Methods for the Detection of Myeloperoxidase

Incorporation by Cross-Reference

The present application claims priority from Australian provisional patent application number 2022902287, filed on 12 August 2022, the entire content of which is incorporated herein by cross-reference.

Technical Field

The present invention relates generally to the fields of biology, biochemistry, and medicine. Specifically, the present invention relates to methods and kits for the determination of oxidative status and/or the detection of myeloperoxidase in a faecal sample. The methods and kits described herein may find particular application in diagnosing and/or determining the severity of inflammatory bowel disease (IBD) and/or infectious colitis.

Background

Inflammatory bowel disease (IBD) is the collective term for a group of chronic non- infectious conditions involving inflammation of the gastrointestinal tract, with the colon and small intestine generally the most severely affected. The term “IBD” primarily includes Crohn's disease (which can affect any segment of the gastrointestinal tract from the mouth to the anus), ulcerative colitis (which is limited to the colon), and indeterminate colitis. Symptoms of IBD tend to be persistent and include abdominal pain, diarrhea, rectal bleeding and weight loss. A chronic relapsing pattern is common and morbidity (including declining mental well-being) is high.

The cause of IBD is not completely understood, but it is generally thought to be a result of an uncontrolled immune response triggered by a combination of genetic and environmental factors. Treatments for IBD are generally aimed at reducing the severity of symptoms or inducing a period of remission as the condition is technically incurable. Current methods of treatment include anti-inflammatory drugs and diet and lifestyle changes. Surgical intervention is used in more severe cases.

IBD imposes health and economic burdens on countries worldwide and the prevalence of the condition has increased substantially in many regions of the world in the last decade. Most diagnoses occur prior to thirty years of age and symptoms typically peak during the second to fourth decade, often disrupting what would normally be the most productive time of adulthood.

Infectious colitis is an acute or chronic form of colitis which is caused by a pathogen (i.e., a bacterium, virus, fungus and/or parasite). Many symptoms of infectious colitis mimic those of IBD and the condition also increasingly contributes to significant morbidity and mortality worldwide.

Original tests used in the diagnosis and management of IBD were invasive, uncomfortable, painful and often expensive, such as an endoscopy with concomitant muscle biopsy. In recent years there has been a focus on replacing such tests with the use of noninvasive biomarkers for diagnosis and for monitoring disease progression, monitoring responses to treatment and predicting potential relapses. The use of biomarkers has the potential to reduce costs and to save time, discomfort and inconvenience. Biomarkers also have the potential to provide a faster diagnosis of infectious colitis when compared to methods which seek to identify a pathogen.

Several biomarkers have been proposed for use in the diagnosis and monitoring of infectious colitis and/or IBD, with enhanced oxidative stress and the immune enzyme myeloperoxidase emerging as promising candidates. However, a method of diagnosis and/or monitoring IBD and/or infectious colitis which is rapid, low-cost and effectively correlates with disease severity has yet to be developed. A need exists for assays, kits and methods which can be used for noninvasive rapid diagnosis and monitoring of IBD and/or infectious colitis by individuals.

Summary of the Invention

The present invention alleviates at least one of the problems outlined above by providing methods and kits for determining the oxidative status and/or the level of myeloperoxidase in a faecal sample. The methods and kits enable rapid and/or effective and/or simple and/or low-cost determination of oxidative status and/or myeloperoxidase levels. In some embodiments of the invention, determination of oxidative status and/or myeloperoxidase levels may be achieved with the use of a smartphone camera, allowing users to use the methods and kits in the comfort of their own home, if required. The methods and kits of the invention may find application in diagnosing and/or monitoring the severity of inflammatory bowel disease (IBD) and/or infectious colitis. The present invention relates at least in part to the following embodiments:

Embodiment 1. A method for determining the total oxidative status of a faecal sample, the method comprising:

(i) mixing:

(a) the faecal sample;

(b) an inorganic salt of chloride, bromide and/or iodide;

(c) a substance capable of chemiluminescence, fluorescence and/or a colorimetric reaction; and

(d) hydrogen peroxide, to provide a reaction mixture; and

(ii) quantifying the level of chemiluminescence, fluorescence and/or a colorimetric reaction generated by the reaction mixture, wherein the level of chemiluminescence, fluorescence and/or a colorimetric reaction is used to determine the total oxidative status of the faecal sample.

Embodiment 2. A method of determining the level of myeloperoxidase in a faecal sample, the method comprising:

(i) mixing:

(a) the faecal sample;

(b) an inorganic salt of chloride, bromide and/or iodide;

(c) a substance capable of chemiluminescence, fluorescence and/or a colorimetric reaction and

(d) hydrogen peroxide, to provide a reaction mixture; and

(ii) quantifying the level of chemiluminescence, fluorescence and/or a colorimetric reaction generated by the reaction mixture, wherein the level of chemiluminescence, fluorescence and/or a colorimetric reaction is used to determine the level of myeloperoxidase in the faecal sample.

Embodiment 3, The method of embodiment 1 or embodiment 2, wherein the level of chemiluminescence, fluorescence and/or a colorimetric reaction is quantified using a smartphone camera.

