WO2012156465A1

WO2012156465A1 - Methods and kits for detecting diagnosing and monitoring diseases

Info

Publication number: WO2012156465A1
Application number: PCT/EP2012/059167
Authority: WO
Inventors: Christiaan Roelant; Kenny De Meirleir
Original assignee: Protea Biopharma Nv
Priority date: 2011-05-18
Filing date: 2012-05-16
Publication date: 2012-11-22

Abstract

The invention provides methods and kits for detecting whether a patient suffers from a Th1 or Th2 mediated disease. The method comprises contacting a sample of a body fluid with at least one redox indicator dye, and the determination whether the patient suffers from a Th1 or Th2 mediated disease is based on the nature of a color change resulting from the direct reaction of the redox indicator dye(s) with a metabolite in the body fluid sample.

Description

METHODS AND KITS FOR DETECTING, DIAGNOSING AND

MONITORING DISEASES

FIELD OF THE INVENTION

The present invention relates to methods and kits for detecting, diagnosing and monitoring diseases. The methods and kits are based on the direct reaction of redox indicator dyes with metabolites in body fluids, more particularly in urine. More particularly, the methods and kits of the present invention are useful in detecting whether a patient suffers from a Th1 or Th2 mediated disease.

BACKGROUND

T helper cells (Th cells) are a sub-group of lymphocytes, a type of white blood cell, and play an important role in establishing and maximizing the capabilities of the immune system. Th cells are specifically involved in activating and directing other immune cells, and may differentiate into two major subtypes of cells known as Th1 and Th2 cells. Whereas Th1 cells are critically involved in the generation of effective cellular immunity, Th2 cells are instrumental in the generation of humoral and mucosal immunity and allergy.

Diseases and particularly immune-mediated disorders typically involve deregulation of the Th1/Th2 balance, and can often be classified as Th1 or Th2 mediated (Th1 or Th2 dominant). Several chronic inflammatory diseases have been described as Th1 mediated diseases, i.e. multiple sclerosis, inflammatory bowel disease, Crohn's disease, diabetes, and rheumatoid arthritis. Diseases reported as being Th2 mediated include chronic fatigue syndrome, allergic rhinitis, acute eczema, asthma, urticaria and dermatitis.

In general, immune-mediated disorders are difficult to treat. Several therapies specifically aim to restore the Th1/Th2 balance by downregulating Th1 activity and upregulating Th2 activity, or vice versa. Obviously, this requires an accurate diagnosis of the disease, as inappropriate treatment results in a greater Th1/Th2 imbalance.

However, it is often difficult to obtain a specific diagnosis. Indeed, many diseases and conditions share common symptoms such as fatigue. Laboratory test results may help but are often inadequate to confirm a diagnosis. Therefore, there is a need for broad spectrum assays and kits, which allow for detecting whether a patient suffers from a Th1 or Th2 mediated disease.

Tetrazolium dyes have been used in the art to determine mitochondrial activity of cells, based on the direct transformation of the dye into a formazan in mitochondria. This has been used to determine the "redox status" of cells. WO2009/069528 suggests the use of this assay to determine, based on the redox status of cells obtained from a patient, an indication on the fatigue status of said patient. This disclosure in no way suggests the methods of the present invention.

SUMMARY OF THE INVENTION

The present invention is based on the observation that Th1 and Th2 mediated diseases can be detected and discriminated via analysis of metabolites found in body fluids. Specifically, it has been found that Th1 and Th2 mediated diseases cause an aberrant production of metabolites that are excreted in the body fluids and that the presence of these metabolites can be detected by contacting the body fluid of patients with a redox indicator dye such as a tetrazolium-based compound. Accordingly, in one aspect, the present invention provides in vitro methods for detecting whether a patient suffers from a Th1 or Th2 mediated disease by contacting a sample of a body fluid of said patient with at least one redox indicator dye at a pH-value between 5 and 1 1. The determination whether said patient suffers from a Th1 or Th2 mediated disease is based on the nature or the color change resulting from the direct reaction of the redox indicator dye with a metabolite in the body fluid sample. In particular embodiments, these methods comprise the steps of (i) obtaining a color reagent by dissolving at least one redox indicator dye compound in a buffer solution, wherein the pH- value of the buffer solution is between 5 and 1 1 , (ii) contacting the sample with the color reagent obtained in step (i), and (iii) determining the color of the color reagent after it has been contacted with the sample, whereby the color is indicative of Th1 or Th2 mediated disease infection. In particular embodiments, in step (iii) the color of the color reagent is determined within two minutes, and preferably within one minute after it has been contacted with the sample, whereby the color is indicative of Th2 mediated disease infection. In particular embodiments, in step (iii) the color of the color reagent is determined (again) 3 to 15 minutes after it has been contacted with the sample, whereby the color is indicative of Th1 mediated disease infection. In particular embodiments, in step (iii) the color of the color reagent is determined within one minute after it has been contacted with the sample, and the color of the reagent is determined again between 3 to 15 minutes after it has been contacted with the sample.

In further particular embodiments step (iii) of the method further comprises comparing the color of the color reagent with a reference.

The methods of the invention are particularly suitable for the analysis of urine. However, the analysis of other body fluids is also envisaged.

In particular embodiments of the methods of the invention described herein, the redox indicator dye is a tetrazolium-based compound, more specifically at least one redox indicator dye is used, of which one or all are selected from the group consisting of MTT, XTT, INT, MTS and NBT.

In a further aspect, the present invention provides tools and kits for detecting and discriminating indications of Th1 and Th2 mediated disease infection in body fluids, comprising at least one redox indicator dye, a buffer, a reaction container, a vial for collecting body fluid and a pipetting device.

