclass (IgA1 or IgA2) and the subtype "m," "d," or "s" (overall, there are six subtypes contained within the two subclasses: dIgA, mIgA, sIgA, dIgA2, mIgA2, and sIgA2).

[0032] As used herein, the term "dIgA" refers to a dimeric or higher polymeric form of IgA bound together by the IgA J chain. A representative diagram of the structure is shown in Figure 1A. As used herein, the term "mIgA" refers to monomeric IgA.

[0033] As used herein, the term "sIgA" refers to secretory IgA (a dimeric or higher polymeric form of IgA that binds to the polymeric Ig receptor (pIgR) and is subsequently released from the cell after cleavage of the pIgR into secretory components). The interaction with pIgR is dependent on the presence of the J chain. A representative diagram of the interaction is shown in Figure 1A.

[0034] As used herein, the term "IgA2" refers to IgA isotype 2. In some embodiments, the term refers to monomeric IgA2 (mIgA2). In some embodiments, dimers and / or higher polymeric forms (dIgA2) may also be present. In one embodiment, 80% or less dIgA2, or 70% or less dIgA2, or 60% or less dIgA2, or 50% or less dIgA2, or 40% or less dIgA2, or 30% or less dIgA2, or 20% or less dIgA2 is present in the sample after pre-reaction with a binding agent that detects dIgA, such as CSC.

[0035] IgA antibodies (total IgA antibodies) can be used to detect all forms of IgA. In the methods and kits described herein, after other fractions of the IgA pool have been removed, complexed, or detected by other means (e.g., by another antibody targeting a specific fraction of the IgA pool, such as an antibody targeting IgA2), IgA antibodies can be used to detect the remaining fraction of IgA. For example, after dIgA and IgA2 are complexed with antibodies or other molecules, such as chimeric secretory components (CSCs) specific for dIgA and IgA2, respectively, IgA antibodies can be used to detect mIgA.

[0036] As used herein, "normal levels of alanine aminotransferase 1 (ALT-1) or alanine aminotransferase" refers to a subject who has a negative result when tested with an ALT1 POC test (see Chinese Patent Application No. CN2016 / 10878590.7 "Point of care assays").

[0037] Reference to a "subject" includes humans and a wide range of mammals, higher primates, or other animals, including wild and domestic animals, pets, pests, and potential vectors of emerging infectious diseases. In one embodiment, the subject is a mammal. In one embodiment, the mammal is a human. In one embodiment, the subject is a higher primate. In one embodiment, the higher primate is a human, monkey, or ape. In relation to a subject, the subject may be suspected of or diagnosed with a disease, condition, infection, or exposure to a factor associated with one or more of liver disease, intestinal barrier dysfunction, and cirrhosis. In one embodiment, the subject may have been diagnosed with a disease or condition that may result in intestinal barrier dysfunction and / or cirrhosis. In one embodiment, the cirrhosis is in a subject diagnosed with or suspected of having (non-alcoholic fatty liver disease) NAFLD. In one embodiment, the subject is diagnosed with or suspected of having (non-alcoholic steatohepatitis) NASH. In one embodiment, the subject is diagnosed with or suspected of having viral hepatitis. In one embodiment, the subject is diagnosed with or suspected of having hepatitis B. In one embodiment, the subject is diagnosed with or suspected of having hepatitis C. In one embodiment, the subject is diagnosed with hepatitis D (in addition to being diagnosed with hepatitis B). In one embodiment, the subject is diagnosed with or suspected of having HIV.

[0038] The term "intestinal barrier dysfunction," also referred to as "leaky gut," "increased intestinal permeability," or "leaky gut syndrome," refers to a digestive condition in which the permeability of the intestinal barrier increases, causing components from within the digestive tract (e.g., bacteria, toxins (e.g., LPS), proteins, and amino acids) to leak through the intestinal wall and into the circulatory system. Some of the components that leak into the bloodstream may ultimately accumulate in the liver and contribute to cirrhosis. There are several causative / contributing factors for intestinal barrier dysfunction, including, but not limited to, diet, microbial imbalance in the digestive tract, antibiotic exposure, bacterial infection, viral infection (e.g., HIV and hepatitis), cirrhosis, inflammatory bowel disease, Crohn's disease, cardiovascular disease, pulmonary disease, and autoimmune disease. In one embodiment, intestinal barrier dysfunction is associated with cirrhosis. In one embodiment, intestinal barrier dysfunction is associated with HIV infection. In one embodiment, intestinal barrier dysfunction is associated with hepatitis.

[0039] As used herein, the term "cirrhosis," also known as "liver cirrhosis" or "hepatic cirrhosis," refers to a chronic disease of the liver characterized by cell degeneration, inflammation, and fibrous thickening of tissue. Cirrhosis is characterized by irreversible scarring of the liver. Subjects with cirrhosis can be treated to prevent / delay further development of scar tissue in the liver or treated with liver transplantation. Cirrhosis can be caused by one or more of alcohol, NAFLD, NASH, viral hepatitis, HIV, unknown cause, primary biliary cirrhosis, and primary sclerosing cholangitis. In one embodiment, the cirrhosis is caused by alcoholism. In one embodiment, the cirrhosis is in a subject diagnosed with NAFLD. In one embodiment, the subject is diagnosed with NASH. In one embodiment, the subject is diagnosed with hepatitis. In one embodiment, the subject is diagnosed with hepatitis B. In one embodiment, the subject is diagnosed with hepatitis C. In one embodiment, the subject is diagnosed with hepatitis D (in addition to being diagnosed with hepatitis B). In one embodiment, the subject is diagnosed with HIV. Clinically, diagnosing a subject with cirrhosis is an indication for priority treatment (e.g., in a subject with hepatitis B).

[0040] A "biological sample" suitable for the methods and kits described herein includes any sample containing or suspected of containing an antibody to be detected, including, but not limited to, a biological fluid (e.g., whole blood or a fraction thereof, plasma, serum, or gingival crevicular fluid). In one embodiment, the sample is whole blood. In one embodiment, the sample is any sample containing IgA antibodies. In one embodiment, the sample is a whole blood fraction, e.g., whole blood depleted of IgM and / or IgG. In one embodiment, the sample is purified or partially purified. In one embodiment, the sample is plasma. In one embodiment, the sample is serum. In one embodiment, gingival crevicular fluid. In one embodiment, the biological sample is collected from the subject at two additional time points. In one embodiment, the sample is obtained in proximity to a point-of-care device. In one embodiment, the sample is previously obtained from the subject. In one embodiment, the sample is stored at about 4°C, about 15°C, or about 24°C for a period of time before use. In one embodiment, the sample is dried, lyophilized, or flash-frozen.

[0041] Assay Type In one aspect, the present invention provides a method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining the dIgA level and mIgA level and the ratio thereof in a biological sample from the subject, and comparing the ratio with a threshold value. In one embodiment, the method comprises detecting intestinal barrier dysfunction in the subject. In one embodiment, the method comprises detecting liver cirrhosis in the subject.

[0042] The methods are suitable for in vitro use and can be performed in assay formats known to those skilled in the art, including immunoassays, chromatographic assays, or homogeneous assays.

[0043] As used herein, "immunoassay" refers to an assay that uses immunoglobulins or their parts to detect and quantify desired biomarkers, such as IgA and its subtypes and subclasses. The immunoassay can be one of a variety of immunoassay formats known to those skilled in the art. A wide range of immunoassay techniques are available, such as those described in Wild D. "The Immunoassay Handbook" Nature Publishing Group, 4th Edition, 2013, and subsequent inventions.

[0044] Electrochemiluminescence (ELICA), enzyme-linked immunosorbent assay (ELISA), fluorescent immunosorbent assay (FIA), and Luminex LabMAP immunoassay are examples of suitable assays for detecting biomarker levels. In one example, a binding agent (e.g., an antibody or protein L) is bound to a support surface, and an additional binding reagent / antibody containing a detectable group is bound to the antibody or a substrate to which the antibody binds. Examples of detectable groups include, but are not limited to, fluorescent dyes, enzymes, epitopes for binding to a second binding reagent (e.g., when the second binding reagent / antibody is a mouse antibody detected by a fluorescently labeled anti-mouse antibody), such as an antigen or a member of a binding pair, such as biotin. The surface may be a planar surface, such as a typical grid-type array (for example, but not limited to, a 96-well plate and a planar microarray), or may be a non-planar surface, such as coated bead array technology, in which each "species" of bead is labeled with, for example, a fluorescent dye (such as the Luminex technology described in U.S. Patent Nos. 6,599,331, 6,592,822, and 6,268,222), or quantum dot technology (for example, as described in U.S. Patent No. 6,306,610). Such assays may also be considered laboratory information management systems (LIMS).

[0045] Lateral flow assays, and more recently non-lateral flow and microfluidic technologies, provide useful setups for biological assays. Such assays can be qualitative, quantitative, or semi-quantitative. In microfluidic devices, small volumes of liquid move through microchannels generated, for example, in chips or cartridges. A wide range of detection reagents are available, including metal nanoparticles, colored, or luminescent materials. Resonance-enhanced adsorption (REA) of bioconjugated metal nanoparticles offers rapid processing times and other advantages. These devices have been combined with barcode technology to identify the patient and analyte being tested. Computer software and hardware for evaluating input data are encompassed by this disclosure. Point-of-care devices and arrays, as well as high-throughput screening methods, are also contemplated.

[0046] Qualitative assays that provide intermediate or definitive diagnoses require an integration threshold, gate, or window that allows for scoring of samples as likely to have or not have the condition. Instrument readers and software are often used to collate the data and process it through a diagnostic algorithm or decision tree.