Embodiment 4, The method of any one of embodiments 1 to 3, further comprising using the oxidative status and/or the level of myeloperoxidase to diagnose and/or monitor the status of inflammatory bowel disease (IBD) and/or infectious colitis. Embodiment 5, The method of any one of embodiments 1 to 4, wherein the substance capable of chemiluminescence, fluorescence and/or a colorimetric reaction is luminol, 2-[6-(4’-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, Taurine tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), o-dianisidine, guaiacol and/or 10-acetyl-3,7-dihydroxyphenoxazine.

Embodiment 6, The method of embodiment 5, wherein the substance capable of chemiluminescence is luminol.

Embodiment 7, The method of embodiment 6, wherein the luminol is present in the reaction mixture at a concentration of between 25 pM and 10 mM, between 1 mM and 9 mM, between 2 mM and 8 mM, between 3 mM and 7 mM, between 4 mM and 6 mM, or between 4.5 mM and 5.5 mM.

Embodiment 8, The method of any one of embodiment 1 to 7, wherein the inorganic salt of chloride, bromide and/or iodide is present in the reaction mixture at a concentration of between 100 pM and 300 mM, between 500 pM and 200 mM, between 1 mM and 100 mM, between 10 mM and 90 mM, between 20 mM and 80 mM, between 30 mM and 70 mM, between 40 mM and 60 mM, or between 45 mM and 55 mM.

Embodiment 9, The method of any one of embodiments 1 to 8, wherein the hydrogen peroxide is present in the reaction mixture at a concentration of between 100 pM and 300 mM, between 200 pM and 200 mM, between 300 pM and 100 mM, between 500 pM and 50 mM, between 1 mM and 10 mM, between 2 mM and 5 mM, or between 2 mM and 3 mM.

Embodiment 10. The method of any one of embodiments 1 to 9, wherein the reaction mixture further comprises a catalase inhibitor and/or a heme inhibitor.

Embodiment 11. The method of embodiment 10, wherein the catalase inhibitor and/or heme inhibitor is 3 -Aminotriazole and/or Sodium Azide.

Embodiment 12. The method of any one of embodiments 1 to 11, wherein the reaction mixture further comprises DNase I and/or DNase II.

Embodiment 13, The method of embodiment 12, wherein the DNase is present in the reaction mixture at a concentration of between 0.005 pg/pL and 10 pg/pL, between 0.01 pg/pL and 8 pg/pL, between 0.02 pg/pL and 5 pg/pL, between 0.03 pg/pL and 3 pg/pL, between 0.04 pg/pL and 2 pg/pL, or between 0.05 pg/pL and 1 pg/pL.

Embodiment 14, The method of any one of embodiments 1 to 13, wherein the reaction mixture further comprises a cyclodextrin. Embodiment 15. The method of embodiment 14, wherein the cyclodextrin is alpha-cyclodextrin, beta-cyclodextrin and/or gamma-cyclodextrin.

Embodiment 16, The method of embodiment 14 or embodiment 15, wherein the cyclodextrin and the substance capable of chemiluminescence, fluorescence and/or a colorimetric reaction are present in the reaction mixture in a ratio of between 0.5: 1 and 20: 1, or approximately 1 : 1.

Embodiment 17, The method of any one of embodiments 1 to 16, wherein the reaction mixture further comprises a polymethacrylate.

Embodiment 18, The method of embodiment 17, wherein the polymethacrylate and the substance capable of chemiluminescence, fluorescence and/or a colorimetric reaction form nanoparticles.

Embodiment 19, The method of any one of embodiments 2 to 18, further comprising:

(iii) repeating (i), wherein the reaction mixture further comprises a myeloperoxidase inhibitor;

(iv) quantifying the level of chemiluminescence, fluorescence and/or a colorimetric reaction generated by the reaction mixture provided in (iii);

(v) subtracting the value obtained in (iv) from the value obtained in (ii) to obtain the level of myeloperoxidase in the faecal sample.

Embodiment 20, The method of embodiment 19, wherein the myeloperoxidase inhibitor is selected from the group consisting of: AZD3421, AZD2134, Sodium Azide, 4- amino benzoic acid hydrazine, AZD5904, PF-06282999, N-Acetyl lysyltyrosylcysteine amide, PF-1355, MPO-IN-28, 4-Methylesculetin, and any combination thereof.

Embodiment 21, A kit when used for the method of any one of embodiments 1 to 20, the kit comprising:

(i) an inorganic salt of chloride, bromide and/or iodide;

(ii) a substance capable of chemiluminescence, fluorescence and/or a colorimetric reaction; and

(iii) hydrogen peroxide.

Embodiment 22, The kit of embodiment 21, further comprising a smartphone camera.

Embodiment 23, The kit of embodiment 21 or embodiment 22, wherein the substance capable of chemiluminescence, fluorescence and/or a colorimetric reaction is luminol, 2-[6-(4’-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, Taurine tetramethylbenzidine (TMB), tetramethylbenzidine (TMB), 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid (ABTS), o-dianisidine, guaiacol and/orlO-acetyl-3,7- dihydroxyphenoxazine.

