WO2025096715A1 - Compositions and methods for the detection and treatment of acute pancreatitis - Google Patents

Abstract

Disclosed are methods for identifying and/or treating an individual having, or likely to have, acute pancreatitis. In aspects, the methods comprise detecting Elastase 2A and/or 2B in a sample obtained from the individual, for example via detection of Elastase 2A and/or 2B protein and/or an mRNA encoding Elastase 2A and/or 2B. Further disclosed are kits for detecting Elastase 2A and/or 2B.

Description

COMPOSITIONS AND METHODS FOR THE DETECTION AND TREATMENT OF ACUTE PANCREATITIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international application that claims priority to and benefit of U.S. Provisional Application Serial No. 63/595,382, entitled Compositions and Methods for the Detection and Treatment of Acute Pancreatitis, filed November 2, 2023, the contents of which are incorporated in their entirety for all purposes.

REFERENCE TO SEQUENCE LISTING

[0002] A Sequence Listing submitted as an ST.26 standards compliant XML file via Patent Center is hereby incorporated by reference. The file name of the XML file for the Sequence Listing is CHMC_2023-1006_SL.xml, the date of the creation of the XML file is October 29, 2024, and the size of the XML file is 8,656 bytes.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0003] This invention was made with government support under DK118190 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0004] Acute pancreatitis (AP) is an inflammatory pancreatic disorder, with recent studies reporting increasing global incidence over time in pediatric and adult populations. Despite the burden and implications of inflammatory pancreatic disorders, the diagnosis of AP remains challenging. AP in children is an emerging problem, with few published data regarding diagnostic methods, and should be studied separately from adults. Serum lipase, a biomarker identified in the early 1900s, has limited specificity, yet it remains the standard molecular diagnostic for AP.3 Misdiagnosis of AP has major consequences, requiring patients to undergo invasive endoscopic interventions or surgeries that may not be warranted. Another unmet need in clinical practice is the absence of a reliable point-of-care test for AP that can be used in real time to facilitate timely management and tailored interventions for AP. BRIEF SUMMARY

[0005] Disclosed are methods for identifying and/or treating an individual having, or likely to have, acute pancreatitis. In aspects, the methods comprise detecting Elastase 2A and/or 2B in a sample obtained from the individual, for example via detection of Elastase 2A and/or 2B protein and/or an mRNA encoding Elastase 2A and/or 2B. Further disclosed are kits for detecting Elastase 2A and/or 2B.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0007] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0008] FIG. 1A-1E. FIG. 1A: Two experiment cohorts were selected to separate patients with AP from a variety of control individuals, including patients with CP, FRCs, and HCs. FIG. IB: A data-independent acquisition mass spectrometry strategy to capture the urine proteome that separates protein detection (of a sample pool) from protein quantification (of individual samples). Using this approach, more than 2000 proteins were detected from a detailed 6-injection proteomics analysis of a sample pool, forming a library for this experiment. With the library, quantified 1694 proteins were quantified with measured signals in every sample of at least one group. FIG. 1C: Volcano plot showing significant proteins (FDR, <0.05; pink) in the discovery cohort when comparing AP patients relative to other control individuals. Five proteins (CELA2A/B, AMY2A, REGIA, SBSN, and GDF15) in black were also significantly increased in every pairwise FDR- controlled test vs each individual control population, making them potential candidates for biomarker screening. CRP, a common inflammation marker, is also indicated in black. FIG. ID: Box plots representing fold changes for key AP-specific candidate protein biomarkers. Bold lines show median log2 intensities, and boxes indicate the interquartile range. Whiskers show 5% and 95% points, and dots indicate each individual measurement. Fold changes are indicated relative to the HC median. (E) Receiver operating characteristic curves for CELA2A/B and AMY2A for predicting AP diagnosis in 100 randomly selected training and testing pools. Bold lines indicate the median ensemble receiver operating characteristic curve, and shaded regions indicate the interquartile prediction range. AUC, area under the curve.

[0009] FIG. 2A-2B. FIG. 2A: Volcano plot showing pairwise significant proteins (FDR < 0.05, pink) in the discovery cohort when comparing AP patients relative to CP patients, HCs, and FRCs. FIG. 2B: Validation cohort results demonstrating differentiation between AP and control groups for elastase 2A, different amylase paralogs, and CRP protein expression. Bold lines show median log2 intensities, and boxes indicate the interquartile range. Whiskers show 5% and 95% points, and dots indicate each individual measurement. Fold changes are indicated relative to the HC median.

DETAILED DESCRIPTION

DEFINITIONS

[0010] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The methods may comprise, consist of, or consist essentially of the elements of the compositions and/or methods as described herein, as well as any additional or optional element described herein or otherwise useful in the diagnosis and/or treatment of acute pancreatitis.

[0011] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth. [0012] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0013] As used herein, the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0014] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some aspects, the terms refer to humans. In further aspects, the terms may refer to children.

Acute Pancreatitis

[0015] Acute pancreatitis (AP) is a common inflammatory disease of the exocrine pancreas. AP causes severe abdominal pain and multiple organ dysfunction and may lead to pancreatic necrosis and persistent organ failure. Overall global incidence of AP is 30-40 cases per 100,000 population per year. The global incidence is rising, although studies suggest rates are currently more stable in Asia. AP leads to significant short- and long-term morbidity, which in a significant minority causes prolonged debility, recurrent disease, and pancreatic exocrine and/or endocrine insufficiency. Quality of life can be significantly impaired as a result of chronic pain and the socio- economic consequences of prolonged hospitalization. Presentations of pancreatitis include epigastric or diffuse abdominal pain (80-95%), nausea and vomiting (40-80%), abdominal distension, fever, breathlessness, irritability, and impaired consciousness, with pyrexia, low oxygen saturation, tachypnoea, tachycardia, hypotension, abdominal guarding, ileus and/or oliguria. A diagnosis of acute pancreatitis requires two out of three criteria: (1) abdominal pain consistent with pancreatitis, (2) a serum amylase or lipase three or more times the upper limit of normal, and (3) findings consistent with pancreatitis on cross-sectional abdominal imaging. See, e.g., Szatmary, Peter et al. “Acute Pancreatitis: Diagnosis and Treatment.” Drugs vol. 82,12 (2022): 1251-1276. doi:10.1007/s40265-022-01766-4.

[0016] Disclosed are methods for identifying an individual having, or likely to have, acute pancreatis, in which Elastase 2A is detected in a sample obtained from the individual. In further aspects, disclosed are methods for identifying an individual having, or likely to have, acute pancreatis, in which Elastase 2B is detected in a sample obtained from the individual. In further aspects, disclosed are methods for identifying an individual having, or likely to have, acute pancreatis, in which both Elastase 2A and Elastase 2A is detected in a sample obtained from the individual.

[0017] In further aspects, disclosed are methods of treating an individual having, or likely to have, acute pancreatis, in which Elastase 2A is detected in a sample obtained from the individual, wherein, based on this detection, a treatment for AP is administered to the individual. In further aspects, disclosed are methods for treating an individual having, or likely to have, acute pancreatis, in which Elastase 2B is detected in a sample obtained from the individual, wherein, based on this detection, a treatment for AP is administered to the individual. In further aspects, disclosed are methods for treating an individual having, or likely to have, acute pancreatis, in which both Elastase 2A and Elastase 2A is detected in a sample obtained from the individual, wherein, based on this detection, a treatment for AP is administered to the individual.