[0047] The Luminex LabMAP system can be used for bead-based immunoassays. The LabMAP system incorporates polystyrene microspheres internally stained with two spectrally distinct fluorescent dyes. Using precise ratios of these fluorescent dyes, arrays consisting of different sets of microspheres with specific spectral addresses are created. Each set of microspheres can carry a different reactant on its surface. Because the sets of microspheres can be distinguished by their spectral addresses, they can be combined, allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel. A third fluorescent dye attached to a reporter molecule quantifies biomolecular interactions occurring on the microsphere surface. Microspheres are individually interrogated in a rapidly flowing fluid stream as they pass through two separate lasers in the Luminex analyzer. High-speed digital signal processing classifies the microspheres based on their spectral addresses and quantifies the surface reactions in a matter of seconds per sample.

[0048] In one embodiment, the assay is a homogeneous assay, i.e., the assay format allows assay measurements to be performed by a simple mix-and-read procedure without the need to process the sample by separation or washing. Such an assay does not include an immunosorbent solid phase step. In one embodiment, the homogeneous assay is time-resolved Forster resonance energy transfer (FRET).

[0049] In one embodiment, the assay is a flow cytometry-, bead array-, lateral flow-, cartridge-, microfluidic-, or immunochromatography-based method, etc. In one embodiment, the assay is a point-of-care assay. In one embodiment, the point-of-care assay reader is an Axxin AX-2X model reader, or an equivalent or modified device. For example, the device can be modified to include LEDs and filters of wavelengths appropriate for the assay of interest.

[0050] Agents to determine dIgA, mIgA, or IgA2 levels The agent for determining the level of IgA or its subclass or subtype in a sample can be any binding agent known to those skilled in the art that binds to IgA or its subclass or subtype and forms a detectable complex.The binding agent can conveniently be an antibody or its antigen-binding fragment.Other suitable binding agents are known in the art, and include antigen-binding constructs such as binding proteins, binding peptides, affimers, affibodies, aptamers, nanobodies, and mimetics.

[0051] Methods for producing antigen-specific binding agents, including antibodies and their derivatives, analogs, and mimetics, are well known in the art. Polyclonal antibodies can be generated by immunization of animals. Monoclonal antibodies can be prepared according to standard (hybridoma) methodologies. Antibody derivatives and analogs, including humanized antibodies, can be prepared recombinantly by isolating DNA fragments from DNA encoding a monoclonal antibody and subcloning the appropriate V regions into an appropriate expression vector according to standard methods. Phage display and aptamer technologies have been described in the literature and allow for the in vitro clonal amplification of antigen-specific binding reagents with very low affinity cross-reactivity. Phage display reagents and systems are commercially available, including the Recombinant Phage Antibody System (RPAS) available from Amersham Pharmacia Biotech, Inc. of Piscataway, New Jersey, and the pSKAN phagemid display system available from MoBiTec, LLC of Marco Island, Florida. Aptamer technology is described, for example, but not limited to, U.S. Patent Nos. 5,270,163, 5,475,096, 5,840,867, and 6,544,776. Those skilled in the art can select binding agents for use in the methods described herein.

[0052] In one embodiment, the binding agent is an antibody, or antigen-binding fragment or derivative thereof, an antigen-binding construct such as an affimer, affibody, aptamer, nanobody, and mimetic, or a ligand or binding portion thereof. In one embodiment, the binding agent is immobilized on a support, such as a lateral flow test strip.

[0053] Suitable binding agents for determining dIgA levels include any binding agent that specifically or nonspecifically binds to dIgA and forms a detectable dIgA complex. In one embodiment, the binding agent is nonspecific and binds to other forms of IgA that can be removed from the sample by binding with another binding agent prior to use with the dIgA binding agent. In one embodiment, the binding agent is specific for dIgA. In one embodiment, the binding agent is a dIgA antibody. In one embodiment, the binding agent is an anti-dIgA1 antibody, an anti-dIgA2 antibody, or a combination thereof. In one embodiment, the binding agent is an antibody that binds to the J chain of dIgA (e.g., anti-IgA J chain, LifeSpan Biosciences catalog number LS-B12942 or OriGene Technologies catalog number AM20272PU-M). In one embodiment, the binding agent is pIgR. As used herein, the term "pIgR" refers to the polymeric Ig receptor, including recombinant and modified forms thereof. In some embodiments, the pIgR is the pIgR described in Applicant's earlier application WO / 2014 / 071456. In some embodiments, the pIgR is produced in glycan-deficient cells, such as glycan-deficient CHO cells, to enhance preferential binding to dIgA over IgM. In one embodiment, the pIgR is modified to bind to dIgA but not substantially bind IgM due to removal of human domain 1. In one embodiment, human domain 1 is replaced with rabbit domain 1. In some embodiments, the recombinant pIgR is derived from a primate, such as a human pIgR, and comprises at least one immunoglobulin-like domain derived from a non-primate, such as rabbit, mouse, or rat. In some embodiments, the recombinant pIgR comprises the amino acid sequence set forth in SEQ ID NO:2, or SEQ ID NO:4, or SEQ ID NO:6, or SEQ ID NO:12, or SEQ ID NO:14, or SEQ ID NO:16, as described in WO / 2014 / 071456, or a dIgA-binding portion thereof, or a dIgA-binding variant thereof. In one embodiment, the pIgR is a chimeric secretory component.The chimeric secretory component comprises rabbit domain 1 and human domains 2-5 as set forth in SEQ ID NO: 5 or SEQ ID NO: 6 of WO / 2014 / 071456. In one embodiment, the pIgR is conjugated to a detectable tag.

[0054] Suitable binding agents for determining mIgA levels include any binding agent that binds to mIgA and forms a detectable mIgA complex. In one embodiment, the binding agent is specific for mIgA. In one embodiment, the binding agent is an anti-mIgA antibody. In one embodiment, the binding agent is an anti-IgA antibody (e.g., mouse anti-human IgA Sigma catalog number: I0636). In one embodiment, the binding agent is Protein L. The term "Protein L" refers to the original immunoglobulin (Ig) binding protein derived from the bacterium Peptostreptococcus magnus. Protein L can bind to the kappa light chain of an antibody without interfering with the antibody's antigen-binding site. It can bind to all classes of Ig (IgG, IgM, IgA, IgE, and IgD). The term is intended to encompass modified and recombinant versions of Protein L. Protein L can be obtained, for example, from ThermoFisher Scientific (Protein L, catalog number 21189).

[0055] Suitable binding agents for determining mIgA1 levels include any binding agent that binds to mIgA and forms a detectable mIgA complex. In one embodiment, the binding agent is specific for mIgA1. In one embodiment, the binding agent is an anti-mIgA1 antibody. For example, an antibody that binds to the same region of the IgA molecule occupied by the J chain in dIgA would be expected to be more specific for mIgA than for dIgA.

[0056] Suitable binding agents for determining IgA2 levels include any binding agent that binds to IgA2 and forms a detectable IgA complex. In one embodiment, the binding agent is specific for IgA2. In one embodiment, the binding agent is specific for mIgA2. In one embodiment, the binding agent is an anti-human IgA2 monoclonal antibody (e.g., Nordic MUbio, catalog number MAHu / IgA2).

[0057] In one embodiment, the antibodies described herein may be conjugated to a detectable tag.

[0058] As used herein, the terms "binds" or "binding" refer to the interaction between a binding agent (e.g., a protein or its antigen-binding domain) and an antigen, meaning that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, antibodies recognize and bind to specific protein structures, rather than proteins in general. If an antibody binds to epitope "A," the presence of a molecule containing epitope "A" (or free, unlabeled "A") in a reaction containing labeled "A" and the antibody will reduce the amount of labeled "A" that binds to the antibody.

[0059] As used herein, the terms "specifically bind" or "specific for X" should be interpreted to mean that a binding agent of the present disclosure reacts or associates with a particular antigen(s) or cells expressing the antigen more frequently, more rapidly, for longer duration, and / or with higher affinity than it reacts or associates with alternative antigens or cells. For example, a protein that specifically binds to an antigen binds the antigen with higher affinity (e.g., 20-fold, or 40-fold, or 60-fold, or 80-fold to 100-fold, or 150-fold, or 200-fold higher affinity), avidity, more readily, and / or longer duration than it binds to other antigens, e.g., other subclasses of IgA, or antigens commonly recognized by polyreactive natural antibodies (i.e., naturally occurring antibodies known to bind to a variety of antigens naturally found in humans). By reading this definition, it is also understood that, for example, a protein that specifically binds to a first antigen may or may not specifically bind to a second antigen.

[0060] In one embodiment, the method comprises forming a dIgA complex before the mIgA complex, hi one embodiment, the IgA2 complex is formed before the mIgA complex.

[0061] In one embodiment, the method comprises contacting the sample with a dIgA binding agent to form a detectable dIgA complex, followed by contacting the sample with an mIgA binding agent to form a detectable mIgA complex, hi one embodiment, the method comprises contacting the sample with a dIgA binding agent to form a detectable dIgA complex, followed by contacting the sample with an IgA2 binding agent to form a detectable IgA2 complex, followed by contacting the sample with an mIgA binding agent to form a detectable mIgA complex.

[0062] In one embodiment, the method comprises contacting the sample with pIgR or an anti-IgA J chain antibody to form a detectable dIgA complex, followed by contacting the sample with Protein L, anti-IgA, or anti-mIgA antibody to form a detectable mIgA complex. In one embodiment, the method comprises contacting the sample with pIgR or an anti-IgA J chain antibody to form a detectable dIgA complex, followed by contacting the sample with an anti-IgA2 antibody to form a detectable IgA2 complex, followed by contacting the sample with Protein L, anti-IgA, or anti-mIgA antibody to form a detectable mIgA complex.

[0063] In one embodiment, the method comprises contacting the sample with a dIgA-binding agent to form a detectable dIgA complex, followed by contacting the sample with an mIgA1-binding agent to form a detectable mIgA1 complex. In one embodiment, the method comprises contacting the sample with a dIgA-binding agent to form a detectable dIgA complex, followed by contacting the sample with an IgA2-binding agent to form a detectable IgA2 complex, followed by contacting the sample with an mIgA1-binding agent to form a detectable mIgA complex.