Embodiment 24, The kit of embodiment 23, wherein the substance capable of chemiluminescence, fluorescence and/or a colorimetric reaction is luminol.

Embodiment 25, The kit of any one of embodiments 21 to 24, further comprising a catalase inhibitor and/or a heme inhibitor.

Embodiment 26, The kit of embodiment 25, wherein the catalase inhibitor and/or heme inhibitor is 3 -Aminotriazole and/or Sodium Azide.

Embodiment 27, The kit of any one of embodiments 21 to 26, further comprising any one or more of: a DNase, a cyclodextrin, and a polymethacrylate.

Definitions

As used in this application, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “sample” also includes a plurality of samples.

As used herein, the term “comprising” means “including”. Variations of the word “comprising”, such as “comprise” and “comprises,” have correspondingly varied meanings. Thus, for example, a method “comprising” step ‘A’ and step ‘B’ may consist exclusively of step ‘A’ and step ‘B’ or may include one or more steps before and/or after ‘A’ and step ‘B’ and/or one or more intervening steps between step ‘A’ and step ‘B’.

As used herein, the term “subject” includes any animal of economic, social or research importance including bovine, equine, ovine, primate, avian and rodent species. Hence, a “subject” may be a mammal such as, for example, a human, or a non-human mammal.

As used herein, the term “kit” refers to any delivery system for delivering materials. Such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., labels, reference samples, supporting material, etc. in appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing an assay etc.) from one location to another. For example, kits may include one or more enclosures, such as boxes, containing the relevant reaction reagents and/or supporting materials. As used herein, the term “about” when used in reference to a recited numerical value includes the recited numerical value and numerical values within plus or minus ten percent of the recited value.

As used herein, the term “plurality” means more than one. In certain specific aspects or embodiments, a plurality may mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or more, and any numerical value derivable therein, and any range derivable therein.

As used herein, the term “between” when used in reference to a range of numerical values encompasses the numerical values at each endpoint of the range.

As used herein, the term “more than” when used in reference to a numerical value will be understood to mean “greater than or equal to”.

As used herein, the term “less than” when used in reference to a numerical value will be understood to mean “less than or equal to”.

As used herein, the term “chemiluminescence” will be understood to mean the generation of electromagnetic radiation as light by the release of energy from a chemical reaction involving the oxidation of a substrate.

As used herein, the term “fluorescence” will be understood to mean the generation of electromagnetic radiation as light by a substance that has been activated to absorb or generate light or by a substance that has absorbed another form of electromagnetic radiation.

As used herein, the term “colorimetric reaction” will be understood to mean any chemical reaction which produces a coloured compound in which the concentration of the coloured compound is proportional to its absorption or transmission of light and therefore can be quantified.

As used herein, the term “smartphone” will be understood to mean any mobile phone that includes advanced functionality beyond making phone calls and sending text messages.

As used herein, the term “inflammatory bowel disease (IBD)” will be understood to mean any chronic non-infectious condition involving inflammation of the gastrointestinal tract. Non-limiting examples of such conditions include Crohn's disease, ulcerative colitis and indeterminate colitis. As used herein, the term “infectious colitis” will be understood to mean any acute or chronic form of colitis which is caused by a pathogen (i.e., a bacterium, virus, fungus and/or parasite).

As used herein, the terms “oxidative status” and “total oxidative status” will be understood to mean the overall oxidative potential of a cell/tissue, which in turn will be understood to mean the imbalance between the production of oxidants and the levels of antioxidants within said cell/tissue.

As used herein, the term “inhibitor” will be understood to mean any molecule which prevents the action of an enzyme in such a way as to prevent binding by the substrate, or by prevention of the reaction even if the substrate can still bind. The prevention of the action of the enzyme by the inhibitor may be partial or complete.

As used herein, the term “DNase” will be understood to mean any molecule, for example, an enzyme, that degrades DNA. The degradation of the DNA by the molecule may be partial or complete.

As used herein, the term “cyclodextrin” will be understood to mean a cyclic oligomer of glucose.

As used herein, the term “nanoparticles” will be understood to mean particles with a diameter of between 1 and 250 nanometres capable of chemical/compound encapsulation and vehicular transport of encapsulated chemical/compound.

Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art.

For the purposes of description, all documents referred to herein are hereby incorporated by reference in their entirety unless otherwise stated.

Brief Description of the Figures

Preferred embodiments of the present invention will now be described by way of example only, with reference to the accompanying figures wherein:

Figure l is a graph providing a time-series capture of the Myeloperoxidase Luminol Reaction (MPOLR) assay in faecal samples from healthy mice (blue line) and mice with experimental colitis (DSS) (orange and yellow lines). The specific myeloperoxidase (MPO) inhibitor, AZD3241 or AZD2314 (sourced commercially) was used to differentiate background luminosity from other oxidants/peroxidase activities (yellow line) and the subtraction of the two intensities (orange - yellow) revealed the true specific MPO activity (insert - top right corner).

Figure 2 provides images of smartphone detection of luminosity following the MPOLR assay in murine faecal samples. CTRL = non-treated control mice; DSS = experimental murine colitis. ‘No treatment = no MPO inhibitor’.