More particularly, the invention provides kits for detecting and discriminating indications of Th1 and Th2 mediated disease infection in a body fluid of a patient, which kits comprise at least one redox indicator dye, a buffer with a pH between 5 and 1 1 , a reaction container, a vial for collecting a body fluid and a pipetting device. Optionally the kit further comprises instructions for performing the method as described herein for detecting whether a patient suffers from a Th1 or Th2 mediated disease. With these components, such kits contain all the necessary reagents and tools for performing the detection, and thus are ideally suited for self-testing or testing at home or in a (non-specialized) medical practice. More particularly, such kits are specifically designed for detecting and discriminating indications of Th1 and Th2 mediated disease infection in urine.

In particular embodiments, the kits of the present invention comprise a redox indicator dye which is a tetrazolium-based compound.

The kits according to the invention comprise a reaction container which can be a fluid reaction container or a carrier. In particular embodiments, the kits are provided as diagnostic kits suitable for detecting, diagnosing and/or monitoring Th1 and Th2 mediated disease infection. More particularly the kits are provided as diagnostic kits suitable for detecting, diagnosing and/or monitoring a disease related to inflammatory, infectious and/or chronic immune disease. More particularly the kits are provided for identifying and monitoring a 'disease state' (more particularly one which can be diagnosed as one of the diseases listed above), which can be prior to identification of the disease state (i.e. to determine/confirm the presence of a physiological problem) or for monitoring purposes after the nature of the disease has been identified (e.g. to determine efficiency of therapeutic regimen).

Accordingly, a further aspect of the present invention is the use of the kits as described herein for detection whether a patient suffers from a Th1 or Th2 mediated disease. More particularly, a further aspect of the present invention is the use of the kits for determining the therapeutic regimen of a patient suffering from general symptoms of fatigue, said method comprising determining whether said patient is suffering from a Th1 or Th2 mediated disorder using the method as described herein and adjusting said therapeutic regimen accordingly.

DETAILED DESCRIPTION The present invention will be described with respect to particular embodiments but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope.

Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-10% or less, preferably +1-5% or less, more preferably +/-1 % or less, and still more preferably +/-0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The terms or definitions used herein are provided solely to aid in the understanding of the invention.

The term "Th1 mediated disease" as used herein refers to a disease which involves an increased Th1 activity and a decreased Th2 activity. Th1 mediated diseases include, but are not limited to chronic inflammation and autoimmune diseases, multiple sclerosis, inflammatory bowel disease, Crohn's disease, diabetes, Konigs disease, Bechterev syndrome and rheumatoid arthritis

The term "Th2 mediated disease" as used herein refers to a disease which involves an increased Th2 activity and a decreased Th1 activity. Th2 mediated diseases include, but are not limited to chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME), allergic rhinitis, acute eczema, asthma, urticaria, ulcerative colitis, food intolerance, blastocystis infection and dermatitis.

The terms "Th1 " and "Th2" as used herein refer to 2 subtypes of T helper cells (Th cells) also known as Type 1 and Type 2 helper T cells, respectively.

A first aspect of the present invention provides in vitro methods for detecting aberrant metabolite production indicative of Th1 or Th2 mediated disease infection. The detection occurs in a body fluid of a patient by contacting a sample of the body fluid with at least one compound which is a redox indicator dye, such as a tetrazolium-based compound, under conditions which allow direct reduction of the redox indicator dye by metabolites in the body fluids. It has been found that the urine of patients suffering from a variety of diseases contains (levels of) metabolites not found in urine of healthy controls. For Th2 mediated diseases, these metabolites are particularly sulfur-containing metabolites, most particularly H₂S metabolites such as thiols.

Detection of these metabolites in body fluids can be used to detect, diagnose and/or monitor the presence, evolution and/or severity of diseases, more particularly Th1 and Th2 mediated diseases. Moreover, detection of these metabolites can be used to discriminate between Th1 and Th2 mediated diseases. The methods of the present invention can thus be used for detecting, diagnosing and/or monitoring the aberrant presence of metabolites per se or as a parameter for a disease state such as, but not limited to a disease state selected from inflammation, infectious diseases, chronic immune diseases and diseases with unknown etiology such as ME, fibromyalgia and the like. Detection of these metabolites in body fluids can be used to determine or diagnose the presence of a disease state and/or monitor the evolution and/or severity of a disease state or a particular disease, e.g. to monitor the efficacy of treatment etc. More particularly, for diseases which are "silent", i.e. which do not show clearly recognizable symptoms, the methods can be used to identify a physiological problem which may need medical attention. Finally, for patients suffering from symptoms which are undefined or are not necessarily physiological, such methods can be used to demonstrate the presence of a physiological symptom. Accordingly, in one aspect, the present invention provides methods for detecting metabolites in a body fluid by contacting a sample of the body fluid with at least one reducible dye at a pH-value between 5 and 1 1 . In particular embodiments, the sample is contacted with the dye at a pH which is above the pKa value of the dye.

In particular embodiments, these methods comprise the steps of (i) obtaining a color reagent by dissolving at least one reducible dye compound in a buffer solution, wherein the pH-value of the buffer solution is between 5 and 1 1 , (ii) contacting the sample with the color reagent obtained in step (i), and (iii) determining the color of the color reagent after it has been contacted with the sample. More particularly, the sample is contacted with the color reagent without adding any additional reducing agents which ensures that the reduction of the color reagent occurs by direct reaction of the sulfur- containing metabolites in the body fluid. Thus the methods of the invention provide the detection in one homogenous reaction, directly between the metabolites of the sample and the reducible dye.