[0064] In one embodiment, the detectable complexes referred to herein are directly detectable or indirectly detectable.

[0065] In one embodiment, the detectable complex described herein can be directly detected. In one embodiment, the binding agent is conjugated to a detectable marker or microparticle containing a detectable marker that provides a detectable signal. In one embodiment, the detectable marker is selected from one or more of colloidal gold, magnetic agents, colored latex, carboxycellulose, carbon nanoparticles, and fluorescent labels. In one embodiment, the detectable marker is a visually detectable marker.

[0066] In one embodiment, the detectable complex is indirectly detectable (can be detected by the addition of an additional reagent that binds to one or more of: i) the binding agent, ii) the IgA subclass or subtype bound by the binding agent, iii) the complex, and iv) combinations thereof).

[0067] In one embodiment, the reagent for detecting the complex is a reagent for detecting IgA or a subclass or subtype thereof present in the complex. In one embodiment, the reagent is an antibody for detecting IgA or a subclass or subtype thereof prepared / derived from a species different from that of the subject from which the biological sample was obtained. For example, if the subject is a human, the reagent detects human IgA or a subclass or subtype thereof (e.g., an anti-human IgA antibody prepared / derived from another species, e.g., mouse, goat, rabbit, donkey, etc.). For example, if the subject is a monkey, the reagent detects monkey IgA or a subclass or subtype thereof (e.g., an anti-monkey IgA antibody prepared / derived from another species, e.g., mouse, goat, rabbit, donkey, human, etc.).

[0068] In one embodiment, the subject is a human and the reagent is selected from one or more of anti-human IgA colloidal gold, anti-human IgA1 colloidal gold, and anti-human IgA2 colloidal gold. In one embodiment, the subject is a human and the reagent is anti-human IgA colloidal gold. In one embodiment, the subject is a human and the reagent is anti-human IgA1 colloidal gold. In one embodiment, the subject is a human and the reagent is anti-human IgA2 colloidal gold.

[0069] In one embodiment, the subject is a monkey and the reagent is selected from one or more of anti-monkey IgA colloidal gold, anti-monkey IgA1 colloidal gold, and anti-monkey IgA2 colloidal gold. In one embodiment, the subject is a monkey and the reagent is anti-monkey IgA colloidal gold. In one embodiment, the subject is a monkey and the reagent is anti-monkey IgA1 colloidal gold. In one embodiment, the subject is a monkey and the reagent is anti-monkey IgA2 colloidal gold.

[0070] In one embodiment, the detectable complex comprises one or more of colloidal gold, a magnetic agent, colored latex, carboxycellulose, carbon nanoparticles, and a fluorescent label.

[0071] Threshold Based on the disclosure of this specification, those skilled in the art will understand that one or more levels of dIgA level, mIgA level, mIgA1 level, IgA2 level, the ratio of dIgA level to mIgA level, the ratio of mIgA level to dIgA level, the ratio of dIgA level to mIgA1 level, and the ratio of mIgA1 level to dIgA level can be compared with a threshold value to determine whether a subject has intestinal barrier dysfunction and / or liver cirrhosis.In some embodiments, comparison with a threshold value can be used to determine whether a subject has intestinal barrier dysfunction.In some embodiments, comparison with a threshold value can be used to determine whether a subject has liver cirrhosis.

[0072] The term "threshold" refers to a value, range, or cutoff that must be met, exceeded, or not exceeded to determine whether a subject has intestinal barrier dysfunction and / or cirrhosis.

[0073] In one embodiment, the present application illustrates or describes the invention using a "disease" threshold, i.e., a threshold level that meets or exceeds the threshold at which a subject is assessed as having intestinal barrier dysfunction and / or cirrhosis. As will be understood by those skilled in the art, the levels and / or ratios described herein in healthy or diseased subjects are useful for assessing the absence or level of intestinal barrier dysfunction and / or cirrhosis in such subjects.

[0074] The threshold may be set relative to a control or standard processed simultaneously with the standard, or the threshold may be predetermined based on a data set generated using the particular reagents and platform for a given embodiment of the test.

[0075] In one embodiment, the threshold is a color intensity that can be visually assessed. In one embodiment, the color intensity can be expressed as a number or a range of numbers.

[0076] In one embodiment, the threshold is set relative to a control, which includes any sample or group of samples that can be used to establish a knowledge base of data from a subject or subjects with a known disease state.

[0077] In one embodiment, the levels of one or more of dIgA, mIgA, mIgA1, IgA2, the ratio of dIgA levels to mIgA levels, the ratio of mIgA levels to dIgA levels, the ratio of dIgA levels to mIgA1 levels, and the ratio of mIgA1 levels to dIgA levels can be compared to threshold levels in one or more populations / control subject groups selected from a cohort of normal subjects in which it has been predetermined that the subjects do not have one or more of intestinal barrier dysfunction, liver disease, cirrhosis, NAFLD, HASH, hepatitis B, and hepatitis C, and a population recently infected with or newly diagnosed with a disease or condition that causes intestinal barrier dysfunction, liver disease, NAFLD, HASH, hepatitis B, and hepatitis C.

[0078] The threshold value can be selected to provide acceptable ability to predict intestinal barrier dysfunction and / or liver cirrhosis in subjects.In an exemplary embodiment, receiver operating characteristic (ROC) curve is calculated by plotting the value of one or more variables versus their relative frequency in two groups (arbitrarily referred to as "disease" and "normal").For any of the markers listed above, the distribution of the subject levels in the two groups is likely to overlap.Under such conditions, test level does not completely distinguish between "disease" and "normal" with 100% accuracy, and the overlapping area indicates that the test cannot distinguish between groups.Therefore, in some embodiments, a threshold value or range is selected above (or below) that the test is considered "positive", and below that that the test is considered "negative".

[0079] In one embodiment, the threshold is set as the mean of the control group, the mean of the control group plus one standard deviation, the mean of the control group plus two standard deviations, the mean of the control group plus three standard deviations, or a preselected level in the control group. In one embodiment, the threshold is set as the mean of the control group plus one standard deviation. In one embodiment, the threshold is set as the mean of the control group plus two standard deviations. In one embodiment, the threshold is set as the mean of the control group plus three standard deviations.

[0080] Alternatively, or in addition, a threshold value may be established by obtaining levels of IgA or its subclasses or subtypes from the same patient and comparing them with subsequent results. In these embodiments, the individual essentially serves as their own "control." In addition, the individual may serve as a control, for example, with low levels of cirrhosis (e.g., Child-Pugh A), allowing the progression or effectiveness of a treatment regimen to be measured over time. For markers that increase with disease severity, an increase over time in the same patient can indicate worsening or development of the disease, or risk of disease, or failure of a treatment regimen, while a decrease or maintenance of the value over time can indicate remission, inhibition of progression of the disease state, or success of a treatment regimen. A variety of additional controls will be routinely applied by those skilled in the art.

[0081] In some embodiments, different thresholds may be required for populations with different diseases / conditions and / or different stages of a disease / condition. Internal standards reflecting the thresholds described herein can be included as internal controls in the methods and kits described herein.

[0082] In one embodiment, in the method described herein, the difference from the threshold indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject.In one embodiment, the difference is an increase relative to the threshold.The term "elevated" or "elevating" or "elevated" refers to having a higher or larger protein level or the ratio of the levels of two proteins compared to the threshold.In one embodiment, the protein level or the ratio of two proteins is increased by at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300% compared to the threshold.In one embodiment, the difference is a decrease relative to the threshold. The terms "decrease" or "decreases" or "decreased" refer to eliminating, decreasing, or having a lower level of a protein or the ratio of two protein levels compared to a threshold value. In one embodiment, the protein level or the ratio of two proteins is reduced by at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100% compared to the threshold value.

[0083] In some embodiments, the assay comprises two or more threshold values.

[0084] In one embodiment, an elevated dIgA to mIgA ratio or an elevated dIgA1 to mIgA1 ratio or dIgA2 to mIgA2 ratio compared to a threshold indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject. In one embodiment, a decreased mIgA to dIgA ratio or mIgA1 to dIgA1 ratio or mIgA2 to dIgA2 ratio compared to a threshold indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject.

[0085] In one embodiment, a dIgA to mIgA ratio equal to or greater than a threshold of about 0.5 to 0.8 indicates intestinal barrier dysfunction and / or cirrhosis in a subject. In one embodiment, a dIgA to mIgA ratio equal to or greater than a threshold of about 0.6 to 0.7 indicates intestinal barrier dysfunction and / or cirrhosis in a subject. In one embodiment, a dIgA to mIgA ratio equal to or greater than a threshold of about 0.65 indicates intestinal barrier dysfunction and / or cirrhosis in a subject. In one embodiment, a dIgA to mIgA ratio equal to or greater than a threshold of about 1 indicates intestinal barrier dysfunction and / or cirrhosis in a subject with HIV. In one embodiment, a dIgA to mIgA ratio equal to or greater than a threshold of about 0.5 indicates intestinal barrier dysfunction and / or cirrhosis in a subject with hepatitis B.

[0086] In one embodiment, an mIgA to dIgA ratio below a threshold of about 1.2 to 2.5 indicates intestinal barrier dysfunction and / or cirrhosis in a subject. In one embodiment, an mIgA to dIgA ratio below a threshold of about 1.5 to 2.2 indicates intestinal barrier dysfunction and / or cirrhosis in a subject. In one embodiment, an mIgA to dIgA ratio below a threshold of about 1.54 indicates intestinal barrier dysfunction and / or cirrhosis in a subject. In one embodiment, an mIgA to dIgA ratio below a threshold of about 1 indicates intestinal barrier dysfunction and / or cirrhosis in a subject with HIV, or an mIgA to dIgA ratio below a threshold of about 2 indicates intestinal barrier dysfunction and / or cirrhosis in a subject with hepatitis B.