Figure 3 provides an image of chemiluminescence detection of MPO spiked faecal samples (1 pg/mL). This representative image was taken following exposure for 300s.

Figure 4 provides an image of chemiluminescence detection of healthy human faecal samples spiked with MPO (100 pg/g). 1 = Faecal sample + MPO spike; 2 = Faecal sample + MPO spike + AZD3241.

Figure 5 provides a graph highlighting the relationship between MPO levels and chemiluminescence in a closed system using the novel methods of detection of the invention.

Figure 6 provides an image of chemiluminescence detection of faecal MPO using the luminol substrate complexed with alpha cyclodextrins.

Figure 7 provides an image of chemiluminescence detection faecal MPO using luminol or luminol-encapsulated nanoparticles (Eudragit) in faecal material from mice subjected to mild experimental colitis.

Figure 8 provides a graph of the rate of chemiluminescence decay when detecting faecal MPO using luminol or luminol-encapsulated nanoparticles (Eudragit) in faecal material from mice subjected to mild colitis.

Detailed description

The present inventors have developed methods and kits which enable rapid and/or effective and/or simple and/or low-cost determination of oxidative status (which may be the flux of tissue damaging oxidant production) and/or myeloperoxidase levels. The methods and/or kits may be used to determine the oxidative status and/or the level of myeloperoxidase in a biological sample, for example, a faecal sample.

The sample, for example, a faecal sample, may be taken from a subject which may be any animal (for example, a mammal), including, but not limited to, humans, non-human primates, canines, felines, and rodents. See Deda et al., J. Pharm. Biomed. Anal., 2015; 113: 137-150 for a comprehensive overview of faecal sample preparation suitable for a range of subsequent methods of analyses. In some embodiments of the invention, the method comprises providing a reaction mixture. The reaction mixture may include the sample to be tested for oxidative status and/or myeloperoxidase levels, for example, a faecal sample. Chloride may be added to the reaction mixture. In some embodiments, an inorganic salt of chloride may be added. The chloride may be added in the form of an ionic compound, for example, sodium chloride. Other non-limiting examples of chloride-containing compounds which may be added to the reaction mixture include potassium chloride, calcium chloride, magnesium chloride and ammonium chloride. In some embodiments of the invention, bromide and/or iodide may be added to the reaction mixture. Inorganic salts of chloride, bromide and/or iodide could be added. The chloride, bromide and/or iodide and/or inorganic slats thereof may be present in the reaction mixture at a concentration of between 100 pM and 300 mM, between 500 pM and 200 mM, between 1 mM and 100 mM, between 10 mM and 90 mM, between 20 mM and 80 mM, between 30 mM and 70 mM, between 40 mM and 60 mM, or between 45 mM and 55 mM.

The reaction mixture may include a substance capable of chemiluminescence. In some embodiments of the invention, the substance capable of chemiluminescence is luminol. The chemiluminescent properties of luminol were first discovered in 1928 and have become widely known and commonly used by those skilled in the art. For a review of ways in which the chemiluminescent properties of luminol can be utilized, see Khan, Appl. Biochem. Biotechnol., 2014; 173(2): 333-355. In some embodiments of the invention, the luminol is present in the reaction mixture at a concentration of between 25 pM and 10 mM, between 1 mM and 9 mM, between 2 mM and 8 mM, between 3 mM and 7 mM, between 4 mM and 6 mM, or between 4.5 mM and 5.5 mM.

Other non-limiting examples of substances capable of generating indirect chemiluminescent reactions include peroxyoxalates which generate oxalic acid, which in the presence of a fluorescent compound produces a secondary chemiluminescent reaction dependent on the generation of oxalic acid.

The reaction mixture may include a substance capable of fluorescence. This includes but is not limited to 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid solution which generates fluorescence upon oxidation by the myeloperoxidase-specific oxidant, HOC1.

The reaction mixture may include a substance capable of forming a colorimetric reaction. Many chemical compounds are capable of being oxidised in the presence of H2O2 and peroxidases (for example, myeloperoxidase) and following oxidation form a colorimetric reaction capable of being observed in the visible light spectrum. Non-limiting examples of such compounds include tetramethylbenzidine (TMB), 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid (ABTS), o-dianisidine, guaiacol, 10-acetyl-3,7- dihydroxyphenoxazine that are oxidised to yield products detected by colourimetry.

In some embodiments of the invention, the substance capable of chemiluminescence, fluorescence and/or a colorimetric reaction is luminol, 2-[6-(4’-amino)phenoxy-3H- xanthen-3-on-9-yl]benzoic acid, Taurine - tetramethylbenzidine (TMB), 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid (ABTS), o-dianisidine, guaiacol and/or 10-acetyl-3,7- dihydroxyphenoxazine.

The reaction mixture may comprise hydrogen peroxide, which is commonly used by persons skilled in the art in chemiluminescence reactions and is very easy to source. The hydrogen peroxide may be present in the reaction mixture at a concentration of between 100 pM and 300 mM, between 200 pM and 200 mM, between 300 pM and 100 mM, between 500 pM and 50 mM, between 1 mM and 10 mM, between 2 mM and 5 mM, or between 2 mM and 3 mM.