[0018] In further aspects, kits for the detection of one or both of Elastase 2 A and Elastase 2B are disclosed, the kits being useful for the diagnosis and/or treatment of AP in an individual in need thereof. In aspects, the individual is a pediatric patient. In aspects, the individual is an adult patient. In aspects, the individual is an individual presenting with one symptom of pancreatitis, for example at least one of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever. In aspects, the individual is an individual presenting with two symptoms of pancreatitis, for example at least one of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever. In aspects, the individual is suspected of having acute pancreatitis. In aspects, the individual is an individual presenting with three symptoms of pancreatitis, for example at least one of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.

Sample Preparation

[0019] In aspects, the biological sample for detection of the biomarker may be selected from blood, urine, saliva, cerebrospinal fluid, tissue biopsies, swabs (nasal, throat, or buccal), plasma, serum, and combinations thereof. In aspects, the biological sample is urine. In aspects, the biological sample is previously frozen urine. In aspects, the biological sample is freshly obtained (e.g., never frozen, and/or obtained within an hour of testing) at the time of subjecting the sample to the disclosed methods.

[0020] Methods of preparing biological samples for detection of a biomarker protein and/or nucleic acid are known, and the sample obtained from the individual can be subjected to one or more processes prior to detection of the biomarker. Exemplary methods include, for example: centrifugation to separate plasma or scrum from blood; filtration or centrifugation to remove particulates from urine; collection and possible dilution or stabilization of saliva samples using specialized devices; filtration of cerebrospinal fluid collected via lumbar puncture; homogenization or lysis of tissue biopsies to release cellular contents; elution of swabs in buffer solutions; and treatments such as dilution, filtration, or enzymatic digestion for other bodily fluids. In aspects, the sample is a urine sample, and is not subjected to any processes prior to detection of the biomarker.

Elastase 2A and Elastase 2B

[0021] The disclosed methods employ the detection of one or both of Elastase 2A and Elastase 2B for the determination of whether an individual has, or is likely to have acute pancreatitis (AP). Elastase 2A and 2B are a serine proteases secreted by the pancreas. Enzymatically, serine proteases digest other proteins, where Elastase 2A and 2B have chymotrypsin-like activity, cleaving proteins at hydrophobic amino acids. The proteins arc often referenced by their full names: chymotrypsin- like elastase family member 2A and 2B, or their gene names: CELA2A and CELA2B. From a sequence perspective, Elastase 2A and 2B are nearly identical and diverge by only a few amino acids (88.1% identity).

[0022] The canonical protein sequence for CEL A2A before the signal peptide is removed is:

[0023] MIRTLLLSTLVAGALSCGDPTYPPYVTRVVGGEEARPNSWPWQVSLQYSSNG KWYHTCGGSLIANSWVLTAAHCISSSRTYRVGLGRHNLYVAESGSLAVSVSKIVVHKD WNSNQISKGNDIALLKLANPVSLTDKIQLACLPPAGTILPNNYPCYVTGWGRLQTNGAV PDVLQQGRLLVVDYATCSSSAWWGSSVKTSMICAGGDGVISSCNGDSGGPLNCQASDG RWQVHGIVSFGSRLGCNYYHKPSVFTRVSNYIDWINSVIANN. (SEQ ID NO: 1, CELA2A + Signal Sequence). The signal peptide for CELA2A is MIRTLLLSTLVAGALS (SEQ ID NO: 2) (l-16aa) and it has a propeptide CGDPTYPPYVTR (SEQ ID NO: 3) (17-28aa). The complete function of the propeptide is not clear'. The active protein is 29-269aa.

[0024] The canonical protein sequence for CELA2B before the signal peptide is removed is:

[0025] MIRTLLLSTLVAGALSCGVSTYAPDMSRMLGGEEARPNSWPWQVSLQYSSN GQWYHTCGGSLIANSWVLTAAHCISSSGIYRVMLGQHNLYVAESGSLAVSVSKIVVHK DWNSDQVSKGNDIALLKLANPVSLTDKIQLACLPPAGTILPNNYPCYVTGWGRLQTNG ALPDDLKQGQLLVVDYATCSSSGWWGSTVKTNMICAGGDGVICTCNGDSGGPLNCQA SDGRWEVHGIGSLTSVLGCNYYYKPSIFTRVSNYNDWINSVIANN. (SEQ ID NO: 4, CELA2B + Signal Sequence) The signal peptide for CEL2B is MIRTLLLSTLVAGALS (SEQ ID NO: 2) (l-16aa).

[0026] The cDNA sequence for CELA2A is:

[0027] ACAGAACTCCCACGGACACACCATGATAAGGACGCTGCTGCTGTCCACTT TGGTGGCTGGAGCCCTCAGTTGTGGGGACCCCACTTACCCACCTTATGTGACTAGGG TGGTTGGCGGTGAAGAAGCGAGGCCCAACAGCTGGCCCTGGCAGGTCTCCCTGCAG TACAGCTCCAATGGCAAGTGGTACCACACCTGCGGAGGGTCCCTGATAGCCAACAG CTGGGTCCTGACGGCTGCCCACTGCATCAGCTCCTCCAGGACCTACCGCGTGGGGCT GGGCCGGCACAACCTCTACGTTGCGGAGTCCGGCTCGCTGGCAGTCAGTGTCTCTAA

GATTGTGGTGCACAAGGACTGGAACTCCAACCAAATCTCCAAAGGGAACGACATTG

CCCTGCTCAAACTGGCTAACCCCGTCTCCCTCACCGACAAGATCCAGCTGGCCTGCC

TCCCTCCTGCCGGCACCATTCTACCCAACAACTACCCCTGCTACGTCACGGGCTGGG

GAAGGCTGCAGACCAACGGGGCTGTTCCTGATGTCCTGCAGCAGGGCCGGTTGCTG

GTTGTGGACTATGCCACCTGCTCCAGCTCTGCCTGGTGGGGCAGCAGCGTGAAAACC

AGTATGATCTGTGCTGGGGGTGATGGCGTGATCTCCAGCTGCAACGGAGACTCTGGC

GGGCCACTGAACTGTCAGGCGTCTGACGGCCGGTGGCAGGTGCACGGCATCGTCAG

CTTCGGGTCTCGCCTCGGCTGCAACTACTACCACAAGCCCTCCGTCTTCACGCGGGT

CTCCAATTACATCGACTGGATCAATTCGGTGATTGCAAATAACTAACCAAAAGAAGT

CCCTGGGACTGTTTCAGACTTGGAAAGGTCACAGAAGGAAAATAATATAATAAAGT

GACAACTATGCAAATCA. (SEQ ID NO: 5, CEL A2A cDNA)

[00281 The cDNA sequence for CELA2B is:

[0029] ACAGAACTCCCACGGACACACCATGATTAGGACCCTGCTGCTGTCCACTT

TGGTGGCTGGAGCCCTCAGTTGTGGGGTCTCCACTTACGCGCCTGATATGTCTAGGA

TGCTTGGAGGTGAAGAAGCGAGGCCCAACAGCTGGCCCTGGCAGGTCTCCCTGCAG

TACAGCTCCAATGGCCAGTGGTACCACACCTGCGGAGGGTCCCTGATAGCCAACAG

CTGGGTCCTGACGGCTGCCCACTGCATCAGCTCCTCCGGGATCTACCGCGTGATGCT

GGGCCAGCATAACCTCTACGTTGCAGAGTCCGGCTCGCTGGCCGTCAGTGTCTCTAA

GATTGTGGTGCACAAGGACTGGAACTCCGACCAGGTCTCCAAAGGGAACGACATTG

CCCTGCTCAAACTGGCTAACCCCGTCTCCCTCACCGACAAGATCCAGCTGGCCTGCC

TCCCTCCTGCCGGCACCATTCTACCCAACAACTACCCCTGCTACGTCACGGGCTGGG

GAAGGCTGCAGACCAACGGGGCTCTCCCTGATGACCTGAAGCAGGGCCAGTTGCTG

GTTGTGGACTATGCCACCTGCTCCAGCTCTGGCTGGTGGGGCAGCACCGTGAAGACG

AATATGATCTGTGCTGGGGGTGATGGCGTGATATGCACCTGCAACGGAGACTCCGGT

GGGCCGCTGAACTGTCAGGCATCTGACGGCCGGTGGGAGGTGCATGGCATCGGCAG

CCTCACGTCGGTCCTTGGTTGCAACTACTACTACAAGCCCTCCATCTTCACGCGGGT

CTCCAACTACAACGACTGGATCAATTCGGTGATTGCAAATAACTAACCAAAAGAAG

TCCCTGGGACTGTTTCAGACTTGGAAAGGTCACAGAAGGAAAATAATATTATATAA

AGTGACAACTATGCAAA. (SEQ ID NO: 6, CELA2B cDNA) [0030] In aspects, the method comprises detecting Elastase 2A and/or 2B in a sample obtained from the individual. In aspects, the detecting comprises detecting at least a portion of Elastase 2A. In aspects, the detecting comprises detecting at least a portion of Elastase 2B. In aspects, the detecting comprises detecting a conserved region of Elastase 2A. In aspects, the detecting comprises detecting a conserved region of Elastase 2B. In aspects, the detecting comprises detecting a region of SEQ ID NO: 1. In aspects, the detecting comprises detecting a region of SEQ ID NO: 4. In aspects, the detecting comprises detecting a region of SEQ ID NO: 5. In aspects, the detecting comprises detecting a region of SEQ ID NO: 6. In aspects, the detection of SEQ ID NO: 1 comprises detection of a region that is not the signal sequence. In aspects, the detection of SEQ ID NO: 4 comprises detection of a region that is not the signal sequence.

Methods of detecting biomarker

[0031] The disclosed methods comprise the step of detecting one or both of Elastase 2A and Elastase 2B in a sample. In aspects, the method comprises detecting one or both of Elastase 2A and Elastase 2B protein. In aspects, the method comprises detecting one or both of Elastase 2A and Elastase 2B expression levels, comprising detecting an mRNA level of one or both of Elastase 2A and Elastase 2B. Various methods are known in the art for detection of protein and/or mRNA, as described below.

Protein Detection Assays

[0032] Enzyme-Linked Immunosorbent Assay (ELISA). In aspects, the disclosed biomarker may be detected via Enzyme-Linked Immunosorbent Assay (ELISA). ELISA methods are generally known in the art. In brief, an antibody is used to bind the biomarker of interest. A biological sample, such as blood, urine, cell lysate, or serum, is introduced to a surface coated with a capture antibody specific to the target biomarker. After binding, a secondary antibody conjugated to an enzyme, such as, for example, horseradish peroxidase (HRP), is added. Upon substrate introduction, the enzyme catalyzes a reaction that produces a detectable signal, typically colorimetric or chemiluminescent, which correlates with the biomarker's concentration in the sample. In aspects, the ELISA is used for quantitative detection. In further aspects, the ELISA is used for qualitative detection. [0033] Western Blot. In aspects, the disclosed biomarker may be detected via Western blot. Western blot methods arc generally known in the art. In brief, proteins in a biological sample, such as blood or tissue lysate, are separated based on molecular weight using gel electrophoresis and then transferred onto a membrane, such as nitrocellulose or PVDF. The membrane is subsequently probed with a primary antibody specific to the target biomarker. After binding, a secondary antibody conjugated to an enzyme, such as, for example, horseradish peroxidase (HRP), is applied. Upon the addition of a suitable substrate, the enzyme catalyzes a reaction that produces a detectable signal, typically chemiluminescent, which correlates with the presence of the biomarker on the membrane. In aspects, Western blot is used for both qualitative detection and semi-quantitative analysis of the biomarker.

[0034] Immunohistochemistry (IHC). In aspects, the disclosed biomarker may be detected via Immunohistochemistry (IHC). IHC methods are generally known in the art. In brief, IHC involves the detection of a biomarker directly within tissue sections or cell samples. The sample is first fixed and then exposed to a primary antibody specific to the target biomarker. Following this, a secondary antibody conjugated to an enzyme, such as, for example, horseradish peroxidase (HRP), or a fluorescent tag is introduced. Upon addition of a suitable substrate, the enzyme catalyzes a reaction that generates a colorimetric signal, or the fluorescence is detected, indicating the presence and localization of the biomarker within the sample. In aspects, IHC provides both qualitative detection and spatial localization of the biomarker in tissues.

[0035] Mass Spectrometry (MS). In aspects, the disclosed biomarker may be detected via Mass Spectrometry (MS). MS methods are generally known in the art. In brief, proteins from a biological sample, such as blood or tissue lysate, are first digested into smaller peptides using an enzyme like trypsin. The resulting peptides are then ionized and introduced into a mass spectrometer, where they are separated based on their mass-to-charge (m/z) ratio. The mass spectrometer detects these ionized peptides, generating a mass spectrum that allows for the identification and quantification of the biomarker based on its unique peptide profile. In aspects, MS provides highly sensitive and accurate quantitative detection of the biomarker, as well as detailed structural information about the protein. [0036] Surface Plasmon Resonance (SPR). In aspects, the disclosed biomarker may be detected via Surface Plasmon Resonance (SPR). SPR methods arc generally known in the art. In brief, SPR detects biomolecular interactions in real time without the need for labeling. A biological sample containing the biomarker of interest is passed over a sensor surface coated with a capture molecule, such as an antibody specific to the biomarker. Binding of the biomarker to the capture molecule causes a change in the refractive index at the sensor surface, which is detected as a shift in the resonance angle of the surface plasmon wave. This shift is directly proportional to the amount of biomarker bound, allowing for both qualitative and quantitative analysis. In aspects, SPR provides sensitive and real-time detection of the biomarker, enabling the measurement of binding kinetics and concentration.