[0087] In one embodiment, elevated levels of IgA2 relative to a threshold value indicate intestinal barrier dysfunction and / or liver cirrhosis in a subject. In one embodiment, IgA2 levels equal to or greater than a threshold value of 3500 DA indicate intestinal barrier dysfunction and / or liver cirrhosis in a subject.

[0088] In one embodiment, if the mIgA level is elevated relative to the threshold, the dIgA level is compared to the dIgA threshold, and a difference from the dIgA threshold indicates intestinal barrier dysfunction and / or cirrhosis in the subject; if the mIgA level is decreased relative to the threshold, the ratio of the dIgA level to the mIgA level is determined and compared to the ratio threshold, and a difference from the ratio threshold indicates intestinal barrier dysfunction and / or cirrhosis in the subject. In one embodiment, the dIgA and mIgA are dIgA1 and mIgA1. In one embodiment, the dIgA and mIgA are dIgA2 and mIgA2. In one embodiment, the dIgA is measured using an anti-IgA antibody. In one embodiment, the mIgA threshold is 5000 DA.

[0089] As used herein, "DA" is the absorbance unit provided by a device that measures absorbance, such as the Axxin AX-2X reader. Based on the information provided herein, one of skill in the art can readily determine equivalent threshold values ​​for equivalent and similar devices. In an alternative embodiment, the results of the assay can be converted to mg / mL.

[0090] Diagnostic sensitivity and specificity As used herein, "diagnostic sensitivity" refers to the ability of a diagnostic test to correctly identify those with a disease. As used herein, "diagnosis" refers to the ability of a method to detect a disease (true positive rate). specificity " is a disease It refers to the ability of a diagnostic method to correctly identify those who do not have it (true negative rate).

[0091] In one embodiment, the methods described herein have a diagnostic sensitivity of about 50 to about 98% for intestinal barrier dysfunction. In one embodiment, the methods have a diagnostic sensitivity of about 60 to about 95% for intestinal barrier dysfunction. In one embodiment, the methods have a diagnostic sensitivity of about 70 to about 90% for intestinal barrier dysfunction. In one embodiment, the methods have a diagnostic sensitivity of about 75 to about 90% for intestinal barrier dysfunction. In one embodiment, the methods have a diagnostic sensitivity of about 75 to about 85% for intestinal barrier dysfunction. In one embodiment, the methods described herein have a diagnostic sensitivity of at least 85% for intestinal barrier dysfunction. In one embodiment, the methods described herein have a diagnostic sensitivity of at least 80% for intestinal barrier dysfunction.

[0092] In one embodiment, the method described herein has a diagnostic sensitivity for cirrhosis of about 50 to about 98%. In one embodiment, the method has a diagnostic sensitivity for cirrhosis of about 60 to about 95%. In one embodiment, the method has a diagnostic sensitivity for cirrhosis of about 70 to about 90%. In one embodiment, the method has a diagnostic sensitivity for cirrhosis of about 75 to about 90%. In one embodiment, the method has a diagnostic sensitivity for cirrhosis of about 75 to about 85%. In one embodiment, the method described herein has a diagnostic sensitivity for cirrhosis of at least 85%. In one embodiment, the method described herein has a diagnostic sensitivity for cirrhosis of at least 80%.

[0093] In one embodiment, the methods described herein provide a diagnostic yield of about 50 to about 98% for intestinal barrier dysfunction. specificity In one embodiment, the method provides a diagnostic yield of about 60 to about 95% for intestinal barrier dysfunction. specificity In one embodiment, the method provides a diagnostic yield of about 70 to about 90% for intestinal barrier dysfunction. specificity In one embodiment, the method provides a diagnostic yield of about 75 to about 90% for intestinal barrier dysfunction. specificity In one embodiment, the method provides a diagnostic yield of about 75 to about 85% for intestinal barrier dysfunction. specificity In one embodiment, the methods described herein provide a diagnostic of at least 85% for intestinal barrier dysfunction. specificity In one embodiment, the methods described herein provide a diagnostic of at least 80% for intestinal barrier dysfunction. specificity It has.

[0094] In one embodiment, the methods described herein provide an about 50 to about 98% diagnostic yield for cirrhosis. specificity In one embodiment, the method has a diagnostic yield of about 60 to about 95% for cirrhosis. specificity In one embodiment, the method has an about 70 to about 90% diagnostic yield for cirrhosis. specificity In one embodiment, the method has an about 75 to about 90% diagnostic yield for cirrhosis. specificity In one embodiment, the method has a diagnostic yield of about 75 to about 85% for cirrhosis. specificity In one embodiment, the methods described herein provide a diagnostic yield of at least 85% for cirrhosis. specificity In one embodiment, the methods described herein provide a diagnostic yield of at least 80% for cirrhosis. specificity It has.

[0095] In one embodiment, if the level of IgA2 has not been determined, the method has one or more of: i) a diagnostic sensitivity of at least 54% for Child-Pugh cirrhosis of A; ii) a diagnostic sensitivity of at least 69% for Child-Pugh cirrhosis of B; and iii) a diagnostic sensitivity of at least 87% for Child-Pugh cirrhosis of C.

[0096] In one embodiment, when the level of IgA2 is determined, the method has one or more of: i) a diagnostic sensitivity of at least 72% for Child-Pugh cirrhosis of A; ii) a diagnostic sensitivity of at least 76% for Child-Pugh cirrhosis of B; and iii) a diagnostic sensitivity of at least 88% for Child-Pugh cirrhosis of C.

[0097] In one embodiment, with respect to Child-Pugh cirrhosis of A, the method has one or more of: i) a diagnostic sensitivity of at least 90% for alcoholic cirrhosis, ii) a diagnostic sensitivity of at least 70% for cryptogenic cirrhosis, iii) a diagnostic sensitivity of at least 40% for hepatitis B cirrhosis, iv) a diagnostic sensitivity of at least 76% for hepatitis C cirrhosis, v) a diagnostic sensitivity of at least 90% for NASH cirrhosis, vi) a diagnostic sensitivity of at least 90% for PBC, and vii) a diagnostic sensitivity of at least 90% for PSC.

[0098] In one embodiment, the method comprises the steps of: detecting stage F4 fibrosis of the liver using an APRI (aspartate aminotransferase-to-platelet ratio index) Degrees and specificity It has.

[0099] Treatment method The present invention relates to a method of treating intestinal barrier dysfunction and / or liver cirrhosis in a subject determined to have such a condition using the methods of the present invention. In one embodiment, the method is for treating intestinal barrier dysfunction. In one embodiment, the method is for treating liver cirrhosis.

[0100] In one embodiment, treatment is selected from surgical intervention (e.g., to remove scar tissue or for a liver transplant), administration of a therapeutic agent, administration of a prophylactic agent, dietary intervention, exercise intervention, or probiotic or microbial intervention such as a "fecal transplant," based on the results.

[0101] In one embodiment, the treatment or prevention is selected from one or more of the following: disulfiram, naltrexone, acamprosate, corticosteroids, prednisone, prednisone and azathioprine, penicillamine, trientine, deferoxamine, ciprofloxacin, norofloxacin, ceftriaxone, ofloxacin, amoxicillin-clavulanate, vitamin K, phytonadione, bumetanide, furosemide, hydrochlorothiazide, chlorothiazide, amiloride, triamterene, and spironolactone.

[0102] In one embodiment, the method includes re-testing at a later time point to determine progression and / or response to treatment.

[0103] kit In various related aspects, the present invention also relates to devices and kits for carrying out the methods described herein. Suitable kits will contain at least some, and preferably all, of the reagents sufficient to carry out at least one of the methods described herein.

[0104] In one aspect, the present invention provides a kit for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, comprising: (i) an agent that binds to dIgA and forms a detectable dIgA complex; and (ii) an agent that binds to mIgA and forms a detectable mIgA complex, wherein (i) specifically binds to dIgA and / or (ii) specifically binds to mIgA.

[0105] In one aspect, the present invention provides a kit for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, comprising (i) an agent that binds to dIgA1 and forms a detectable dIgA complex, and (ii) an agent that binds to mIgA1 and forms a detectable mIgA1 complex, wherein (i) specifically binds to dIgA and / or (ii) specifically binds to mIgA.

[0106] In one aspect, the present invention provides a kit for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, comprising (i) an agent that binds to dIgA2 and forms a detectable dIgA complex, and (ii) an agent that binds to mIgA2 and forms a detectable mIgA2 complex, wherein (i) specifically binds to dIgA and / or (ii) specifically binds to mIgA.

[0107] In one embodiment, the kit further comprises an agent that binds to form a detectable IgA2 complex, hi one embodiment, one or more of the agents is bound to a solid support.

[0108] In one embodiment, the kit further comprises a reagent for detecting one or more of mIgA complexes, dIgA complexes, and IgA2 complexes, hi one embodiment, the reagent is anti-human IgA colloidal gold.

[0109] In one embodiment, the kit comprises a strip, chip, or cartridge for use in a lateral flow assay. In one embodiment, the kit comprises a strip, chip, or cartridge for use in a point-of-care device.

[0110] In one embodiment, the kit includes a test strip for a lateral flow device that includes at least one sample loading area; a) the strip comprises a capture portion comprising an agent that binds to dIgA; b) the strip comprises a capture portion comprising an agent that binds to mIgA; a) is closer to the sample loading area than b) so that the sample contacts a) before b). In one embodiment, the strip further comprises c) a capture moiety between a) and b) that comprises an agent that binds to IgA2.

[0111] The sample can flow through the device, and simultaneously or subsequently, a detection reagent that binds to IgA can flow through the device. Detection of the detection reagent bound to IgA can be performed in a subsequent step, for example, by measuring absorbance. In one embodiment, the detection reagent is selected from one or more of anti-human IgA colloidal gold, anti-human IgA1 colloidal gold, and anti-human IgA2 colloidal gold.