In these methods, the color of the fluid sample obtained after contacting with the color reagent is indicative of the presence of metabolites in the sample of body fluid used. In further particular embodiments step (iii) of the method comprises comparing the color of obtained with the color reagent with a reference. In particular embodiments, the methods of the present invention are based on the detection of metabolites associated with Th2 mediated disease infection, more particularly sulfur-containing metabolites, most particularly H₂S metabolites such as thiols, with a compound which can be reduced, where reduction is associated with a color change of the compound. Such compounds are also referred to herein as "redox indicator dyes". The redox indicator dyes provided by the present invention are particularly dyes which can be reduced by a thiol compound, i.e. thiol-reducible dyes.

Thus, in particular embodiments the invention provides in vitro methods for detecting sulfur-containing metabolites in a body fluid which comprise contacting a sample of the body fluid with at least one thiol-reducible dye at a pH-value between 5 and 1 1 , whereby a color change resulting from the direct reduction of the thiol-reducible dye by metabolites in the body fluid, is indicative of the presence of metabolites in the body fluid.

Preferably, such a thiol-reducible dye is a non-toxic compound such as a tetrazolium-based compound, also referred to as a tetrazolium dye, such as a tetrazolium- based salt. Examples of suitable tetrazolium-based compounds include but are not limited to 2-(2'benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium (BSPT), 2, 3, 5-triphenyl tetrazolium chloride (TTC), 2-benzothiazolyl-(2)-3,5-diphenyl tetrazolium (BTDP), 2,3-di(4- nitrophenyl) tetrazolium (DNP), 2,5-diphenyl-3-(4-styrylphenyl) tetrazolium (DPSP), distyryl nitroblue tetrazolium (DS-NBT), 3,3'-[3,3'-dimethoxy-(1 , 1 '-biphenyl)-4,4'-diyl]-bis[2-(4- nitrophenyl)-5-phenyl(-2H tetrazolium (NBT), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide (XTT), 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), 2-phenyl-3-(4-carboxyphenyl)-5-methyl tetrazolium (PCPM), tetrazolium blue (TB), thiocarbamyl nitroblue tetrazolium (TCNBT), tetranitroblue tetrazolium (TNBT), tetrazolium violet, (TV), 2-benzothiazothiazolyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2- sulfoethylcarbamoyl)phenyl]-2H-tetrazolium (WST-4), and 2,2'-dibenzothiazolyl-5,5'-bis[4- di(2-sulfoethyl)carbamoylphenyl]-3,3'-(3, 3'-dimethoxy- 4,4'-biphenylene) ditetrazolium, disodium salt (WST-5). More particularly, the tetrazolium-based compound is selected from the group consisting of MTT, XTT, INT, MTS and NBT.

The redox indicator dyes used in the context of the present invention are typically light-colored or colorless compounds that undergo a reduction reaction, in the presence of a reducing agent, to yield a highly colored compound, such as, in the case of tetrazolium- based compounds, a highly colored formazan. Typically the color reaction is gradual and quantitative in that the intensity of the colored reaction product is dependent on the amount of reducing agent present. Accordingly, the measure of the color change is indicative of the amount of reducing agent present in the sample. For instance, NBT in unreduced form is yellow in solution and in reduced form is dark blue. It is noted that, in the presence of other colored compounds which may be present in the body fluid to be tested, such as urochrome of urine, the color of the dyes both in unreduced form and in reduced form, may be modified. For instance, shades of blue (e.g. in the reduction of NBT) will typically be observed as shades of green in the presence of urochrome. Accordingly, the color change referred to herein typically relates to the change in the color of the color reagent upon contacting with a test sample compared to a control sample (i.e. color reagent in the presence of body fluid sample not containing metabolites). Methods according to the present invention typically comprise contacting a body sample such as a urine sample with one or more redox indicator dyes or more particularly tetrazolium-based compounds, such as those described above, under conditions which allow reduction of said one or more dyes or compounds and observation of the color formation.

Particular embodiments of the invention accordingly provide in vitro methods, which comprise:

(i) obtaining a color reagent by dissolving at least one redox indicator dye such as a tetrazolium-based compound in a buffer solution, wherein the pH-value of the buffer solution is between 5 and 1 1 ,

(ii) contacting a sample of the body fluid with the color reagent obtained in step (i), in the absence of any other additional reducing agents, and

(iii) determining the color of said color reagent after said contacting step, where the color is indicative of Th1 or Th2 mediated disease infection.

The methods of the invention provide homogenous, single step assays for a first line diagnosis of diseases characterized by similar symptoms, such as fatigue.

The inventors found that the color change of the redox indicator dye(s) due to metabolites characteristic of Th2 mediated disease infection occurs rapidly, i.e. within half a minute after contacting the sample of the body fluid with the color reagent and without the presence of any additional reducing agents. Similar metabolites are also found in body fluids of healthy individuals, although in lower concentration. Therefore, a (more limited) color change of the redox indicator dye generally is also observed in healthy control samples, although less rapidly: usually a color change is observed after three minutes or longer, although the difference between body fluid samples from patients with a Th2 mediated condition and healthy individuals remains visible even after fifteen minutes. Surprisingly, the inventors found that with body fluid samples of patients infected with a Th1 mediated disease, this color change does not occur, or occurs far less rapidly compared to healthy control samples. Therefore, Th1 and Th2 mediated diseases can be discriminated by an analysis of the same metabolites: wherein Th2 mediated diseases are characterized by an increased concentration of the metabolites, Th1 mediated diseases are characterized by a decreased concentration.