[0037] Flow Cytometry. In aspects, the disclosed biomarker may be detected via Flow Cytometry. Flow cytometry methods are generally known in the art. In brief, cells from a biological sample, such as blood or tissue, are first labeled with a fluorescently tagged antibody specific to the target biomarker. The labeled cells are then passed through a laser beam within a flow cytometer, where individual cells are analyzed based on their fluorescence intensity, which corresponds to the amount of biomarker present on or within each cell. The flow cytometer collects data on various parameters, including cell size, complexity, and fluorescence, allowing for both qualitative detection and quantitative analysis of biomarker expression across a large number of cells. In aspects, flow cytometry provides high-throughput and multiparametric detection of the biomarker in a heterogeneous cell population. Luminex Assay: mRNA Detection Assays

[0038] Reverse Transcription Quantitative PCR (RT-qPCR). In aspects, the disclosed biomarker may be detected via Reverse Transcription Quantitative PCR (RT-qPCR). RT-qPCR methods are generally known in the art. In brief, RNA from a biological sample, such as blood or tissue, is first reverse-transcribed into complementary DNA (cDNA) using reverse transcriptase. The resulting cDNA is then amplified using quantitative PCR, where specific primers targeting the biomarker sequence are used. During amplification, fluorescent dyes or probes, such as SYBR Green or TaqMan probes, bind to the DNA, and the fluorescence intensity is measured in real time. The amount of fluorescence correlates with the amount of the biomarker's mRNA originally present in the sample. In aspects, RT-qPCR provides sensitive, specific, and quantitative detection of biomarkcr mRNA expression.

[0039] Northern Blotting. In aspects, the disclosed biomarker may be detected via Northern Blotting. Northern blotting methods are generally known in the art. In brief, RNA is extracted from a biological sample and separated by size using gel electrophoresis. The RNA is then transferred onto a membrane, such as nitrocellulose or nylon, where it is hybridized with a labeled probe that is complementary to the target biomarker’s mRNA. Detection of the labeled probe, typically via radioactive or chemiluminescent methods, allows for the visualization and quantification of the biomarker’s mRNA. In aspects, Northern blotting provides both qualitative and semi-quantitative detection of specific mRNA transcripts.

[0040] In Situ Hybridization (ISH). In aspects, the disclosed biomarker may be detected via In Situ Hybridization (ISH). ISH methods are generally known in the art. In brief, ISH involves the use of a labeled probe that binds to the target biomarker’s mRNA directly within a fixed tissue or cell sample. The labeled probe, which is complementary to the specific mRNA of interest, hybridizes to the target within the tissue, allowing for spatial visualization of biomarker expression. Detection is achieved through either fluorescent (FISH) or colorimetric signals. In aspects, ISH provides both qualitative detection and precise localization of mRNA expression within tissue samples.

[0041] RNA-Seq (Next-Generation Sequencing). In aspects, the disclosed biomarker may be detected via RNA-Seq (Next-Generation Sequencing). RNA-Seq methods are generally known in the art. In brief, RNA is isolated from a biological sample and converted into complementary DNA (cDNA) through reverse transcription. The cDNA is then subjected to high-throughput sequencing, where millions of short reads are generated and aligned to a reference genome. This allows for the quantification of the biomarker’s mRNA expression levels across the entire transcriptome. In aspects, RNA-Seq provides quantitative detection of mRNA with high sensitivity and can also reveal alternative splicing, novel transcripts, and other RNA modifications.

[0042] Microarray. In aspects, the disclosed biomarker may be detected via Microarray analysis. Microarray methods are generally known in the art. In brief, RNA from a biological sample is converted into labeled complementary DNA (cDNA), which is then hybridized to a microarray chip containing thousands of probes corresponding to specific genes, including the target biomarkcr. The level of hybridization at each probe site generates a signal that can be quantified to determine the relative expression levels of the biomarker’s mRNA. In aspects, microarray technology allows for the simultaneous detection and quantification of the biomarker along with other mRNAs, providing high-throughput analysis of gene expression.

[0043] NanoString nCounter. In aspects, the disclosed biomarker may be detected via the NanoString nCounter system. NanoString nCounter methods are generally known in the art. In brief, RNA from a biological sample is hybridized with specific color-coded probes complementary to the target biomarker’s mRNA. These probes are directly counted using a digital detection system without the need for amplification. The resulting counts reflect the absolute quantity of the biomarker’s mRNA in the sample. In aspects, the NanoString nCounter system provides highly multiplexed, quantitative detection of the biomarker and other targets in a single assay, offering a balance of sensitivity and simplicity.

[0044] Digital Droplet PCR (ddPCR). In aspects, the disclosed biomarker may be detected via Digital Droplet PCR (ddPCR). ddPCR methods are generally known in the art. In brief, RNA is first reverse-transcribed into complementary DNA (cDNA), which is then partitioned into thousands of droplets. Each droplet undergoes PCR amplification individually, with some droplets containing the target biomarker’s DNA and others not. Fluorescent probes are used to detect amplification within the droplets, and the number of positive droplets is counted to determine the absolute quantity of the biomarker’s mRNA. In aspects, ddPCR provides highly sensitive, precise, and absolute quantification of mRNA, even in low-abundance samples.

[0045] Branched DNA (bDNA) Assay. In aspects, the disclosed biomarker may be detected via the Branched DNA (bDNA) Assay. bDNA methods are generally known in the art. In brief, RNA from a biological sample is hybridized with specific probes that bind to the target biomarker’s mRNA. Signal amplification is achieved through the addition of secondary "branched" DNA probes that further bind to the primary probes, enhancing the signal without the need for RNA amplification. The signal is typically detected using chemiluminescence, with the intensity correlating to the biomarker’s mRNA concentration. In aspects, the bDNA assay allows for quantitative detection of mRNA with high sensitivity and minimal background noise. Kits

[0046] In aspects, a kit for the detection of the disclosed biomarker is provided. The kit may comprise any of the aforementioned detection methods, such as ELISA and/or a lateral flow assay. In aspects, the kit is a home-based assay. In further aspects, the kit is for self-administration.

[0047] In aspects, the kit for detection of the biomarker comprises a sample receiving region and a capture surface comprising an immobilized detection agent specific for the biomarker. Exemplary detection agents include an antibody or binding fragment thereof specific for the biomarker (as in the case of a protein), or a nucleotide sequence specific to the biomarker (as in the case of mRNA). The detection agent can be an antibody, aptamer, or other molecules with high affinity and specificity for the biomarker. The sample receiving region is designed to accommodate and direct a biological sample towards the capture surface, he sample receiving region may also be configured to handle various types of biological samples, such as that of blood, urine, saliva, tissue extracts, and combinations thereof. This region may include features such as wells, channels, or pads to ensure efficient application and distribution of the sample.

[0048] Upon application of the sample to the sample receiving region, the sample is directed to the capture surface where the biomarker, if present, binds to the immobilized detection agent. This binding event generates a detectable signal, which can be measured using various detection methods. For example, a colorimetric assay may produce a visible color change, while other assays may use fluorescence or electrochemical signals to indicate the presence of the biomarker.

[0049] In aspects, the kit provides a rapid result, the detection of the biomarker occurring in less than 1 hour, or less than 30 minutes, or less than 15 minutes, or less than 10 minutes, or less than 5 minutes. In aspects, the kit further comprises one or more components selected from buffers, reagents, control samples, and combinations thereof.

[0050] In aspects, the kit may be used to carry out one or more of the disclosed methods. For example, in aspects, the method comprises detecting one or both of Elastase Al and Elastase A2, in a biological sample obtained from an individual. In aspects, the biological sample is a urine sample that is contacted with a receiving region of the kit. [0051] In aspects, the kit may be configured for home use. In aspects, the kit may be configured for use in a physician’s office or hospital. The kit may contain a solution or buffer for stabilizing and preserving the biological sample upon collection, such as a saline solution or phosphate- buffered solution. Collection tools, such as swabs, lancets, or needles, may be included for obtaining different types of samples, including saliva, blood, or tissue. The kit may further comprise one or more collection receptacles, such as sterile tubes, vials, or microtiter plates. In aspects, the collection receptacle may comprise labels. The kits may further comprises a collection receptacle designed to be securely sealed for transport. In aspects, absorbent pads or wipes may be provided for cleaning a collection site prior to sample retrieval.