[0112] In one embodiment, the kit can be stored at about 4° C. In one embodiment, the kit can be stored at about 15° C. In one embodiment, the kit can be stored at about 23° C.

[0113] In one embodiment, the kit includes an immunoassay test strip. In one embodiment, the immunoassay test strip includes a sample loading portion including a binding agent and two or more capture portions. In one embodiment, a biological sample is contacted with the binding agent by applying the sample to the sample portion of the immunoassay test strip, which is operably connected to the spaced capture portions of the test strip, whereby components of the sample flow from the test strip sample portion to and through the test strip capture portions, one capture portion including a binding agent for detecting dIgA and a second capture portion including a binding agent for detecting mIgA, at least one of the binding agents specifically detecting their target. In one embodiment, the binding agent for detecting dIgA specifically binds to dIgA. In one embodiment, the binding agent for detecting mIgA specifically binds to mIgA. In one embodiment, the sample is contacted with a capture portion including a binding agent for detecting dIgA followed by contact with a capture portion including a binding agent for detecting mIgA. In a further embodiment, the capture moiety includes a third capture moiety comprising a binding agent for detecting IgA2. In one embodiment, the sample is contacted with a capture moiety comprising a binding agent for detecting dIgA, followed by a capture moiety comprising a binding agent for detecting IgA2, and then by a capture moiety comprising a binding agent for detecting IgA2, followed by a capture moiety comprising a binding agent for detecting mIgA. In one embodiment, the capture moiety is a test line (e.g., the test line shown in Figure 2 or Figure 3).

[0114] In one embodiment, the kit is used to perform some or all of the assays disclosed herein. In one embodiment, the present disclosure enables and provides point-of-care devices capable of performing the methods disclosed and claimed herein. [Example]

[0115] Example 1 - Materials Recombinant Protein L -Supplied by:ThermoFisher Scientific -Catalog number: 21189 -Concentration: 25ug / mL Mouse anti-human IgA -Supplier: Sigma -Catalog number: I0636 -Concentration: 0.5mg / ml Mouse anti-human IgA2 (subclass specific) -Supplied by: Nordic Mubio -Catalog number: MAHu / IgA2 -Concentration: 0.25mg / mL CSC (chimeric secretory component, chimeric polymeric Ig receptor) -Source: In-house (Burnet Institute as described in WO / 2014 / 071456), expressed in mammalian cells -Concentration: 1mg / mL Goat anti-human IgA 40nm gold conjugate -Supplier:BBI Solutions - Catalog number: BA.GAHA40 -Concentration: OD3 Running Buffer -Plasma: PBS+0.5% Tween+0.05% Azide Whole Blood: 1% Triton X-100 in PBS Conjugate Buffer for Drying (BDS, used to dilute gold conjugates for drying) -20% sucrose, 5% trehalose, 0.25% Tween 20, 0.35% PEG, 1% BSA, 2mM EDTA, 10mM borate Patient samples Samples were obtained from patients with various liver disease cohorts, including hepatitis B, hepatitis C, or other causes of cirrhosis, at The Alfred Hospital or St Vincent's Hospital, Melbourne, as well as healthy volunteers at the Burnet Institute and patients with HIV infection but no known diagnosis of liver disease at The Alfred Hospital. All samples were collected with appropriate informed consent.

[0116] Example 2 - Detection of dIgA, mIgA, and IgA2 for intestinal barrier dysfunction and liver cirrhosis Lateral flow test strips were prepared by applying three test lines across a nitrocellulose membrane (NCM) (Vivid 90, Pall Corporation) using an IsoFlow™ dispenser (Imagene Technology) and laminating the NCM with an integrated sample pad and gold conjugate pad (Glass Fiber 8951, AHLSTROM-Munksjo) and an absorbent sink (CF6 absorbent pad, GE Healthcare). Test strips were assembled by laminating the NCM, sample pad, dissolution pad, and absorbent sink onto an adhesive plastic backing card and cutting into 5 mm test strips (BIODOT guillotine). These strips were assembled into disposable plastic housings (Burnet Institute). Exemplary layouts and dimensions of the test strips are shown in Figures 2 and 3.

[0117] The first test strip contains 1 mg / ml of recombinant protein, chimeric secretory component (CSC or pIgR containing domain 1 that binds dIgA and does not substantially bind IgM in a sample, such as from rabbit, and domains 2-5 from human; see WO / 2014 / 071456; produced by the Burnet Institute), which, when reacted with sample plasma, serum blood, or gingival crevicular fluid, forms pIgR-dIgA complexes on the test strip.

[0118] The second test line contains 0.25 mg / ml of anti-human IgA2 monoclonal antibody (Nordic MUbio, catalog number MAHu / IgA2), which when reacted with sample plasma, serum, blood, or gingival crevicular fluid forms an anti-IgA2-IgA2 complex on the test strip.

[0119] The third test line is 0.025 mg / ml of recombinant Protein L (ThermoFisher Scientific, catalog number 21189), which forms a Protein-L:monomeric IgA complex when reacted with sample plasma, serum, blood, or gingival crevicular fluid. Notably, Protein L also binds both IgG and IgM antibody isotypes, so the amount of bound monomeric IgA represents the relative level of IgA compared to other isotypes, as well as the absolute amount of monomeric IgA.

[0120] To perform the assay, 5 microliters of plasma is added to the sample pad of the test strip, followed by one drop (approximately 30 microliters) of PBS containing 0.5% Tween 20 detergent. A similar method is expected to work with serum. Alternatively, 5 microliters of whole blood can be added, followed by one drop of PBS containing 1% Triton X-100 detergent. The sample and buffer are allowed to flow over the strip for 10 minutes.

[0121] Subsequently, four drops of the same buffer are added to a colloidal gold pad containing OD3 of goat anti-human IgA 40 nm gold conjugate (BBI Solutions, UK; catalog number BA.GAHA40) in BDS (20% sucrose, 5% trehalose, 0.25% Tween 20, 1% bovine serum albumin, 2 mM EDTA, 10 mM borate pH 8.6). The rehydrated gold and buffer are allowed to flow over the test strip for 20 minutes to detect complexes containing human IgA antibodies on the first, second, and third test lines.

[0122] After 20 minutes (30-minute total assay time), the test strip should show three visible lines representing (starting from the sample well): dimeric IgA, IgA2, and monomeric IgA. Absence of the third line (monomeric IgA) or all three lines indicates either a failure of the assay or that the patient is IgA-deficient, making the test unusable in these rare individuals (Figure 5A).

[0123] The intensity of each test line is proportional to the amount of the respective analyte and is interpreted visually or, more preferably, using an automated reader such as the Axxin AX-2X reader (Axxin Ltd, Melbourne). The AX-2X is used to obtain a numerical readout of each test line. The workflow for using the Axxin AX-2X is shown in Figure 4, and an example readout is provided in Figure 5A.

[0124] Test interpretation is accomplished in two ways: 1) The relative amounts of dIgA and mIgA, i.e., the numerical ratio of test line intensities of dIgA / mIgA, is calculated, and a positive test result is indicated if this ratio exceeds 0.65 in the example shown.

[0125] 2) The absolute amount of IgA2 is observed, and if this value exceeds 3,500 arbitrary units (AX-2X), a positive test result is indicated.

[0126] The calculation of these cutoffs / thresholds is shown in FIG.

[0127] If either or both of these methods produce a positive test result, the overall test result is considered positive. If both of these methods produce a negative test result, the test result is considered negative. It is possible that other arithmetic formulas can be applied to the test values ​​to provide additional information, such as the absolute amount of dIgA.

[0128] The results of this test in assessing healthy subjects, patients with hepatitis B, and patients with cirrhosis are shown in Figures 6, 7, 8, and 17. This test is suitable for identifying patients with low and high levels of cirrhosis (Child-Pugh A, Child-Pugh B, and Child-Pugh C, see Figure 9), as well as patients with cirrhosis caused by different underlying disease states (see Figures 10 and 17). The importance of testing for cirrhosis is further illustrated when testing the cirrhotic patients in Figures 6, 7, and 8 for alanine aminotransferase 1 (BioPoint ALT1 Rapid Test). As shown in Figure 11 and as is well known in the art, only a small percentage (6.6%) of cirrhotic patients have evidence of liver disease when tested using ALT, the most commonly used biomarker for liver disease.

[0129] Example 3 - dIgA study of intestinal barrier dysfunction and liver cirrhosis Lateral flow test strips were prepared by applying three test lines across a nitrocellulose membrane (NCM) (Vivid 90, Pall Corporation) using an IsoFlow™ dispenser (Imagene Technology) and laminating the NCM with an integrated sample pad and gold conjugate pad (Glass Fiber 8951, AHLSTROM-Munksjo) and an absorbent sink (CF6 absorbent pad, GE Healthcare). Test strips were assembled by laminating the NCM, sample pad, dissolution pad, and absorbent sink onto an adhesive plastic backing card and cutting into 5 mm test strips (BIODOT guillotine). These strips were assembled into disposable plastic housings (Burnet Institute). Exemplary layouts and dimensions of the test strips are shown in Figures 2 and 3.

[0130] The first test strip contains 1 mg / ml of recombinant protein, chimeric secretory component (CSC or pIgR containing domain 1 that binds dIgA and does not substantially bind IgM in a sample, such as from rabbit, and domains 2-5 from human; see WO / 2014 / 071456; produced by the Burnet Institute), which, when reacted with sample plasma, serum blood, or gingival crevicular fluid, forms pIgR-dIgA complexes on the test strip.

[0131] The second test line contains 0.25 mg / ml of anti-human IgA2 monoclonal antibody (Nordic MUbio, catalog number MAHu / IgA2), which when reacted with sample plasma, serum, blood, or gingival crevicular fluid forms an anti-IgA2-IgA2 complex on the test strip.