As indicated hereabove, the methods of the present invention are characterized in that the intensity and/or the timing of the color change is monitored. In particular embodiments, the difference in intensity of the color is monitored at one time point. In particular embodiments, the detection or monitoring of the color change (step (iii)) is performed at between five or fifteen minutes after contacting the sample with the color reagent (step (ii)). After this time, it is usually possible to discriminate healthy conditions, Th1 mediated disease conditions and Th2 mediated disease conditions, based on the intensity of the color of the sample. More particularly, in this context reference can be made to no color reaction (corresponding to an insignificant change of color, as typically observed for patients suffering from Th1 mediated conditions according to the present invention), limited color reaction (corresponding to an observable color change which is not intense, typically observed in samples of patients not suffering from either Th1 or Th2 mediated disease, or healthy patients) and an intense color reaction (corresponding to an strong coloration, typically observed in patients suffering from Th2 mediated disease). Accordingly, in particular embodiments, the invention provides methods for determining whether or not a patient suffers from a th1 or th2 mediated disorder, which comprises (i) obtaining a color reagent by dissolving at least one redox indicator dye such as a tetrazolium-based compound in a buffer solution, wherein the pH-value of the buffer solution is between 5 and 1 1 ,

(ii) contacting a sample of the body fluid of the patient with the color reagent obtained in step (i), and

(iii) determining the color of said color reagent within five to fifteen minutes after said contacting step, where the detection of a strong color change is indicative of Th1 or Th2 mediated disease infection. Indeed, typically in these embodiments, the absence of a significant color change is indicative of a Th1 mediated disorder, a limited (but significant) color change is indicative of the fact that the patient does not suffer from a Th1 or Th2 mediated disorder, and a strong color change is indicative of the fact that a patient suffers from a Th2 mediated disorder. This will be discussed more in detail below.

It is noted that according to the present invention, if the patient suffers from a Th2 mediated disease, the color change will occur in less than one minute after step (ii). Thus, in particular embodiments, step (iii) as described hereabove is performed within two minutes after step (ii), preferably within one minute after step (ii), for example 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 seconds after step (ii). In these embodiments, an observation of a color change of the redox indicator dye in step (iii) is indicative of Th2 mediated disease infection. Accordingly, in particular embodiments, the invention provides methods for determining whether or not a patient suffers from a Th2 mediated disorder, which comprises

(iii) determining the color of said color reagent within 1 minute or less after said contacting step, where the detection of a strong color change is indicative of Th2 mediated disease infection. In further embodiments of the methods of the present invention, the color (change) additionally or alternatively observed at a later time, i.e. more than one minute after contacting the sample with the color reagent (step ii), preferably between 3 and 15 minutes after step (ii), more preferably between five and fifteen minutes after step (ii), even more preferably between ten and fifteen minutes after step (ii). Where no or only a very limited color change is observed between 3 and 15 minutes, this is indicative of Th1 mediated disease infection. Accordingly, in particular embodiments, step (iii) as described hereabove is performed, (and/or additionally performed ), between 3 and 15 minutes after step (ii) preferably between ten and fifteen minutes after step (ii), and even more preferably between ten and fifteen minutes after step (ii). Accordingly, in particular embodiments, the invention provides methods for determining whether or not a patient suffers from a Th1 mediated disorder, which comprises

(iii) determining the color of said color reagent between 10 and 15 minutes after said contacting step, whereby the absence of a (significant) color reaction is indicative of Th1 mediated disease infection.

As will be detailed below, in particular embodiments of the methods of the invention, the color is determined by comparing said color to that of a reference or reference sample.

The color change of the redox indicator dye can be assessed by visual inspection or spectrophotometrically, as explained further in this text, of the fluid mixture (i.e. sample + color reagent). Spectroscopy generally allows detection of more subtle color changes, compared to visual inspection. Spectroscopy also allows quantification of the color change, for example via the Absorbance (Optical density) or Transmission, as known by the person skilled in the art. Because of this quantification, these methods allow for an increased accuracy, particularly when the measurement is performed shortly after step (ii).

As indicated above, the methods of the present invention may comprise an assessment of the color intensity. It is noted that in the present context reference is made to both the assessment of a color "change" and to the assessment of "color". It will be understood by the skilled person that, depending on the circumstances, these concepts can be interchangeable. In the context of the present invention it is envisaged that both absolute color intensity and difference in color compared to the sample prior to contacting with the reagent (or contacted with buffer alone) can be of interest.

In particular embodiments, the methods of the invention comprise comparing the otpical density to that of a healthy control. For example, in particular embodiments, the method according to the invention comprises a measurement of the optical density of the sample is at three minutes after step (ii). It is envisaged, for example that in such a method, an optical density below 2y can be considered indicative of a Th1 mediated disease condition, an optical density equal to or higher than 3y is indicative of a healthy condition, and an optical density higher than 20y is indicative of Th2 mediated disease condition. Herein, "y" is the expected optical density of a healthy control sample directly after step (ii) (time zero).

As a further example, in particular embodiments of the methods of the present invention, the optical density of the sample is made fifteen minutes after step (ii). It can be envisaged that in particular embodiments, an optical density below 4y is considered indicative of a Th1 mediated disease condition, an optical density equal to or higher than 10y is indicative of a healthy condition, and an optical density higher than 50y is indicative of Th2 mediated disease condition. Herein, "y" again is the expected optical density of a healthy control sample directly after step (ii) (time zero).