[0052] In aspects, the kit may also include detailed written instructions or visual guides to ensure proper sample collection and handling, which can be in the form of printed materials or accessible through a QR code linking to instructional videos. The kit may further include reagents necessary for biomarker detection, such as antibodies, primers, or enzyme-based solutions for quantitative assays. Additionally, a small portable reader or device may be included for processing or analyzing the sample, providing immediate feedback on biomarker levels or preliminary classifications.

[0053] For at-home use, the kit may also include pre-labeled shipping materials for sending the collected sample to a laboratory for further analysis, including tamper-proof seals to ensure the integrity of the sample during transport. Additional components may include gloves, alcohol wipes, and biohazard bags for safe handling and disposal of materials. In some embodiments, software access or a mobile application may be provided, allowing users or healthcare professionals to input the results and receive an algorithm-generated classification for guiding further treatment decisions.

Diagnosis of AP

[0054] Based on the detection of one or both of Elastase Al and Elastase A2, a determination of AP risk or a diagnosis of AP is made. For example, an individual may be diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B is at least two times that of a normal control (e.g. an individual who does not have acute pancreatitis). In aspects, an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 5 times that of a normal control. In aspects, an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 10 times that of a normal control. In aspects, an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 20 times that of a normal control. In aspects, an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 30 times that of a normal control. In aspects, an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 40 times that of a normal control. In aspects, an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 50 times that of a normal control. In aspects, an individual is diagnosed with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 60 times that of a normal control.

[0055] In aspects, following detection of one or both biomarkers Elastase Al and Elastase A2 and determination of a level of the biomarker in the sample, values can be optionally normalized to a control value (such as a baseline or reference standard). In aspects, an algorithm is applied to the normalized levels, wherein the algorithm compares the levels to a reference baseline associated with a predefined population or condition. The algorithm may be used to provide a score, such as a a numerical or categorical value, which is indicative of the individual's classification as having AP, likely to have AP, or not having AP, or unlikely to have AP. This classification can be used to determine the subsequent therapeutic regimen for administration to the individual. Additional steps may include preprocessing of the biological sample to remove contaminants or interfering substances, amplification or enrichment of the biomarker signals to enhance detection sensitivity, and validation of the results using a secondary assay or independent biomarkers to confirm the reliability of the classification. Furthermore, the method may involve adjusting the algorithm based on demographic factors, such as age, gender, or disease severity, to improve the accuracy of the classification and the corresponding treatment recommendation.

[0056] In further aspects, the method may be used to, in whole or in pail, exclude a diagnosis of one or more of chronic pancreatis, appendicitis, gastrointestinal infection, and combinations thereof. Administering a Therapy

[0057] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is treated via one or more of the following: fluid resuscitation, pain control, nutritional support, antibiotic use, insulin/plasmapheresis (for hypertriglyceridemia induced AP), low-molecular weight heparan (LMWH), protease inhibitors, and endoscopic retrograde cholangiopancreatography (ERCP).

[0058] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered additional monitoring for development of acute pancreatitis.

[0059] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered fluids, for example IV fluid and/or lactated Ringer’s is administered until electrolyte and fluid balance is restored. In further aspects, the individual may be administered a moderate fluid resuscitation (10 ml/kg bolus in case of hypovolemia, followed by 1.5 ml/kg/h) using Ringers lactate.

[0060] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered electrolytes.

[0061] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered a pre-biotic and/or a probiotic. In aspects, the individual is administered Bifilac and/or Bacillus subtilis and Enterococcus faecium. In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered micro-encapsulated tributyrin (a butyrate prodrug).

[0062] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered an analgesic. In aspects, the analgesic is selected form an opioid, a nonsteroidal anti-inflammatory drug (NSAID), acetaminophen, an epidural, and combinations thereof. In aspects, a combination of acetaminophen, metamizole and opiate is administered.

[0063] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered a feeding tube. [0064] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered an antibiotic. In aspects, the antibiotic is carbapcmcns.

[0065] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered a surgery. In aspects, the surgery is a cholecystectomy.

[0066] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered insulin/plasmapheresis (for hypertriglyceridemia induced AP).

[0067] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered low-molecular weight heparan (LMWH).

[0068] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered a protease inhibitor.

[0069] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered Auxora, an Orai Ca2+ channel inhibitor (in a phase lib trial in the USA (CARPO, NCT04681066 and NCT04195347).

[0070] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered the biologic infliximab in the UK (RAPID-I, NCT03684278)

[0071] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered the peripherally acting p-opioid receptor antagonist methylnaltrexone, which counteracts inhibitory effects of opiates on gut function and immune responses, without affecting analgesia.

[0072] In aspects, the individual is diagnosed as having, or likely to have, acute pancreatitis and is administered an endoscopic cholangiopancreatography (ERCP). ERCP combines upper gastrointestinal endoscopy and x-rays to treat narrowing or blockage of a bile or pancreatic duct.

[0073] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that arc disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

[0074] A prospective cohort consisting of children (aged 0-21 years) presenting with Acute Pancreatitis (AP) compared to control individuals was used. A summary of the overall study design and steps is shown in FIG. 1A. A total of 130 patients were included in the discovery cohort and 29 in the validation cohort. Discovery urine samples were obtained from 28 patients with AP, 50 with chronic pancreatitis (CP), 21 healthy control individuals (HCs), and 31 fracture pain control individuals (FRCs). The validation cohort included 12 patients with AP, 7 patients with CP, and 10 HCs. All AP patients had lipase or amylase elevation of >3 times the upper limit of normal and either presented with abdominal pain or met criteria for imaging findings. The severity of AP was classified using the North American Society for Pediatric Gastroenterology, Hepatology & Nutrition and the International Study Group of Pediatric Pancreatitis: In Search for a Cure criteria. Of the 40 patients with AP, 33 had a mild AP episode, 16 had abdominal imaging performed at the time of presentation, and 17 had imaging later in the course. The most common etiology for AP was idiopathic (36.8%), followed by gallstones (26.3%). CP diagnosis was made using the International Study Group of Pediatric Pancreatitis: In Search for a Cure criteria.