The reaction mixture may comprise additional components including but not limited to high purity water, buffer, Hg ²⁺ , ethylenedi aminetetraacetic acid, 4-Hydroxy-2,3-trans- nonenal, pentetic acid, AZD3241, AZD2134 Sodium Azide and/or 3 -Aminotriazole.

The high purity water used in the preparation of the reaction mixture may be any water substantially free from contaminants. Many types of high purity water are readily available commercially. Additionally or alternatively, the skilled person may prepare high purity water by any one of many well-known methods such as activated carbon, reverse osmosis, ion exchange, filtration and distillation.

The present inventors have identified optimal concentrations of chloride, bromide and/or iodide, the substance capable of chemiluminescence, fluorescence and/or a colorimetric reaction, hydrogen peroxide and/or additional components which may be used in the methods and/or kits of the present invention, some of which are described in the Detailed Description Examples and Claims of the present application. It will be understood that all of the concentrations disclosed for use in the methods and kits are exemplary only.

The reaction mixture may generate chemiluminescence. In some embodiments of the invention, the level of chemiluminescence may be used to determine the level of myeloperoxidase in a sample, for example, a faecal sample. The chemiluminescent reaction may be detectable between the 380-780 nm wavelength.

Luminosity generated in the chemiluminescent reaction may be detected and/or measured and/or quantified using any suitable imaging system. Persons skilled in the art would be aware of a range of systems suitable for imaging luminosity and in some cases analysing the results. The skilled person would also be capable of determining a suitable exposure time for the chemiluminescent reaction. Generally, the exposure time can be expected to range from 10-300 s. The accumulated signal generated may be related to the specific level of active myeloperoxidase enzymic oxidant in the sample.

In some embodiments of the invention, the reaction mixture may generate a colorimetric reaction, where substrates other than luminol are used to detect the presence of HOC1. In these embodiments, the colorimetric reaction may be quantified by imaging in the visible light spectrum. A higher intensity of colorimetric change may be used to determine the level of myeloperoxidase in a sample, for example, a faecal sample.

In some embodiments of the invention, luminosity levels may be used to determine the level of myeloperoxidase in a sample the presence of a specific MPO inhibitor such as AZD321 or AZD2134 (inhibitors of extracellular myeloperoxidase), for example, a faecal sample. In further embodiments, the specific MPO inhibitor is commercially available. In the absence of an MPO inhibitor, a luminosity reading in faecal samples with added H2O2 and Cl may provide a measurement of total oxidative status as other oxidants present in the stool sample may be capable of oxidising luminol, i.e. OH, ONOO, O2 and LOO in a non- enzymic capacity. This may be referred to as the baseline luminosity as MPO-mediated enzymic oxidation of the substrate luminol gives rise to a greater signal above the baseline/background. Other non-limiting examples of MPO inhibitors which may be used with the methods of the invention include Sodium Azide, 4-amino benzoic acid hydrazine, AZD5904, PF-06282999, N-Acetyl lysyltyrosylcysteine amide, PF-1355, MPO-IN-28 and 4-Methylesculetin. MPO inhibitors may be used individually or in any combination.

Determination of luminosity levels, which may be used to determine levels of myeloperoxidase, may be achieved with the use of a smartphone to capture luminosity, allowing users to use the methods and kits in the comfort of their own home, if required. This may be achieved with the use of a smartphone camera.

In some embodiments of the invention, the method is repeated using a reaction mixture which further comprises a myeloperoxidase inhibitor. For example, the myeloperoxidase inhibitor may be AZD3241 or AZD2134. The level of chemiluminescence generated by the reaction mixture including AZD3241 may be measured and/or quantified. The value obtained may be subtracted from the measurement obtained for the reaction mixture without AZD3241 to help to determine the level of myeloperoxidase in the faecal sample.

In further embodiments of the invention, the reaction mixture further comprises a catalase inhibitor and/or a heme inhibitor. The catalase inhibitor and/or heme inhibitor may be Sodium Azide and/or 3 -Aminotriazole.

A DNase may also be present in the reaction mixture. The DNase could be DNase I and/or DNase II. In some exemplary embodiments, the DNase is present in the reaction mixture at a concentration of between 0.005 pg/pL and 10 pg/pL, 0.01 pg/pL and 8 pg/pL, between 0.02 pg/pL and 5 pg/pL, between 0.03 pg/pL and 3 pg/pL, between 0.04 pg/pL and 2 pg/pL, or between 0.05 pg/pL and 1 pg/pL.

Cyclodextrins, for example, alpha-cyclodextrin, beta-cyclodextrin and/or gammacyclodextrin may be used to enhance a chemiluminescence reaction. A non-limiting example of how this could be achieved is by complexing a cyclodextrin, for example, alpha- cyclodextrin, beta-cyclodextrin and/or gamma-cyclodextrin, to luminol prior to the addition of luminol to the reaction mixture. Cyclodextrin/luminol complexes could be created using a 1 : 1 mol/mol ratio of alpha-cyclodextrin and luminol sodium salt. It should be understood that the 1 : 1 ratio is exemplary only. The ratio could be between, for example, 0.5: 1 and 20: 1. Magnetic stirring could be used to entrap the luminol within the complexes.