Reduction of a redox indicator dye by metabolites such as a thiol compound will typically occur only in a certain range of pH values. For tetrazolium-based compounds this implies that the dye should be contacted with the sample at a pH between 5 and 1 1 , depending on the tetrazolium-based compound used. For instance, for NBT, typically the pH is preferably around 8,5 or higher. Accordingly, in certain embodiments, the dye is contacted with the sample at a pH between 8 and 1 1 , more preferably between 8 and 9. In particular embodiments, the dye is contacted with the sample at a pH which is above the pKa value of the dye. Typically the suitable pH is obtained by addition of a buffer having a buffering capacity in the indicated pH range. More particularly, the dye can be dissolved in a buffer with suitable pH and then brought into contact with the sample. Alternatively, dye, buffer and sample are contacted simultaneously. Thus, where reference is made herein to a buffer with a certain pH or pH range, it is intended to refer to the fact that addition of the buffer (either in dry form or in solution) to the dye will ensure a pH of the solution which is about that certain pH value or within that pH range.

Different chemical compounds can be used as buffers and/or to make buffer solutions and are well known to the person skilled in the art. Typically sodium phosphate and potassium phosphate are used. Examples include, but are not limited to solutions of 10mM sodium or potassium phosphate. The buffer may contain or be mixed with other components which improve stability of the reaction product.

Accordingly, suitable buffers for use in the methods according to the present invention have a buffering capacity between a pH of 5 and 1 1. In particular embodiments, the buffers have a buffering capacity between a pH of 8 and 1 1. Non-limiting examples of such buffers are Tris buffer, phosphate buffered saline and diethanolamine buffer and combinations thereof. In particular embodiments, a mixture of buffers is used, such as for example a mixture of phosphate buffer and Tris buffer is used. In more specific embodiments, the tetrazolium-based compounds are dissolved in a mixture of 2 volumes of phosphate buffered saline (100mM) and 1 volume of Tris buffer (100 mM) at a concentration between 1 and 20 mg/ml_, such as between 1 and 10 mg/ml_, wherein the resulting solution has a pH value between 8,0 and 9,0.

In the methods of the present invention, the reduction of the redox indicator dye, such as the tetrazolium-based compound(s), occurs by direct reaction of metabolites in the sample of body fluid with the color reagent comprising at least one redox indicator dye such as a tetrazolium-based compound under buffered conditions. Accordingly, the reduction reaction of the dye is ensured by metabolites present in the sample. No reducing agents need to be added and the metabolites need not be isolated from the sample. Thus, contrary to most prior art assays based on the detection of tetrazolium-based compounds, where the reduction of the tetrazolium based compounds is ensured by the addition of an enzyme (e.g. used as a label for an analyte-specific reagent), in the methods of the present invention, the redox indicator dye is contacted directly with the sample of body fluid and is reduced by metabolites in the sample.

Accordingly, in particular embodiments, the methods according to the present invention do not make use of compounds other than the redox indicator dye, buffer and solvent; in particular the methods do not make use of reducing agents, enzymes, coenzymes or electron transfer agents. In further particular embodiments, the methods according to the present invention do not make use of a combination of reagents, which for example comprises a tetrazolium-based reagent, a flavin-dependent enzyme bound to flavin, an electron transfer agent, and a nitrite salt or for example comprises tetrazolium- based reagent, an aluminum compound, Fe³⁺, a phenazine electron transfer agent and/or a flavin agent.

The methods of the present invention are based on the detection of an unusual production of metabolites by the body when in a diseased state compared to healthy controls, which metabolites are excreted in body fluids. The methods of the present invention are further based on the fact that the production of metabolites is different in Th1 and Th2 mediated diseases. Most particularly the body fluid is urine. More particularly the body fluid is whole body fluid, such as whole urine (see below). Other suitable body fluids include sweat, sputum, prostate fluid, lumbal fluid, vaginal secretion, and serum. The methods of the present invention however are of particular interest in the context of home- monitoring and self-testing. Accordingly, in particular embodiments of the invention, the body fluid is a body fluid which is a readily self-collectable body fluid, such as urine or saliva (sputum).

The sample of body fluid for use in the methods of the present invention is typically a fresh sample of body fluid. More particularly the methods are suitable for use on freshly collected urine, more particularly on a morning urine sample. Contrary to prior art methods relating to the detection of mitochondrial activity, the methods of the present invention are directed at detecting metabolites in body fluid, i.e. not detecting enzymatic activity within the cells. Typically, the body fluid sample as such ('whole') is used directly in the methods of the invention (with the exception of blood, where the use of serum is preferred), as the presence of a limited number of cells in the sample may not interfere. However, under particular circumstances it may be desirable to make use of a pre-treated sample, such as for example but not limited to, a sample of body fluid from which certain molecules, compounds, cells, particles or any other material that could interfere with the reduction reaction of the metabolites with the redox indicator dye, herein generally referred to as "interfering factors" are removed. Methods for removing interfering factors from body fluids such as urine are known to the skilled person and include, but are not limited to sedimentation, filtration, chromatography etc. In particular embodiments, a sedimentation step is performed to remove white blood cells from the body fluid sample to be used in the methods of the invention. In some cases, presence of cells may influence the outcome of the assay. However, in most body fluids (except blood), the number of cells is relatively limited such that removal of the cells is not necessary. If necessary, the amount of color reagent used can be adjusted to ensure that presence of cells does not affect the outcome of the assay (i.e. adjusting the sensitivity of the assay). The methods of the present invention are particularly directed at detection of metabolites in "fluid". This also implies that the detection of the color occurs on the fluid rather than on specific components which may be present therein.

In particular embodiments the methods of the invention are used on a sample of body fluid obtained from a mammal, more particularly from a human.