[0075] Proteomic analysis showed altered levels of urine proteins in AP patients compared to CP patients and control individuals. Briefly, global quantitative proteomics was performed on the discovery cohort urine specimens (FIG. IB). From these, 2137 proteins were identified in a urine chromatogram library and 1694 proteins were quantified across all samples. 105 differential proteins were significantly associated with AP (false discovery rate [FDR], <0.05) with both global analysis of variance testing (FIG. 1C) and pairwise AP vs individual control t tests (patients with CP, HCs, FRCs) (FIG. 2A). Although many consistently significant proteins were downregulated in AP, five proteins showed promise as potential biomarkers: CELA2A/B, AMY2A, REGIA, SBSN, and GDF15 (FIG. ID). AMY2A, a specific isoform of amylase (2A), was consistently up- regulated in AP. Serum lipase was not observed in any urine sample subgroup. CELA2A (elastase 2A), a known active pancreas-derived proteinase, demonstrated a 61-fold increase in the discovery cohort. Elastase 2A and 2B are highly similar gene paralogs, and the mass spectrometry experiments used here cannot differentiate between these putative proteins. However, because elastase 2B has never been detected at the protein level, elastase 2A is referred to as the putative biomarker. Using machine learning to combine elastase 2 A with the conventional biomarker amylase 2A dramatically increased the accuracy of AP diagnosis (FIG. IE) with an area under the curve of 0.91 (±0.03) compared to amylase 2A alone (0.82 ± 0.04). Other combinations including CRP, REGIA, SBSN, and GDF15 did not improve diagnostic accuracy. In a separate validation cohort of 29 patients, elastase 2A continued to outperform amylase 2A and CRP (62-fold increase relative to control groups) (Supplementary FIG. IB). In this analysis, signals from the amylase 2A paralog from amylase IB and 2B could be separated; it was observed that only elevated amylase 2A and 2B indicate AP.

[0076] The AP biomarkers serum amylase and lipase without imaging have poor specificity, with limited application in children. Obtaining imaging as pail of the diagnosis for every AP episode is not always feasible or timely and contributes to high health care costs. To Applicant’s knowledge, this study is the first to investigate urine proteomics in children with AP to identify novel biomarkers that improve diagnosis accuracy. Here, urine elastase 2A was identified as a promising diagnostic marker that can potentially be used as an accurate AP point-of-care test. The study shows a promising role for urinary markers in the diagnosis of AP in children through urinary noninvasive testing for timely and accurate diagnosis.

[0077] Sample Preparation

[0078] Voided urine samples were collected in a sterile urine collection cup (Coviden Precision), aliquoted in 1-1.5-mL amounts and saved in a -80°C freezer until ready for analysis. Urine samples were thawed, and a 400-mL aliquot was used for acetone protein precipitation. For this, 1600 mF of -20°C acetone was added, following by vortexing and an overnight incubation at -20°C. Samples were then centrifuged at 20,000g for 20 minutes to pellet proteins, after which the acetone was carefully removed. The pellet was washed twice with 500 mF of -20°C acetone following a second centrifugation at 20,000g for 20 minutes for each wash. Pellets were air dried for at least 15 minutes and then resuspended in 50-200 mF of 5% sodium dodecyl sulfate and 50 mmol/E triethylammonium bicarbonate buffer. [0079] Proteins were then reduced with 20 mmol/L dithiothreitol for 15 minutes at 65°C and alkylated with 40 mmol/L iodoacctamidc in the dark for 30 minutes at room temperature. Reduced and alkylated proteins were acidified with 1.2% phosphoric acid and mixed with 7x S-trap binding buffer made of 90% methanol and 100 mmol/L triethylammonium bicarbonate. Proteins were loaded onto a ProtiFi Mini S-Trap, washed with 3x 200-mL binding buffer, and then digested overnight at 37°C with a 1:50 ratio of trypsin to substrate. Peptides were eluted according to the manufacturer’s instructions using a mixture of acetonitrile, water, and formic acid. Finally, peptides were dried down using a speed vacuum concentrator.

[0080] Proteomics Data Acquisition

[0081] Peptides were separated with a Thermo Scientific Ulti-Mate 3000 UHPLC using a 2- cm PepMap C18 trap column followed by a 25-cm PepMap EASY-Spray C18 analytical column and emitted into a Thermo Scientific Fusion tandem mass spectrometer. Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in 80% acetonitrile. For each injection, approximately 1 mg was loaded and eluted using a 90-minute gradient from 2% to 35% solvent B, followed by a 30-minute washing gradient. The Thermo Scientific Fusion was configured to acquire mass spectrometry data using data-independent acquisition (DIA) using 38 xl6-m/z-wide windows in a staggered window pattern. DIA windows were placed from 392.43 to 1008.70 m/z using EncyclopeDIA (version 1.12.31 ) with forbidden-zone-optimized window placements and acquired as targeted fragment mass spectra (MS2) using 17,500 resolution and an automatic gain control (AGC) target of 4e5. Selected ion monitoring precursor spectra were placed every 38 scans (1 per cycle) from 390 to 1010 m/z using 60,000 resolution and an AGC target of 4e5.

[0082] In addition, a pool of representative peptides from each sample group was made from sample subaliquots and used for library generation with gas-phase fractionated (GPF) DIA. Following the chromatogram library approach, 6 injections of the pooled peptide sample were performed focused on different m/z regions (ie, 396.43-502.48, 496.48-602.52, 596.52-702.57, 696.57-802.61, 796.61-902.66, and 896.6-1002.70 m/z). Measurements were performed as for normal DIA as described, except MS2 spectra were configured using only 4-m/z-wide staggered windows.

[0083] Global Cohort Data Analysis [0084] Raw files were demultiplexed to deconvolute staggered windows with 1 O-parts-per- million accuracy after peak picking using MSConvcrt in the Protcowizard package (version 3.0.20169). All raw data searches were performed using EncyclopeDIA (version 1.12.31), which was configured to use default settings: 10-parts-per-million precursor, fragment, and library tolerances. EncyclopeDIA was allowed to consider both B and Y ions, and trypsin digestion was assumed. Searches of GPF-DIA injections were performed against a Prosit predicted spectrum library to generate a chromatogram library. To be included in the predicted library, peptides in a downloaded Homo sapiens Uniprot FASTA database (April 25, 2019; 20,415 entries) were required to have either +2H or +3H charge states and fall within 396.43 and 1002.70 precursor m/z, considering up to 1 missed tryptic cleavage. Peptides that were detected in the 6 GPF-DIA injections at a 1% peptide level FDR as judged by Percolator (version 3.01) were compiled into the chromatogram library using instrument-specific fragmentation and retention time values. Searches of the quantitative DIA injections were performed using this chromatogram library, again filtered to a 1% peptide-level FDR by Percolator.

[0085] Quantitative data were normalized by the precursor total ion current in each injection. Peak areas for each peptide were generated by summing peak areas from up to 6 total MS2 transitions. Protein peak areas were similarly generated by summing peptides uniquely assigned to that protein (e.g., not matching anywhere else in the canonical human genome). For each protein, 1-way analysis of variance and 2-sample, 2-tailed t tests were performed to assess the significance of quantitative changes. P values from these tests were FDR-corrected using the Benjamini- Hochberg approach. Proteins were marked as changing quantitatively if they passed a <0.05 FDR threshold. Protein quantitative changes were additionally filtered using t tests (<0.05 FDR) to determine the direction and consistency of the change.

[0086] Machine Learning

[0087] One hundred machine learning-based linear discriminant analysis (LDA) classifiers were created from randomly bootstrapped datasets using selected proteins to construct an ensemble predictor based on prevalidation. This approach aims to combine multiple protein biomarkers to aid in AP diagnosis without the potential bias introduced by constructing only a single classifier. For each LDA classifier, the dataset was randomly divided into half, where 50% of the data were used for training the LDA model, and the remaining 50% were used to test the model. Classifier success was determined using an area under the curve calculation from receiver operator curves. Median prediction accuracy was reported for all classifiers in the aggregate to statistically represent the prediction strength. The final aggregate classifier uses votes from each individual LDA to assign disease predictions.