Another way in which chemiluminescence reactions used in the methods of the invention could be enhanced is by encapsulating a substance capable of chemiluminescence, for example, luminol, in nanoparticles. In one non-limiting embodiment of the invention, luminol is encapsulated in a polymethacrylate, for example, Eudragit, to form carrier nanoparticles. Methods for the synthesis of nanoparticles are well known in the art. See, for example, Rane et al., Micro and Nano Technologies, 2018; Chapter 5.

The invention also provides kits which may be used for the methods of the invention. The kits may include chloride, bromide and/or iodide and/or inorganic salts thereof and/or a substance capable of chemiluminescence and/or hydrogen peroxide and/or a smartphone camera. In some embodiments, the camera may be mounted. Kits of the invention may also include a DNase, a cyclodextrin and/or a polymethacrylate, for example, Eudragit. The DNase may be DNase I and/or DNase II. The cyclodextrin in the kit may be any one or more of alpha-cyclodextrin, beta- cyclodextrin or gamma-cyclodextrin.

The methods and kits of the invention may find application in diagnosing and/or monitoring the severity of inflammation in disorders such as inflammatory bowel disease (IBD). In some embodiments of the invention, luminosity, and therefore levels of myeloperoxidase, may be positively correlated with the severity of IBD. The methods and/or kits of the invention may be used to diagnose IBD, predict the course of the disease and/or predict an impending relapse.

It will be appreciated by persons of ordinary skill in the art that numerous variations and/or modifications can be made to the present invention as disclosed in the specific embodiments without departing from the spirit or scope of the present invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Examples

The present invention will now be described with reference to specific Examples, which should not be construed as in any way limiting.

Example One: Myeloperoxidase Luminol Reaction (MPOLR) assay

Assay mechanism

Myeloperoxidase readily reacts with free Cl- (in the presence of hydrogen peroxide) to form hypochlorous acid (H0C1). H0C1 is a powerful 2 electron oxidant whereby luminol is a substrate and the subsequent oxidation of luminol by H0C1 results in a chemiluminescent reaction detectable in the 380-780 nm wavelength range. The luminosity generated by the reaction of HOC1 and luminol in this Example could be detected by a smartphone camera.

Below is a detailed protocol using mice faecal pellets subjected to experimental colitis (therefore containing myeloperoxidase).

Protocol

Stock Solutions/Consumables

96 well v bottom white plate

H2O2 = (200 pL in 50 mL dEEO; 36 mM) - make daily

NaCl = (5.84g/50ml w/v dEEO; 2 M) (mw: 58.44) • AZD3241 = (5mg/50 mL; 715 pM) - (mw: 253.32) (alternative name - PXS-5398)

- store at -30°C

• Luminol = (5mg in 1.25 mL dH ₂ O; 20 mM) (mw: 199.14) - make daily

• 3 -Aminotriazole = (84mmg/10mL; 100 mM) (mw: 84.08) - store at -30°C

Master mix (procedure for 96-well plate)

• 1.1 mL NaCl (200 mM final cone.)

• L lmL 3-AT (10 mM final cone.)

• 2 mL dH ₂ O; 2 M)

Procedure

• 25 pL of each fecal supernatant sample into 4 wells (Patrick's faecal normalization study samples)

• 38.2 pL of Master Mix into each well

• 25 pL of dH ₂ O or AZD drug (180 pM final cone.) into each well, mixed and incubated for 10 min in an orbital shaker at RT

• 10 pL luminol into each well (2 mM final cone.)

• 2.5 pL H ₂ O ₂ (-475 pM final cone.) is immediately added on the corner of each well and the plate is tapped to allow for the reaction to initiate simultaneously

• Read the wells immediately using Chemidoc with a 300s exposure time and again after 10 mins. NB: All 'working solution' concentrations are calculated at the final concentration of - 100 pL.

Results

The luminosity generated in the reaction was still detectable 25 minutes following the initiation of the reaction with hydrogen peroxide. Control faecal samples (no inflammation) showed no detectable levels of luminosity in any of the treated conditions/groups. In faecal samples obtained from mice following experimental colitis (with inflammation), the luminosity was detectable with as little as 60 s exposure time, but most prominent with a 300 s exposure time. This was markedly elevated from baseline control samples. The specific myeloperoxidase inhibitor, AZD was used to obtain a background reading of luminosity unrelated to myeloperoxidase activity. The AZD group showed a marked reduction in luminosity when compared to the group without an inhibitor (see Figure 1). Sodium azide is a powerful catalase inhibitor and this group was used to detect the background signal generated by the reaction of hydrogen peroxide with Fe ³⁺ (oxidized Hb from haemorrhage). The sodium azide showed a luminosity markedly less intense than the group with no inhibitor or AZD compound. Figure 1 demonstrates the specificity of the assay for faecal myeloperoxidase.

As can be seen in Figure 2, the differences in luminosity between the samples was clearly detectable using a smartphone camera.