[0132] The third test line is 0.025 mg / ml of recombinant Protein L (ThermoFisher Scientific, Catalog No. 21189), which forms a Protein-L:monomeric IgA complex when reacted with sample plasma, serum, blood, or gingival crevicular fluid. Notably, Protein L also binds both IgG and IgM antibody isotypes, so the amount of bound monomeric IgA represents the relative level of IgA compared to other isotypes, as well as the absolute amount of monomeric IgA.

[0133] Plasma (5 microliters) is added to the sample pad of the test strip, followed by one drop (approximately 30 microliters) of PBS containing 0.5% Tween 20 detergent. Serum can be used as an alternative to plasma. Alternatively, 5 microliters of whole blood can be added, followed by one drop of PBS containing 1% Triton X-100 detergent. The sample and buffer are allowed to flow over the strip for 10 minutes.

[0134] Subsequently, four drops of the same buffer are added to a colloidal gold pad containing OD3 goat anti-human IgA 40 nm gold conjugate (BBI Solutions, UK; catalog number A.GAHA40) in BDS (20% sucrose, 5% trehalose, 0.25% Tween 20, 1% bovine serum albumin, 2 mM EDTA, 10 mM borate pH 8.6). The rehydrated gold and buffer are allowed to flow over the test strip for 20 minutes.

[0135] After 20 minutes (30-minute total assay time), the test strip should show three visible lines representing (starting from the sample well): dimeric IgA, IgA2, and monomeric IgA. Absence of the third line (monomeric IgA) or absence of all three lines indicates either a failure of the assay or that the patient is IgA-deficient, making the test unusable in these rare individuals (Figure 5B).

[0136] The intensity of each test line is proportional to the amount of the respective analyte and is interpreted visually or, more preferably, using an automated reader such as the Axxin AX-2X reader (Axxin Ltd, Melbourne). The AX-2X is used to obtain a numerical readout of each test line. The workflow for using the Axxin AX-2X is shown in Figure 4, and an example readout is provided in Figure 5B.

[0137] Test interpretation is accomplished in two ways: 1) The relative amounts of dIgA and mIgA, i.e., the numerical ratio of test line intensities of dIgA / mIgA, is calculated, and a positive test result is indicated if this ratio exceeds 0.65 in the example shown.

[0138] 2) The absolute amount of IgA2 is observed, and if this value exceeds 3,500 arbitrary units (AX-2X), a positive test result is indicated.

[0139] The calculation of these cutoffs / thresholds is shown in FIG.

[0140] If either or both of these methods produce a positive test result, the overall test result is considered positive. If both of these methods produce a negative test result, the test result is considered negative. It is possible that other arithmetic formulas can be applied to the test values ​​to provide additional information, such as the absolute amount of dIgA.

[0141] In the above example, dimeric IgA was captured with CSC and detected with colloidal gold anti-IgA, which detects both the IgA1 and IgA2 isotypes of IgA. To determine whether it was possible to detect dIgA1 and dIgA2 separately, similar test strips were constructed using CSC capture as before, except that primary antibodies against IgA1 and IgA2 (Nordic MUbio catalog numbers MAHu / IgA1 (5 μg / ml) and MAHu / IgA2 (25 μg / ml), respectively) and colloidal gold-conjugated goat anti-mouse antibody (BBI, OD3) were used (schematically depicted in FIG. 13). The reactivity of the dIgA test strips was measured using an Axxin AX-2X reader.

[0142] The results (Figure 12) show that both dIgA1 and dIgA2 levels are significantly elevated in cirrhotic patients relative to healthy controls, suggesting that dIgA1 and / or dIgA2 can be used as surrogates for total dIgA in determining leaky gut / cirrhosis using the outlined method. The very low dIgA2 reactivity observed in most healthy control subjects compared with the strong reactivity in most cirrhotic patients may offer some advantages, for example, in developing test types where detection of any visible test line of dIgA2 (rather than comparison with dIgA and mIgA) may provide an indication of leaky gut / cirrhosis.

[0143] In the above example, monomeric IgA is captured by Protein L, which also captures IgG and IgM, meaning that there is competition between mIgA present in the sample and the IgG and IgM also present in the sample. Therefore, the level of mIgA detected using Protein L reflects both the absolute concentration of mIgA and the relative concentrations of mIgA, IgG, and IgM in the sample. This may be particularly relevant in the case of HIV infection and other conditions associated with hypergammaglobulinemia (which in the case of HIV infection is due to generalized immune activation, leading to leaky gut). Alternatively, it may be preferable to detect mIgA independently of the levels of IgG and IgM in such samples.

[0144] To investigate this, test strips were constructed as before, except that mouse anti-human IgA (Sigma, Cat. No.: I0636; 0.5 mg / ml) was used and compared with Protein L (0.05 mg / ml).

[0145] The results (Figure 14) show that for the healthy control samples, the use of alternative anti-IgA capture resulted in similar mIgA signals for the majority of samples (6 / 8), but 2 / 8 samples showed much higher apparent levels of mIgA when captured by anti-IgA rather than Protein L, reflecting a lack of competition for anti-IgA capture in these samples. In contrast, 7 / 8 HIV-infected patient samples showed much higher apparent levels of mIgA when captured by anti-IgA rather than Protein L, reflecting a lack of competition for anti-IgA capture in these samples, where higher levels of IgG and IgM (due to HIV infection) increased the level of competition for Protein L.

[0146] When used on samples from cirrhotic patients (Figure 15), 17 / 24 patients had similar apparent levels of mIgA using either anti-IgA or Protein L as the mIgA capture reagent, but 7 / 24 samples had significantly higher levels of mIgA using anti-IgA capture. This indicates that anti-IgA can be used as an alternative to Protein L for mIgA capture, and by inference, other IgA capture reagents can also be used.

[0147] When comparing dIgA / mIgA ratios using anti-IgA versus Protein L capture reagents (Figure 16; note that in this case, an arbitrary cutoff of 1.0 was assigned to the dIgA / mIgA ratio for comparison purposes), the use of anti-IgA capture assays specificity It was found that the sensitivity improved, with 1 / 8 healthy controls and 3 / 8 HIV patients testing negative with the anti-IgA assay but positive with the Protein L assay, whereas the use of anti-IgA capture somewhat reduced the sensitivity, with 4 / 24 cirrhotic patients testing negative with the anti-IgA assay but positive with the Protein L assay. Therefore, further improvements to the assay could be made, for example, by using both anti-IgA and Protein L in the same or separate test lines, or by using alternative reagents for the capture of mIgA. specificity This may be done to balance the requirements for speed and sensitivity.

[0148] Example 4 - Discussion The methods presented in Examples 2 and 3 offer a highly reproducible, non-invasive test (requiring only 5 μl of blood) and provide an objective numerical scale (using an AX-2X instrument or similar), with a positive result for cirrhosis being the detection of one or both of the dIgA / mIgA ratio and IgA2 levels above a cutoff. The cutoff can be determined using healthy controls, the mean + 1 or 2 standard deviations. In practice, a control sample, one or more standards, or a dose-response curve can be provided for comparison.

[0149] The methods described herein are suitable for detecting cirrhosis in both patients with diagnosed liver disease who are being monitored for progression (HBV, HCV, nonalcoholic fatty liver disease or NAFLD), and for population-wide screening for NAFLD and its more severe form, NASH. In resource-rich settings, this allows for triage of patients for more detailed cirrhosis investigation (ultrasound, fibroscan, and other radiological tests) before therapeutic intervention, while in resource-poor settings where cirrhosis is difficult to diagnose, it allows access to HBV antiviral therapy (where cirrhosis is the preferred treatment indication) and appropriate management of HCV antiviral therapy (where cirrhotic patients are seen by specialists, while non-cirrhotic patients are seen in general practice).

[0150] NAFLD affects approximately 25% of the world's population but is typically not diagnosed until advanced disease (NASH and grade B cirrhosis). NAFLD, along with other metabolic diseases related to obesity, is on the rise, and as treatments become available, population-based screening will be in high demand within the next few years. The methods described herein can be combined with other tests, such as liver disease tests, to provide a more complete picture of a subject's liver health. For example, the methods / tests can be used in combination with ALT / ALT1 tests (e.g., ALT1 test, Nanjing BioPoint), enhancing the results of these tests by detecting liver disease at an earlier stage.

[0151] It will be apparent to those skilled in the art that numerous variations and / or modifications may be made to the present invention as illustrated in the specific embodiments without departing from the spirit or scope of the invention as generally described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0152] This application claims priority from Australian Provisional Application No. 2020 / 900194, entitled "Detecting gut barrier dysfunction and / or cirrhosis," filed on January 24, 2020, the entire contents of which are incorporated herein by reference.

[0153] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0154] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and should not be construed as an admission that any or all of such matters form part of the prior art or were common general knowledge in the art relevant to the present invention by virtue of existing prior to the priority date of each claim of this application. The present invention includes the following embodiments. [1] A method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, comprising determining dIgA and mIgA levels and their ratio in a biological sample from the subject, and comparing the ratio with a threshold value. [2] A method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, comprising determining dIgA1 or dIgA2 levels and mIgA1 or mIgA2 levels and their ratio in a biological sample from the subject, and comparing the ratio with a threshold value. [3] The method according to [1] or [2] above, wherein a difference from the threshold value indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject. [4] The method according to any one of [1] to [3] above, wherein an elevated dIgA to mIgA ratio, an elevated dIgA1 to mIgA1 ratio, or an elevated dIgA2 to mIgA2 ratio compared to a threshold value indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject. [5] The method according to any one of [1] to [4] above, wherein a decreased mIgA to dIgA ratio, a decreased mIgA1 to dIgA1 ratio, or a decreased mIgA2 to dIgA2 ratio compared to a threshold value indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject. [6] The method according to any one of the above [1], [3], or [4], wherein a dIgA to mIgA ratio equal to or greater than a threshold of 0.65 indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject. [7] The method of any of the above [1], [3], or [4], wherein a dIgA to mIgA ratio equal to or greater than a threshold of 1 indicates intestinal barrier dysfunction and / or liver cirrhosis in a subject with HIV, or a dIgA to mIgA ratio equal to or greater than a threshold of 0.5 indicates intestinal barrier dysfunction and / or liver cirrhosis in a subject with hepatitis B. [8] The method according to any one of the above [1], [3], or [5], wherein an mIgA to dIgA ratio below a threshold of 1.54 indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject. [9] The method of any of the above [1], [3], or [5], wherein an mIgA to dIgA ratio below a threshold of 1 indicates intestinal barrier dysfunction and / or liver cirrhosis in a subject with HIV, or a dIgA to mIgA ratio below a threshold of 2 indicates intestinal barrier dysfunction and / or liver cirrhosis in a subject with hepatitis B.