[0088] Validation Cohort Data Analysis

[0089] The validation cohort protein samples were measured using the same DIA proteomics approach as the global cohort. However, rather than analyze the entire global proteome, specific peptides for targeted quantitative interpretation were selected using Skyline (version 22.2.0.527). Again, a predicted spectrum library was generated by Prosit to aid in the analysis of all potential +2H and +3H peptides from AMY1A, AMY1B, AMY2A, AMY2B, CRP, and CELA2A. Peak areas were calculated and compared using analysis of variance.

[0090] All percentages and ratios are calculated by weight unless otherwise indicated.

[0091] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0092] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0093] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.” [0094] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0095] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

[0096] Illustrative Combinations

[0097] The following examples relate to various non-exhaustive ways in which the teachings herein may be combined or applied. The following examples are not intended to restrict the coverage of any claims that may be presented at any time in this application or in subsequent filings of this application. No disclaimer is intended. The following examples are being provided for nothing more than merely illustrative purposes. It is contemplated that the various teachings herein may be arranged and applied in numerous other ways. It is also contemplated that some variations may omit certain features referred to in the below examples. Therefore, none of the aspects or features referred to below should be deemed critical unless otherwise explicitly indicated as such at a later date by the inventors or by a successor in interest to the inventors. If any claims are presented in this application or in subsequent filings related to this application that include additional features beyond those referred to below, those additional features shall not be presumed to have been added for any reason relating to patentability. [0098] Example 1

[0099] A method for identifying and/or treating an individual having, or likely to have, acute pancreatitis, comprising detecting Elastase 2A and/or 2B in a sample obtained from the individual.

[0100] Example 2

[0101] The method of example 1 , the detecting comprising detecting an Elastase 2A and/or 2B protein.

[0102] Example 3

[0103] The method of example 1, the detecting comprising detecting an mRNA encoding Elastase 2 A and/or 2B.

[0104] Example 4

[0105] The method of example 1 wherein the individual is suspected of having acute pancreatitis.

[0106] Example 5

[0107] The method of example 1 or 2 wherein the individual presents with a symptom selected from at least one of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.

[0108] Example 6

[0109] The method of any preceding example wherein the individual presents with a symptom selected from at least two of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.

[0110] Example 7

[0111] The method of any preceding example wherein the individual presents with a symptom selected from at least three of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever. [0112] Example 8

[0113] The method of any preceding example wherein the sample is a urine sample.

[0114] Example 9

[0115] The method of any preceding example, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least two times that of a Elastase 2A and/or 2B level of a normal control (e.g. an individual who does not have acute pancreatitis).

[0116] Example 10

[0117] The method of any preceding example, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 5 times that of a Elastase 2A and/or 2B level of a normal control, or at level of Elastase 2A and/or 2B that is at least 10 times that of a Elastase 2 A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 20 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 30 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 40 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 50 times that of a Elastase 2A and/or 2B level of a normal control is detected.

[0118] Example 11

[0119] The method of any preceding example, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 60 times that of a Elastase 2A and/or 2B level of a normal control is detected.

[0120] Example 12

[0121] The method of any preceding example, wherein the sample is a urine sample obtained from the individual, and wherein the urine sample is not treated or modified chemically prior to the detection. [0122] Example 13

[0123] The method of any preceding example, wherein the detection is carried out via an antibody-based detection method.

[0124] Example 14

[0125] The method of any preceding example, wherein the detection is carried out via a lateral flow assay.

[0126] Example 15

[0127] The method of any preceding example, wherein the detection is carried out via an ELISA.

[0128] Example 16

[0129] The method of any preceding example, wherein the detection is carried out via a homebased assay.

[0130] Example 17

[0131] The method of any preceding example, wherein the detection is self-administered.

[0132] Example 18

[0133] The method of any preceding example, wherein the detection is carried out via a test comprising a region for receiving sample, the sample being a urine sample, an antibody that binds Elastase 2A and/or 2B, a surface that allows the sample to come into contact with the antibody; and a signal that is visible when Elastase 2A and/or 2B is present in the sample and binds to the antibody.

[0134] Example 19

[0135] The method of any preceding example, further comprising detection of CRP.

[0136] Example 20 [0137] The method of any preceding example, further comprising detection of AMY2A.

[0138] Example 21

[0139] The method of any preceding example, further comprising excluding a diagnosis of one or more of chronic pancreatis, appendicitis, gastrointestinal infection.

[0140] Example 22

[0141] The method of any preceding example, wherein the detection occurs within 24 hours of a symptom presenting.

[0142] Example 23

[0143] The method of any preceding example, further comprising diagnosing the individual as having acute pancreatitis based on a level of Elastase 2A and/or 2B as compared to a control value.

[0144] Example 24

[0145] The method of any preceding example, further comprising treating the individual within 24 hours of a diagnosis of acute pancreatitis.

[0146] Example 25

[0147] The method of example 22 wherein the treatment is selected from IV fluid and/or lactated Ringer’s.

[0148] Example 26

[0149] The method of example 13 wherein IV fluid and/or lactated Ringer’s is administered until electrolyte and fluid balance is restored.

[0150] Example 27

[0151] A method of detecting and/or treating a pancreatitis flare in an individual, comprising detecting a level of Elastase 2A and/or 2B in a sample obtained from the individual. [0152] Example 28

[0153] The method of example 27 wherein the individual is diagnosed with chronic pancreatitis.

[0154] Example 29

[0155] The method of example 27 or 28 wherein the individual is suspected of having a pancreatitis flare.

[0156] Example 30

[0157] The method of any of examples 27 through 29, wherein the individual presents with a symptom selected from at least three of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.

[0158] Example 31

[0159] The method of any of examples 27-30, wherein the sample is a urine sample.

[0160] Example 32

[0161] The method of any of examples 27-31, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least two times that of a Elastase 2A and/or 2B level of a normal control (e.g. an individual who does not have acute pancreatitis).

[0162] Example 33

[0163] The method of any of examples 27-31, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 5 times that of a Elastase 2A and/or 2B level of a normal control, or at level of Elastase 2A and/or 2B that is at least 10 times that of a Elastase 2 A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 20 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 30 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 40 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 50 times that of a Elastase 2A and/or 2B level of a normal control is detected.

[0164] Example 34

[0165] The method of any of examples 27-31, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 60 times that of a Elastase 2A and/or 2B level of a normal control is detected.

[0166] Example 35

[0167] The method of any of examples 27-34, wherein the sample is a urine sample obtained from the individual, and wherein the urine sample is not treated or modified chemically prior to the detection.

[0168] Example 36

[0169] The method of any of examples 27-35, wherein the detection is carried out via an antibody based detection method.

[0170] Example 37

[0171] The method of any of examples 27-35, wherein the detection is carried out via a lateral flow assay.

[0172] Example 38

[0173] The method of any of examples 27-35, wherein the detection is carried out via an ELISA.

[0174] Example 39

[0175] The method of any of examples 27-35, wherein the detection is carried out via a homebased assay.

[0176] Example 40 [0177] The method of any of examples 27-39, wherein the detection is self-administered.