As will be detailed below, the methods of the present invention can be carried out either in solution or on a solid carrier. In particular embodiments, the methods are carried out in solution in a reaction vial, typically in volumes of 50μΙ-2000μΙ. In these embodiments, the methods of the invention comprise contacting a suitable volume of collected body fluid sample with the redox indicator dye and buffer solution, which is optionally pre-mixed as a "color reagent". The relative amounts of the different reagents to be combined can be determined by the skilled person using positive and negative control samples (see below). In particular embodiments, the methods of the invention comprise contacting 2 volumes of body fluid such as urine with 1 volume of color reagent (e.g. tetrazolium-based compound dissolved in appropriate buffer).

The methods of the present invention are based on the determination of the color of a redox indicator dye, which color is indicative of the presence of metabolites in a sample. After contacting the sample of body fluid with the color reagent comprising at least one redox indicator dye, the color of the color reagent is determined (i.e. where the method is carried out in solution, this may imply determining the color of the reaction solution). Determining the color of the resulting color reagent can for example be performed by comparing it with a reference. Such a reference can for example be the color of a control. For instance a negative control can be obtained by reacting a sample of the same body fluid of a healthy individual with the redox indicator dye (under the same conditions as those used for the test sample). Additionally or alternatively, positive controls can be obtained by reacting a sample of the same body fluid of a patient diagnosed with a particular disease with the redox indicator dye (under the same conditions as used for the test sample) or by spiking a sample of a healthy individual with metabolites. Additionally or alternatively, the color of the resulting reagent can be compared with the colors of a color chart or a color code, assigning a particular color to a healthy state or a disease state. Such a color chart or color code can also comprise a series of shades, wherein the different shades indicate different levels of severity of the disease. In further particular embodiments the color of the color reagent is determined spectrophotometrically. casein further particular embodiments, the optical density of the resulting reagent at one or more wavelengths can be compared to threshold values. In particular embodiments, and especially when the color reagent comprises MTT, the color of the color reagent is determined spectrophotometrically at one or more wavelengths between 540 and 640, preferably between 610 and 570 nm, more preferably between 600 and 580 nm, even more preferably between 595 and 585 nm, for example at 590 nm. It will be understood by the skilled person that where detection of color is not possible, conversion into e.g. scales of gray of the color reaction is also possible. A further aspect of the present invention provides tools and kits for carrying out the methods of the present invention.

In particular embodiments, kits are provided comprising at least one redox indicator dye such as a tetrazolium-based compound, a suitable buffer, a vial for collecting a body fluid, a pipetting device and a reaction container. As detailed below, the term reaction container encompasses both fluid reaction containers and carriers.

The kits and tools of the present invention are based on the detection of metabolites with a redox indicator dye, more particularly a tetrazolium-based compound which can be reduced by a thiol compound, whereby reduction is associated with a color change of the compound.

Different kits and tools are envisaged which are suitable for carrying out the methods of the present invention, and the physical format in which the reagents are presented are not critical to the invention. According to particular embodiments, the kits comprise a color reagent comprising the at least one redox indicator dye.

For use in the methods and kits of the present invention, the redox indicator dye may be in any form that is suitable for undergoing a reduction reaction with metabolites by directly contacting the dye with a sample of a body fluid. For example, the dye may be present as either a wet or a dry composition. By wet composition is meant a fluid composition, typically an aqueous composition, such as a buffer solution as described herein. By dry compositions is meant a composition that is not fluid, i.e., in dry form, such as a composition that is substantially free of uncombined water. Such a dry form may comprise or may not comprise constituents of a buffer, which, upon contacting with water or another solution, can be dissolved therein (e.g. powdered buffer solution).

Accordingly, in particular embodiments of the kits of the present invention, the at least one redox indicator dye may be dissolved in the buffer solution so as to provide a ready-to use solution that can be directly contacted with the sample of body fluid. Alternatively, the redox indicator dye and/or the buffer or the color reagent may be provided in powder form. Independently thereof, the dye and buffer may thus be provided in separate modules or compartments for mixing prior to usage, i.e. prior to contacting with the urine sample, or may be provided as one reagent.

In particular embodiments, the kits according to the present invention may further comprise a vial for collecting a body fluid and a pipetting device. In more particular embodiments kits are provided for urine testing which comprise a vial for collecting urine. After a sample is collected in the provided vial, the pipetting device can be used to add a suitable volume of the body fluid sample directly to the reaction container comprising one or more other reagents of the kit. Examples of suitable collection vials are disposable canisters with a cap and are known to the skilled person. Similarly, suitable pipetting devices are typically disposable (e.g. plastic) pipettes for volumes of 50-2000μΙ and are known to the skilled person. Where the volume of collectable body fluid is limited (e.g. sweat or saliva), the collection vial may also be a device particularly suited for collecting the body fluid (e.g. optionally comprising swabs or other devices). Alternative vials suitable for collecting different types of body fluids are known in the art.

In particular embodiments, kits are provided which are configured for carrying out the methods of the invention in solution, i.e. where the body fluid, such as urine, is contacted with the buffer and dye in one reaction container, which is a fluid container. In particular embodiments, kits are provided which comprise a fluid reaction container comprising therein a color reagent (i.e. a redox indicator dye readily dissolved in a suitable buffer), a vial for collecting a body fluid and a pipetting device. Contacting the urine sample with the color reagent is ensured by pipetting an appropriate volume of the collected urine directly into the reaction container.

Suitable reaction containers are known to the skilled person and the size and shape thereof is not critical to the invention. In particular embodiments, the reaction container is a fluid container. Typically, such a reaction container is a recipient of 50μΙ- 2000μΙ, such as an eppendorf, cuvette, a multiwell plate, or any other such recipient.