Prophetic Example Two: Myeloperoxidase TMB (3,3\5,5'-Tetramethylbenzidine) Reaction

Assay mechanism

Myeloperoxidase readily reacts with free Cl- (in the presence of hydrogen peroxide) to form hypochlorous acid (H0C1). H0C1 is a powerful 2 electron oxidant which oxidises taurine amine residues resulting in the formation of the HOCl-specific taurine chloroamine - a substrate for TMB. Oxidation of TMB via taurine chloroamine results in the formation of 3,3',5,5'-tetramethylbenzidine diamine. The diamine causes a colorimetric reaction to form a blue product and this product is detectable at the 370 - 650 nm wavelength (visible light). The reaction can then be halted by the addition of 2 M sulfuric acid or 20 pg/mL catalase.

The colorimetric reaction could be captured on a smartphone device and the intensity could be determined via image analysis software, where the intensity is proportional to the concentration of MPO.

Protocol

Stock Solutions/Consumables

• 96 well v bottom white plate

• H2O2 = (200 pL in 50 mL dLLO; 36 mM) - make daily

• NaCl = (5.84g/50ml w/v dFLO; 2 M) (mw: 58.44)

• AZD3241 = (5mg/50 mL; 715 pM) - (mw: 253.32) (alternative name - PXS-5398) - store at -30°C

• 0.5 mM TMB

• 3 -Aminotriazole = (84mmg/10mL; 100 mM) (mw: 84.08) - store at -30°C

• 2 mM Taurine

Master mix (procedure for 96-well plate)

• 1.1 mL NaCl (200 mM final cone.)

• L lmL 3-AT (10 mM final cone.) 2 mL dH ₂ O; 2 M)

2mM Taurine

Procedure

• 25 pL of each faecal supernatant sample into 4 wells (Patrick's faecal normalization study samples)

• 38.2 pL of Master Mix into each well

• 25 pL of dH ₂ O or AZD drug (180 pM final cone.) into each well, mixed and incubated for 10 min in an orbital shaker at RT

• 2.5 pL H ₂ O ₂ (-475 pM final cone.) is immediately added on the corner of each well and the plate is tapped to allow to incubate for 20 min at room temperature.

• 10 pL TMB into each well (0.5 mM final cone.) and allowed to incubate for a future 10 min.

• The reaction is halted with the addition of 2 M sulfuric acid or 20 pg/mL catalase.

• The colorimetric reaction in the wells is captured via a smartphone camera and the intensity is determined via image analysis software.

Expected results

A colorimetric reaction would occur that could be visualized by eye (605 nm) in the yellow-green visible light spectrum in faecal samples which contains endogenous MPO, while faecal samples devoid of MPO would undergo little/no colorimetric change. Following 40 min incubation at room temperature, the addition of sulfuric acid to stop the reaction would cause a color shift to the purple visible spectrum (405 nm). The addition of catalase instead would cause no colorimetric shift. The intensity of the colorimetric reaction (either at 605 or 405 nm) would be proportional to the activity of MPO.

Example Three: DNase enhances chemiluminescence detection of faecal MPO Method

To determine whether DNase enhances chemiluminescence detection of faecal MPO, stool homogenate was prepared at 10% w/v dH ₂ O from healthy mice and was either spiked with human exogenous MPO (4 pg/mL) + Dnase (0.05 pg/pL), human exogenous MPO (4 pg/mL) or not spiked (control). The mixtures were centrifuged for 2 min at 14,800 r.p.m and the collected supernatant was further diluted to % total volume. To simulate full MPO extraction, some conditions were subjected to mice faecal supernatant spiking with 1 pg/mL human exogenous MPO. A list of all experimental conditions is provided in Table 1. The final concentrations of additives in each well were 200mM NaCl and lOmM 3 -AT (catalase inhibitor). Luminol 2mM w/v dELO was mixed with varying H2O2 concentrations and simultaneously incubated before immediate detection of chemiluminescence using the BioRad ChemiDoc Touch for 300s, with accumulative images taken every 15 secs.

Table 1. MPO spiking conditions

Results

The results of chemiluminescence detection are shown in Figure 3. A comparison of conditions 7 & 9 revealed a significant increase in chemiluminescence with the addition of DNase. Extracellular MPO is typically bound to DNA in Neutrophil Extracellular Trap (NET) structures and this may reduce MPO bioavailability in the assay without DNase. DNase cleaves and releases MPO to react in the methods of the invention to increase oxidation of luminol.

Example Four: MPO activity is detected at pathophysiological levels in human faecal samples spiked with MPO

Method

Stool homogenate was prepared at 10% w/v dELO from healthy mice and was spiked with human exogenous MPO (10 pg/mL) + Dnase (0.05 pg/pL). AZD3241 180 pM was incubated in one group as an inhibitor of MPO. The mixtures were centrifuged for 2 min at 14,800 r.p.m and the collected supernatant was further diluted to 14 total volume. The final concentrations of additives in each well were 200mM NaCl and lOmM 3 -AT (catalase inhibitor). Luminol 2mM w/v dELO was mixed with 200 mM H2O2 and simultaneously incubated before immediate detection of chemiluminescence using the BioRad ChemiDoc Touch with an exposure of 60 s. Results

Figure 4 shows the detection of faecal MPO activity in spiked human faecal samples using the method of detection of the invention (200 mM NaCl; 200 mM H2O2; 100 mM 3- AT; 100 pM AZD3241). There was minimal background activity as confirmed by MPO inhibitor subtraction (group 2).