[0178] Example 41

[0179] The method of any of examples 27-40, wherein the detection is carried out via a test comprising a region for receiving sample, the sample being a urine sample, an antibody that binds Elastase 2A and/or 2B, a surface that allows the sample to come into contact with the antibody; and a signal that is visible when Elastase 2A and/or 2B is present in the sample and binds to the antibody.

[0180] Example 42

[0181] The method of any of examples 27-41, further comprising detection of CRP.

[0182] Example 43

[0183] The method of any of examples 27-42, further comprising detection of AMY2A.

[0184] Example 44

[0185] The method of any of examples 27-43, further comprising excluding a diagnosis of one or more of chronic pancreatis, appendicitis, gastrointestinal infection.

[0186] Example 45

[0187] The method of any of examples 27-44, wherein the detection occurs within 24 hours of a symptom presenting.

[0188] Example 46

[0189] The method of any of examples 27-45 wherein the individual presents with at least one of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.

[0190] Example 47 [0191] A kit for detecting Elastase 2A and/or 2B, comprising a sample receiving region and a surface comprising a test region comprising an immobilized detection agent specific for Elastase 2A and/or 2B.

[0192] Example 48

[0193] A method comprising contacting the sample receiving region of the kit of example 47 with a sample from an individual.

[0194] Example 49

[0195] The method of example 48 wherein the sample is a urine sample.

Claims

CLAIMS What is claimed is:

1. A method for identifying and/or treating an individual having, or likely to have, acute pancreatitis, comprising detecting Elastase 2A and/or 2B in a sample obtained from the individual.

2. The method of claim 1, the detecting comprising detecting an Elastase 2A and/or 2B protein.

3. The method of claim 1, the detecting comprising detecting an mRNA encoding Elastase 2 A and/or 2B.

4. The method of claim 1 wherein the individual is suspected of having acute pancreatitis.

5. The method of claim 1 or 2 wherein the individual presents with a symptom selected from at least one of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.

6. The method of any preceding claim wherein the individual presents with a symptom selected from at least two of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.

7. The method of any preceding claim wherein the individual presents with a symptom selected from at least three of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.

8. The method of any preceding claim wherein the sample is a urine sample.

9. The method of any preceding claim, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least two times that of a Elastase 2A and/or 2B level of a normal control (e.g. an individual who does not have acute pancreatitis).

10. The method of any preceding claim, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 5 times that of a Elastase 2A and/or 2B level of a normal control, or at level of Elastase 2A and/or 2B that is at least 10 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 20 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 30 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 40 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 50 times that of a Elastase 2A and/or 2B level of a normal control is detected.

11. The method of any preceding claim, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 60 times that of a Elastase 2A and/or 2B level of a normal control is detected.

12. The method of any preceding claim, wherein the sample is a urine sample obtained from the individual, and wherein the urine sample is not treated or modified chemically prior to the detection.

13. The method of any preceding claim, wherein the detection is carried out via an antibodybased detection method.

14. The method of any preceding claim, wherein the detection is carried out via a lateral flow assay.

15. The method of any preceding claim, wherein the detection is carried out via an ELISA.

16. The method of any preceding claim, wherein the detection is carried out via a homebased assay.

17. The method of any preceding claim, wherein the detection is self-administered.

18. The method of any preceding claim, wherein the detection is carried out via a test comprising a. a region for receiving sample, the sample being a urine sample, b. an antibody that binds Elastase 2A and/or 2B, c. a surface that allows the sample to come into contact with the antibody; and d. a signal that is visible when Elastase 2A and/or 2B is present in the sample and binds to the antibody.

19. The method of any preceding claim, further comprising detection of CRP.

20. The method of any preceding claim, further comprising detection of AMY2A.

21. The method of any preceding claim, further comprising excluding a diagnosis of one or more of chronic pancreatis, appendicitis, gastrointestinal infection.

22. The method of any preceding claim, wherein the detection occurs within 24 hours of a symptom presenting.

23. The method of any preceding claim, further comprising diagnosing the individual as having acute pancreatitis based on a level of Elastase 2A and/or 2B as compared to a control value.

24. The method of any preceding claim, further comprising treating the individual within 24 hours of a diagnosis of acute pancreatitis.

25. The method of claim 22 wherein the treatment is selected from IV fluid and/or lactated Ringer’s.

26. The method of claim 13 wherein IV fluid and/or lactated Ringer’s is administered until electrolyte and fluid balance is restored.

27. A method of detecting and/or treating a pancreatitis flare in an individual, comprising detecting a level of Elastase 2A and/or 2B in a sample obtained from the individual.

28. The method of claim 27 wherein the individual is diagnosed with chronic pancreatitis.

29. The method of claim 27 or 28 wherein the individual is suspected of having a pancreatitis flare.

30. The method of any of claims 27 through 29, wherein the individual presents with a symptom selected from at least three of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.

31. The method of any of claims 27-30, wherein the sample is a urine sample.

32. The method of any of claims 27-31, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least two times that of a Elastase 2A and/or 2B level of a normal control (e.g. an individual who does not have acute pancreatitis).

33. The method of any of claims 27-31, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 5 times that of a Elastase 2A and/or 2B level of a normal control, or at level of Elastase 2A and/or 2B that is at least 10 times that of a Elastase 2 A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 20 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 30 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 40 times that of a Elastase 2A and/or 2B level of a normal control, or a level of Elastase 2A and/or 2B that is at least 50 times that of a Elastase 2A and/or 2B level of a normal control is detected.

34. The method of any of claims 27-31, further comprising diagnosing an individual with acute pancreatitis when a level of Elastase 2A and/or 2B that is at least 60 times that of a Elastase 2A and/or 2B level of a normal control is detected.

35. The method of any of claims 27-34, wherein the sample is a urine sample obtained from the individual, and wherein the urine sample is not treated or modified chemically prior to the detection.

36. The method of any of claims 27-35, wherein the detection is carried out via an antibody based detection method.

37. The method of any of claims 27-35, wherein the detection is carried out via a lateral flow assay.

38. The method of any of claims 27-35, wherein the detection is carried out via an ELISA.

39. The method of any of claims 27-35, wherein the detection is carried out via a home-based assay.

40. The method of any of claims 27-39, wherein the detection is self-administered.

41. The method of any of claims 27-40, wherein the detection is carried out via a test comprising a. a region for receiving sample, the sample being a urine sample, b. an antibody that binds Elastase 2 A and/or 2B, c. a surface that allows the sample to come into contact with the antibody; and d. a signal that is visible when Elastase 2A and/or 2B is present in the sample and binds to the antibody.

42. The method of any of claims 27-41, further comprising detection of CRP.

43. The method of any of claims 27-42, further comprising detection of AMY2A.

44. The method of any of claims 27-43, further comprising excluding a diagnosis of one or more of chronic pancreatis, appendicitis, gastrointestinal infection.

45. The method of any of claims 27-44, wherein the detection occurs within 24 hours of a symptom presenting.

46. The method of any of claims 27-45 wherein the individual presents with at least one of a family history of acute pancreatitis, chronic pancreatitis, vomiting, abdominal pain, and fever.

47. A kit for detecting Elastase 2 A and/or 2B, comprising a. a sample receiving region and b. a surface comprising a test region comprising an immobilized detection agent specific for Elastase 2 A and/or 2B.

48. A method comprising contacting the sample receiving region of the kit of claim 47 with a sample from an individual.

49. The method of claim 48 wherein the sample is a urine sample.