In particular embodiments, kits are provided which are configured for carrying out the methods of the invention on a reagent container which is a carrier. The term carrier as used herein refers to a material through which a fluid reagent can pass. Examples of suitable materials are known to the person skilled in developing diagnostic test-kits and include bibulous or non-bibulous materials. By bibulous is meant a material that exhibits preferential retention of one or more components as would occur, for example, in materials capable of absorbing or "imbibing" one or more components, as occurs in chromatographic separations. Examples of bibulous materials include, but are not limited to: nylon, untreated forms of paper, nitrocellulose and the like which result in chromatographic separation of components contained in liquids which are passed therethrough.

Alternatively, the substrate may be non-bibulous. Non-bibulous substrates include inert porous matrices which provide a support for the various members of the signal producing system, described infra, and may have a positive charge. These matrices are generally configured to provide a location for application of a physiological sample, e.g., blood, and detection of the chromogenic product produced by the dye of the signal producing system. As such, the matrix is typically one that is permissive of aqueous fluid flow through it and provides sufficient void space for the chemical reactions of the signal producing system to take place.

In particular embodiments, the reagent container is provided in the form of a test strip. In particular, the invention encompasses (dry) strips for detecting Th1 and Th2 disease infections. In the broadest sense, the reagent test strip includes a solid support and typically a dry dye, more particularly a dry tetrazolium-based compound present thereon. The dye is either coated on or associated with the material of the reagent container.

In particular embodiments, the dry dye is provided in the form of a dry color reagent, which is made up of all of the compounds necessary to produce a detectable signal in the presence of the metabolite(s). More particularly, the test strip comprises a dry color reagent comprising e.g. a tetrazolium-based compound and a buffer having a pH-value between 5 and 1 1 , described in greater detail above. Additionally or alternatively, the test strip comprises only the dye and the dye is contacted with the buffer prior to adding the sample or the urine sample is mixed with a suitable buffer solution prior to contacting with the test strip. Thus, kits according to these embodiments may or may not include the buffer separately.

The test strips are envisaged to be configured in a similar way to the test strips known for other diagnostic uses. More particularly, the test strip may comprise a detection zone comprising the dye (or color reagent) which is distal on the strip and a sample application zone proximally on the strip whereby upon application of the sample of body fluid in the application zone, the urine will be moved (typically by capillary forces) through the strip material and will reach the detection zone where metabolites present in the sample will react with the color reagent.

In particular embodiments the reagent container may be in the form of a carrier that is affixed to a solid support. The support may be a plastic, e.g. polystyrene, nylon, or polyester, or metallic sheet or any other suitable material known in the art. The strip may also be configured in more complex arrangements, e.g., where the test pad is present between the support and a surface layer, where one or more reagents employed in sample processing may be present on the surface layer. In addition, flow paths or channels may be present on the test strip, as is known in the art.

Alternatively the reaction container may be in the form of a carrier provided in a container, such as a porous matrix in a longitudinal vial (e.g. column). Typically, in these embodiments a sample will flow vertically through the carrier as a result of gravity or by application of a force.

The dimensions and porosity of the material of the carrier may vary greatly, where the matrix may or may not have a porosity gradient, e.g., with larger pores near or at the sample application region and smaller pores at the detection region. The matrix may be configured as a membrane test pad and be affixed to a solid support, where the support may be a plastic (e.g., polystyrene, nylon or polyester) or metallic sheet or any other suitable material known in the art.

The carriers may be fabricated employing any convenient protocol. One convenient protocol is to contact at least the test pad portion of the strip with an aqueous composition that includes the redox indicator dye and a buffer solution having a pH value between 5 and 1 1. Conveniently, the test pad may be immersed in the aqueous composition, maintained therein for a sufficient period of time and then dried, whereby the test pad of the reagent test strip which has associated therewith the reagent composition is produced.

In particular embodiments, the kits provided according to the invention further provide tools for interpreting the obtained result, i.e. the (lack of) color reaction observed in the reaction container. Indeed, the reagents of kits of the invention are configured such that the color observed in the reaction container is indicative of the presence of certain metabolites in the sample, and thus indicative of Th1 or Th2 mediated disease infection. Determining the color of the resulting color reagent (and/or interpreting the result of the assay) can for example be performed by comparing the observed color with a reference. Accordingly, the kits according to the present invention may further comprise a color reference. Such a color reference can for example be a color chart or a color code, typically comprising different shades corresponding to the colors obtained with different concentrations of metabolite present in the body sample. In particular embodiments references are provided assigning a particular color to a healthy state or a (particular stage of a) disease state. Such a color chart or color code can also comprise different shades of the color reaction, with an indication of how the colors are indicative of different levels of severity of disease. Such references may be provided in the form of one or more reaction containers containing different shades of the dye or may be provided on paper reproducing the different shades of the dye. In particular embodiments, the kits according to the present invention may further comprise a second color reference, wherein one color reference is for detecting or monitoring Th1 mediated disease infection and the other for Th2 mediated disease infection. In other embodiments, the kits according to the present invention may further comprise one color reference with two legends, one for detecting or monitoring Th1 mediated disease infection and the other for Th2 mediated disease infection. In addition to above mentioned components, the kits of the present invention may typically further include instructions for using the components of the kit to practice the methods according to the invention. The instructions for practicing the methods of the invention are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.

Typically, the kits of the present invention do not comprise any additional reducing agents and the metabolites need not be isolated from the sample. Thus, contrary to most prior art kits based on the detection of tetrazolium-based compounds, where the reduction of the tetrazolium based compounds is ensured by the addition of an enzyme (e.g. used as a label for an analyte-specific reagent), in the kits of the present invention, the redox indicator dye is contacted directly with the urine sample and is reduced by metabolites in the sample.