[10] The method according to any one of [1] or [3] to [9] above, wherein the dIgA level in the sample is determined by contacting the sample with pIgR, an anti-dIgA antibody, or an anti-IgA J chain antibody to form a detectable dIgA complex.

[11] The method according to

[10] above, wherein the pIgR is a chimeric secretory component.

[12] The method according to any one of [1] or [3] to

[11] above, wherein the mIgA level in the sample is determined by contacting the sample with an anti-mIgA antibody to form a detectable mIgA complex.

[13] The method according to any one of [2] to [5] above, wherein the mIgA1 level in the sample is determined by contacting the sample with an anti-mIgA1 antibody to form a detectable mIgA complex.

[14] The method according to any one of [1] or [3] to

[11] above, wherein the mIgA level is determined in a sample depleted of dIgA and IgA2 by contacting the sample with protein L or anti-IgA to form a detectable mIgA complex.

[15] The method according to any one of [2] to [5] or

[13] above, wherein the mIgA1 level is determined in a sample depleted of dIgA and IgA2 by contacting the sample with protein L or anti-IgA to form a detectable mIgA complex.

[16] and (ii) determining a level of IgA2 in the sample. (i) an elevated dIgA to mIgA ratio compared to threshold, a decreased mIgA to dIgA ratio compared to threshold, an elevated dIgA1 to mIgA1 ratio compared to threshold, a decreased mIgA1 to dIgA1 ratio compared to threshold, an elevated dIgA2 to mIgA2 ratio compared to threshold, a decreased mIgA2 to dIgA2 ratio compared to threshold, and (ii) an elevated IgA2 level compared to a threshold, wherein one or both of these indicate intestinal barrier dysfunction and / or liver cirrhosis in the subject.

[17] The method according to

[16] above, wherein an IgA2 level above a threshold of 3500 DA indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject.

[18] The method according to

[16] or

[17] above, wherein the IgA2 level in the sample is determined by contacting the sample with an anti-IgA2 antibody to form a detectable IgA2 complex.

[19] The method according to any one of [1] or [2] to

[18] above, comprising contacting the sample with pIgR, an anti-dIgA antibody, or an anti-IgA J chain antibody to form a detectable dIgA complex, and subsequently contacting the sample with Protein L, an anti-IgA antibody, or an anti-mIgA antibody to form a detectable mIgA complex.

[20] The method according to any one of [1] or [2] to

[19] above, comprising contacting the sample with pIgR, an anti-dIgA antibody, or an anti-IgA J chain antibody to form a detectable dIgA complex, subsequently contacting the sample with an anti-IgA2 antibody to form a detectable IgA2 complex, and subsequently contacting the sample with Protein L, an anti-IgA antibody, or an anti-mIgA antibody to form a detectable mIgA complex.

[21] The method according to any one of

[10] to

[20] above, wherein one or more of the mIgA complex, dIgA complex, and IgA2 complex are detected by a reagent that binds to IgA.

[22] The method according to

[21] above, wherein the reagent that binds to IgA is selected from one or more of anti-human IgA colloidal gold, anti-human IgA1 colloidal gold, and anti-human IgA2 colloidal gold.

[23] The method according to any one of

[10] to

[22] above, wherein the detectable complex comprises one or more of colloidal gold, a magnetic agent, colored latex, carboxycellulose, carbon nanoparticles, and a fluorescent label.

[24] The method according to any one of the above [1] to

[23] , wherein the biological sample is selected from whole blood, plasma, serum, or gingival crevicular fluid.

[25] The method according to any one of [1] to

[24] above, wherein the cirrhosis is the result of alcohol, NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis), viral hepatitis, HIV, unknown cause, primary biliary cirrhosis, and / or primary sclerosing cholangitis.

[26] The method according to any one of the above-mentioned [1] to

[25] , wherein the liver cirrhosis has Child-Pugh classification of A, Child-Pugh classification of B, or Child-Pugh classification of C.

[27] The method according to any one of the above [1] to

[26] , wherein the method has a diagnostic sensitivity of at least 80% for liver cirrhosis.

[28] The method according to any one of the above [1] to

[27] , wherein the method has a diagnostic specificity of at least 85% for liver cirrhosis.

[29] If the level of IgA2 has not been determined, the method further comprises: i) a diagnostic sensitivity of at least 54% for Child-Pugh cirrhosis of A; ii) a diagnostic sensitivity of at least 69% for Child-Pugh cirrhosis of B, and iii) A diagnostic sensitivity of at least 87% for cirrhosis according to the Child-Pugh classification of C.

[30] If the level of IgA2 is determined, the method further comprises: i) a diagnostic sensitivity of at least 72% for Child-Pugh cirrhosis of A; ii) a diagnostic sensitivity of at least 76% for Child-Pugh cirrhosis of B, and iii) a diagnostic sensitivity of at least 88% for cirrhosis according to the Child-Pugh classification of C.

[31] For Child-Pugh cirrhosis of A, the method comprises: i) a diagnostic sensitivity of at least 90% for alcoholic cirrhosis; ii) diagnostic sensitivity of at least 70% for cryptogenic cirrhosis; iii) diagnostic sensitivity of at least 40% for hepatitis B cirrhosis; iv) diagnostic sensitivity of at least 76% for hepatitis C cirrhosis; v) diagnostic sensitivity of at least 90% for NASH cirrhosis; vi) a diagnostic sensitivity of at least 90% for PBC; and vii) a diagnostic sensitivity of at least 90% for PSC.

[32] The method according to any one of the above [1] to

[31] , wherein the method is suitable for use in a point-of-care (POC) device.

[33] The method according to any one of the above [1] to

[32] , wherein the method comprises a chromatographic assay, an enzyme-linked immunosorbent assay, a fluorescent immunosorbent assay, a radioimmunosorbent assay, or a homogeneous assay.

[34] The method according to any one of the above [1] to

[33] , wherein the method comprises a lateral flow format.

[35] performed on a lateral flow device comprising a test strip containing at least one sample loading area; a) the strip comprises a capture portion comprising an agent that binds to dIgA; b) the strip comprises a capture portion comprising an agent that binds to mIgA; The method according to any one of the above [1] to

[34] , wherein a) is closer to the sample loading area than b) so that the sample contacts a) before b).

[36] The method according to

[35] above, further comprising, between a) and b), a third region of the substrate comprising c) an agent that binds to IgA2.

[37] The method according to

[35] or

[36] above, comprising flowing the sample through the device, and simultaneously or subsequently flowing a detection reagent that binds to IgA through the device, and detecting the detection reagent that has bound to IgA.

[38] The method according to

[37] above, wherein the detection reagent is selected from one or more of anti-human IgA colloidal gold, anti-human IgA1 colloidal gold, and anti-human IgA2 colloidal gold.

[39] The method according to any one of [1] to

[38] above, wherein the subject is a mammal.

[40] The method according to

[39] above, wherein the mammal is a human.

[41] The method according to any one of the above-mentioned [1] to

[40] , wherein the subject has normal levels of alanine aminotransferase 1 (ALT-1) and / or alanine aminotransferase.

[42] The method according to any one of the above [1] to

[41] , wherein the method is used to monitor the progression of cirrhosis in the subject.

[43] A method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining the level of IgA2 in a biological sample from the subject, wherein an elevated level of IgA2 compared to a threshold indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject.

[44] The method according to

[43] above, wherein an IgA2 level above a threshold of 3500 DA indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject.

[45] A method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining the level of dIgA2 in a biological sample from the subject, wherein a level of dIgA2 different from a threshold value indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject.

[46] A method of treating intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising administering to the subject a treatment for intestinal barrier dysfunction and / or liver cirrhosis, wherein the subject has been determined to have intestinal barrier dysfunction and / or liver cirrhosis using a method described in any of [1] to

[45] or

[56] to

[57] above.

[47] 1. A kit for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, comprising: (i) an agent that binds to dIgA and forms a detectable dIgA complex; (ii) an agent that binds to mIgA and forms a detectable mIgA complex; A kit, wherein (i) specifically binds to dIgA and / or (ii) specifically binds to mIgA.

[48] The kit according to

[47] above, further comprising an agent that binds to and forms a detectable IgA2 complex.

[49] The kit according to

[47] or

[48] above, further comprising a reagent for detecting one or more of the mIgA complex, dIgA complex, and IgA2 complex.

[50] The kit according to

[49] above, wherein the reagent is anti-human IgA colloidal gold.

[51] The kit according to any one of the above

[47] to

[50] , wherein one or more of the agents is bound to a solid support.

[52] The kit according to any one of

[47] to

[51] above, wherein the kit comprises a strip, chip, or cartridge for use in a lateral flow assay.

[53] The kit according to any one of

[47] to

[52] above, wherein the kit comprises a strip, chip, or cartridge for use in a point-of-care device.

[54] 1. A test strip for a lateral flow device comprising at least one sample loading area, a) the strip comprises a capture portion comprising an agent that binds to dIgA; b) the strip comprises a capture portion comprising an agent that binds to mIgA; A test strip wherein a) is closer to the sample loading area than b) so that the sample contacts a) before b).

[55] 54. The test strip according to claim 54, wherein the strip further comprises, between a) and b), c) a capture portion comprising an agent that binds to IgA2.