Example Five: MPO activity measured using the methods of the invention has a linear relationship to chemiluminescence at pathophysiological ranges

Method

A reaction mixture containing 200 mM NaCl, 2 mM luminol and 20 mM H2O2 was brought to 100 uL in dJLO. Human exogenous MPO was spiked at 2, 5 and 10 pg/mL, while the control sample contained no MPO. Images were taken with 10s exposure on the BioRad ChemiDoc and densitometry was performed from integrated intensity measurements using the freeware ImageJ. Chemiluminosity (Lum) was graphed as densMPO subtracted by densControl.

Results

Figure 5 shows a linear relationship of MPO detection and chemiluminescence with the methods of the present invention in a closed system (i.e., MPO protein alone with no other biological material present). This shows that MPO levels are directly proportional to chemiluminescence when measured using the methods of the invention.

Example Six: Cyclodextrin-luminol complexes enhance the specificity and intensity of chemiluminescence detection of faecal MPO

Method

Cyclodextrin/luminol complexes (CD/L complexes) were formed using a 1 : 1 ratio of alpha-cyclodextrin and luminol sodium salt and stirred using magnetic stirring overnight for proper entrapment of luminol. The CD/L complex solution was degassed with nitrogen (g). Stool homogenate was prepared at 10% w/v d^O from healthy mice and was spiked with human exogenous MPO (10 pg/mL) + Dnase (0.05 pg/pL). AZD3241 180 pM was incubated in one group as an inhibitor of MPO. The mixtures were centrifuged for 2 min at 14,800 r.p.m and the collected supernatant was further diluted to % total volume. The final concentrations of additives in each well were 200mM NaCl and lOmM 3 -AT (catalase inhibitor). The CD/L complexes were added to each well and the reaction commenced upon addition of 200 mM H2O2. Chemiluminescence detected was measured using BioRad ChemiDoc Touch with an exposure of 60 s.

Results

Sensitivity and specificity of faecal MPO detection in faecal material from mice subjected to mild colitis was enhanced when luminol was complexed with cyclodextrins (Figure 6). This was evident from increased chemiluminescence and decreased background activity in AZD3241 MPO (180 pM) inhibition. Cyclodextrin/luminol complexes enhance the signal-to-noise ratio which increases the ability of the methods to detect true MPO activity in faecal samples and thus increases the potential of the methods for clinical application.

Example Seven: Eudragit RS PO nanoparticle encapsulation of luminol increased the specificity of chemiluminescence detection of faecal MPO while mitigating the rate of chemiluminescence degradation

Method

Luminol was encapsulated in Eudragit RS PO using a quasi -emulsion solvent diffusion oil-in-water approach. An aqueous phase containing 5 mL luminol sodium salt (2 mM) was mixed with 10 mL polyvinyl alcohol (0.4% w/v dFFO) containing 0.02% tween 20 (v/v). Separately, 3 mL 10% Eudragit RS PO was prepared in acetone and added dropwise to the aqueous phase under magnetic mixing for 15 min. The oil-in-water emulsion was sonicated with 75% power for 3 min and topped up with dEEO to a total volume of 40 mL for proper diffusion of acetone. The acetone was then distilled under vacuum pressure and the solution was centrifuged at 15,000 rpm for 1 h to obtain the pellet fraction. The Eudragit nanoparticles pellet was resuspended in 1 mL dEEO.

Stool homogenate was prepared at 10% w/v dEEO from healthy mice and was spiked with human exogenous MPO (10 pg/mL) + Dnase (0.05 pg/pL). AZD3241 180 pM was incubated in one group as an inhibitor of MPO. The mixtures were centrifuged for 2 min at 14,800 r.p.m and the collected supernatant was further diluted to 14 total volume. The final concentrations of additives in each well were 200mM NaCl and lOmM 3 -AT (catalase inhibitor). The Eudragit nanoparticles of luminol were added to each well and the reaction commenced upon addition of 200 mM H2O2. Chemiluminescence detected was measured using BioRad ChemiDoc Touch with an exposure of 60 s and the Tecan Infinite 200 Pro Fluorescence Spectophotometry.

Results Specificity of faecal MPO detection in faecal material from mice subjected to mild colitis was enhanced when luminol was encapsulated in Eudragit nanoparticles (Figure 7). This was evident by decreased chemiluminescence background in AZD3241 MPO (180 pM) inhibition, when compared to ‘assay’ AZD3241 MPO inhibition.

Further, the rate of chemiluminescence decay was markedly reduced with luminol- Eudragit encapsulation (Figure 8). The rate of decay of luminol was almost 1-fold higher when compared to encapsulated luminol.

This Example shows that Eudragit-luminol nanoparticles increase signal-to-noise ratio and have enhanced clinical application due to a greater ability to detect true MPO activity in faecal samples.