[56] 1. A method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining dIgA and mIgA levels in a biological sample from the subject; If the mIgA level is elevated relative to a threshold, the dIgA level is compared to a dIgA threshold, and a difference from the dIgA threshold indicates intestinal barrier dysfunction and / or liver cirrhosis in the subject; If the mIgA level is decreased relative to a threshold, the ratio of the dIgA level to the mIgA level is determined and compared with a ratio threshold, and a difference from the ratio threshold indicates intestinal barrier dysfunction and / or cirrhosis in the subject.

Claims

1. 1. A point-of-care method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, comprising: (i) determining the levels of dIgA and mIgA and the ratio thereof in a biological sample from the subject and comparing the ratio to a threshold, the method comprising contacting the biological sample with a test strip containing an agent that binds to dIgA to form a detectable dIgA complex, followed by contacting the biological sample with an agent that binds to mIgA to form a detectable mIgA complex; or (ii) determining the levels of dIgA1 or dIgA2 and mIgA1 or mIgA2 in a biological sample from the subject, and a ratio thereof, and comparing the ratio with a threshold value, the method comprising the steps of contacting the biological sample with a test strip containing an agent that binds to dIgA1 or dIgA2 to form a detectable dIgA1 complex or dIgA2 complex, and subsequently contacting the biological sample with the agent that binds to mIgA1 or mIgA2 to form a detectable complex. The method.

2. 2. The method of claim 1, wherein a difference from the threshold indicates that the subject has intestinal barrier dysfunction and / or liver cirrhosis.

3. The method of claim 1 or 2, wherein if the dIgA to mIgA ratio or the dIgA1 to mIgA1 ratio or the dIgA2 to mIgA2 ratio is elevated compared to a threshold value, it indicates that the subject has intestinal barrier dysfunction and / or liver cirrhosis.

4. The method of any one of claims 1 to 3, wherein a decrease in the mIgA to dIgA ratio, or the mIgA1 to dIgA1 ratio, or the mIgA2 to dIgA2 ratio compared to a threshold value indicates that the subject has intestinal barrier dysfunction and / or liver cirrhosis.

5. The following characteristics (i) and (ii): (i) if the dIgA to mIgA ratio is equal to or greater than a threshold of 0.65, it indicates that the subject has intestinal barrier dysfunction and / or liver cirrhosis; (ii) if the ratio of dIgA to mIgA is equal to or greater than a threshold of 1, it indicates that the subject with HIV has intestinal barrier dysfunction and / or liver cirrhosis, or if the ratio of dIgA to mIgA is equal to or greater than a threshold of 0.5, it indicates that the subject with hepatitis B has intestinal barrier dysfunction and / or liver cirrhosis; The method of any one of claims 1 to 3, further comprising one or both of:

6. The following characteristics (i) to (v): (i) if the mIgA to dIgA ratio is below a threshold of 1.54, it indicates that the subject has intestinal barrier dysfunction and / or liver cirrhosis; (ii) when the mIgA to dIgA ratio is equal to or less than a threshold of 1, it indicates that the subject with HIV has intestinal barrier dysfunction and / or liver cirrhosis, or when the dIgA to mIgA ratio is equal to or less than a threshold of 2, it indicates that the subject with hepatitis B has intestinal barrier dysfunction and / or liver cirrhosis; (iii) the level of dIgA in the sample is determined by contacting the sample with a pIgR, an anti-dIgA antibody, or an anti-IgA J chain antibody to form a detectable dIgA complex, optionally wherein the pIgR is a chimeric secretory component; (iv) the level of mIgA in the sample is determined by contacting the sample with an anti-mIgA antibody to form a detectable mIgA complex; (v) the mIgA level is determined in a sample depleted of dIgA and IgA2 by contacting the sample with Protein L or an anti-IgA antibody to form a detectable mIgA complex; The method of any one of claims 1 to 5, further comprising one or more of:

7. the level of mIgA1 in the sample is determined by contacting the sample with an anti-mIgA1 antibody, Protein L, or an anti-IgA antibody to form a detectable mIgA complex; The method according to any one of claims 1 to 6.

8. When determining the level of IgA2 in said sample, (i) and (ii): (i) an elevated dIgA to mIgA ratio relative to threshold, a decreased mIgA to dIgA ratio relative to threshold, an elevated dIgAl to mIgAl ratio relative to threshold, a decreased mIgAl to dIgAl ratio relative to threshold, an elevated dIgA2 to mIgA2 ratio relative to threshold, a decreased mIgA2 to dIgA2 ratio relative to threshold, and (ii) elevated IgA2 levels relative to a threshold; The method of any one of claims 1 to 7, wherein one or both of the following indicates that the subject has intestinal barrier dysfunction and / or liver cirrhosis.

9. The method comprises: (i) contacting the sample with pIgR, anti-dIgA antibody, or anti-IgA J chain antibody to form a detectable dIgA complex, and subsequently contacting the sample with Protein L, anti-IgA antibody, or anti-mIgA antibody to form a detectable mIgA complex; or (ii) contacting the sample with pIgR, anti-dIgA antibody, or anti-IgA J chain antibody to form a detectable dIgA complex, followed by contacting the sample with an anti-IgA2 antibody to form a detectable IgA2 complex, followed by contacting the sample with Protein L, anti-IgA antibody, or anti-mIgA antibody to form a detectable mIgA complex. The method according to any one of claims 1 to 8, comprising:

10. The following characteristics (i) to (x): (i) the cirrhosis is the result of one or more of alcohol, NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis), viral hepatitis, HIV, unknown cause, primary biliary cirrhosis, and primary sclerosing cholangitis; (ii) the cirrhosis has a Child-Pugh classification of A, a Child-Pugh classification of B, or a Child-Pugh classification of C; (iii) has a diagnostic sensitivity of at least 80% for cirrhosis; (iv) has a diagnostic specificity of at least 85% for cirrhosis; (v) if the level of IgA2 has not been determined, the method further comprises: a) a diagnostic sensitivity of at least 54% for cirrhosis of the Child-Pugh classification of A; b) a diagnostic sensitivity of at least 69% for Child-Pugh cirrhosis of B; and c) a diagnostic sensitivity of at least 87% for cirrhosis of the Child-Pugh classification of C; (vi) if the level of IgA2 has been determined, the method further comprises: a) a diagnostic sensitivity of at least 72% for cirrhosis of the Child-Pugh classification of A; b) a diagnostic sensitivity of at least 76% for Child-Pugh cirrhosis of B; and c) a diagnostic sensitivity of at least 88% for cirrhosis of the Child-Pugh classification of C; (vii) For cirrhosis of the liver according to Child-Pugh classification A, the method a) a diagnostic sensitivity of at least 90% for alcoholic cirrhosis; b) a diagnostic sensitivity of at least 70% for cryptogenic cirrhosis; c) a diagnostic sensitivity of at least 40% for hepatitis B cirrhosis; d) a diagnostic sensitivity of at least 76% for hepatitis C cirrhosis; e) a diagnostic sensitivity of at least 90% for NASH cirrhosis; f) a diagnostic sensitivity of at least 90% for PBC; and g) a diagnostic sensitivity of at least 90% for PSC; (viii) the method comprises a chromatographic assay, an enzyme-linked immunosorbent assay, a fluorescent immunosorbent assay, a radioimmunosorbent assay, or a homogeneous assay; (ix) the method comprises a lateral flow format; (x) the biological sample is selected from whole blood, plasma, serum, or gingival crevicular fluid; The method of any one of claims 1 to 9, further comprising one or more of:

11. performed on a lateral flow device comprising a test strip containing at least one sample loading area; a) the strip comprises a capture portion comprising an agent that binds to dIgA; b) the strip comprises a capture portion comprising an agent that binds to mIgA; The method of any one of claims 1 to 10, wherein a) is closer to the sample loading area than b) so that the sample contacts a) before b).

12. The following (i) to (iii): (i) between a) and b), c) a third area of ​​the substrate comprising an agent that binds to IgA2; (ii) flowing the sample through the device and simultaneously or subsequently flowing a detection reagent that binds IgA through the device and detecting the detection reagent bound to IgA; (iii) the detection reagent is selected from one or more of anti-human IgA antibody colloidal gold, anti-human IgA1 antibody colloidal gold, and anti-human IgA2 antibody colloidal gold; The method of claim 11 , further comprising one or more of:

13. 1. A method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising: (i) determining the level of IgA2 in a biological sample from the subject, wherein if the level of IgA2 is elevated compared to a threshold, it indicates that the subject has intestinal barrier dysfunction and / or liver cirrhosis; or (ii) determining the level of dIgA2 in a biological sample from the subject, wherein a difference between the threshold and the level of dIgA2 indicates that the subject has intestinal barrier dysfunction and / or liver cirrhosis; method.

14. 1. A test strip for a point-of-care lateral flow device comprising at least one sample loading area, a) the strip comprises a capture portion comprising an agent that binds to dIgA; b) the strip comprises a capture portion comprising an agent that binds to mIgA; A test strip wherein a) is closer to the sample loading area than b) so that the sample contacts a) before b).

15. 1. A method for detecting intestinal barrier dysfunction and / or liver cirrhosis in a subject, the method comprising determining dIgA and mIgA levels in a biological sample from the subject; The method includes contacting the biological sample with an agent that binds dIgA to form a detectable dIgA complex, and subsequently contacting the biological sample with an agent that binds mIgA to form a detectable mIgA complex; If the mIgA level is elevated relative to a threshold, the dIgA level is compared to a dIgA threshold, and a difference from the dIgA threshold indicates that the subject has intestinal barrier dysfunction and / or liver cirrhosis; If the mIgA level is decreased relative to a threshold, the ratio of the dIgA level to the mIgA level is determined and compared with a ratio threshold, and if there is a difference from the ratio threshold, this indicates that the subject has intestinal barrier dysfunction and / or cirrhosis.

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