Accordingly, the kits according to the present invention do not comprise a combination of reagents, which for example comprises a tetrazolium-based reagent, a flavin-dependent enzyme bound to flavin, an electron transfer agent, and a nitrite salt or for example comprises tetrazolium-based reagent, an aluminum compound, a phenazine electron transfer agent and a flavin agent. In particular embodiments the invention accordingly provides kits consisting of a limited number of components which provide all the necessary tools for ensuring detection of metabolites in a body fluid. More particularly these tools are a collection vial for collecting body fluid, a pipette, and a reaction vial which comprises the color reagent dissolved in the appropriate buffer and a packaging insert comprising details on the interpretation of the color reaction. Such kits are particularly suitable for home-testing or testing in a medical practitioner's office.

The methods and kits according to the present invention may be (in vitro) diagnostic methods and diagnostic kits that can be used for detecting, diagnosing and/or monitoring a Th1 or Th2 mediated disease infection. In particular, the methods and kits of the present invention are suitable for detecting, diagnosing and/or monitoring of inflammatory, infectious and/or chronic immune diseases, such as infectious diseases (including viral, bacterial, prion-related infectious diseases), cancers, mental disorders, autism, immunologic disorders (including auto-immune diseases, rheumatoid arthritis and the like), nervous system disorders, disorders of the gut, defective wound healing processes, intoxications (with e.g. heavy metals, pesticides and the like), unexplained syndromes, aging related processes, biological clock related processes and diseases with unknown etiology such as CFS/ME, fibromyalgia and the like. Thus the methods and kits of the present invention can be used to detect the presence of a "disease state" (which may be, but is not limited to any one of the disease states listed above) or to monitor the severity of a previously identified disease state.

The methods and kits of the present invention are useful in alerting patients to seek medical advice and/or in assisting medical staff in recognizing a disease state where a disease may be "silent" and difficult to diagnose.

The methods and kits according to the present invention are broad-spectrum detection tools. These may be combined with one or more additional assays or test kit (components) to further identify the nature of the disease state.

For instance the methods and kits for determining a disease state based on the presence of metabolites in a body fluid may be combined with an assay which determines e.g. the presence of heavy metals in a body fluid sample, which is known to be indicative for CFS/ME. Alternatively and for example, the methods and kits of the present invention may be combined with a test for measuring the presence of organophosphorus compounds in the urine sample, which are known to be indicative for chronic immune diseases.

The present invention is further illustrated by the following non-limiting example:

EXAMPLE

Correlation of MTT reduction by urine with disease classification The method of the present invention was tested on urine obtained from different patients suffering from diseases which are known to be Th1 or Th2 mediated.

For the patients suffering from Th1 mediated diseases, the diseases included Konigs disease (1 patient), rheumatoid Arthritis (12 patients), Bechterev syndrome (ankylosing spondylitis - 2 patients) and lupus (5 patients). For the patients suffering from Th2 mediated diseases, the diseases included CFS/ME (610 patients/tests), ulcerative colitis (10 patients), food intolerance (5 patients), blastoystis infection (2 patients) and autism (25 patients). Also the urine from 73 healthy individuals ("ThO") was tested in the same way.

For the tests, 200 μΙ solution of colorimetric reagent is contacted with 400 μΙ morning urine in a reaction vial. The reaction vial content is mixed and the color change is observed after one, two, three and fifteen minutes. In order to quantify the results, the optical density (O.D.) of the reaction vial content is measured via UV-Vis spectroscopy at a wavelength of 590 nm.

The test results obtained with this method for the healthy controls (ThO), and various Th1 or Th2 mediated diseases conditions are shown in Table 1. From these results, it is clear that after one minute, a significant color change is only observed with Th2 mediated conditions, as no color change is obtained with ThO or Th1 mediated conditions.

After two minutes, a substantial color change is observed with Th2 mediated conditions, whereas still no significant color change is observed with ThO or Th1 conditions. After three minutes, a slight color change is observed with healthy controls, still no color change is observed with Th1 mediated conditions, and a dramatic color change can be seen with

Th2 mediated conditions. After fifteen minutes, a significant color change is observed on healthy controls and still no significant color change is observed with Th1 mediated conditions. With Th2 mediated conditions, the optical density of the reaction vial content exceeds the maximum which can be measured by the spectrophotometer.

Thus, these results show that with the tests according to the present invention, Th2 mediated disease conditions can be detected within one minute by spectroscopy and even by visual inspection. Where the samples show no indication of Th2 infection, a Th1 mediated disease condition and a healthy condition can be discriminated within fifteen minutes by visual inspection. Using spectroscopy, these conditions can be discriminated within two or three minutes.

Table 1 : test results of urine samples of patients diagnosed with various Th1 mediated disease conditons, and healthy controls.

Urine/MTT O.D. (590 nm) after

1 min 2min 3min

Condition: Healthy controls (ThO) n=73 0.035 0.060 0.110 1.200

CFS/ME (Th2) n=610 0.315 0.650 1.200 > 3.000

Ulcerative Colitis (Th2) n=10 0.325 0.920 1.560 >3.000

Food Intolerance (Th2) n=5 0.650 0.955 1.400 >3.000 Blastocystis Infection (Th2) n=2 0.600 1.250 1.800 >3.000

Autistic (Th2) n=25 0.400 0.750 1.150 >3.000

Mean O.D. Th2 0.458 0.905 1.422 >3.000

Konigs disease (Th1 ) n=1 0.030 0.035 0.035 0.035

Rheumatoid Arthritis (Th1 ) n= 12 0.025 0.035 0.035 0.125

Bechterew (Th1 ) n=2 0.030 0.030 0.045 0.040

Lupus (Th1 ) n=5 0.035 0.035 0.040 0.040 Mean O.D. Th1 0.030 0.033 0.038 0.060

Claims

1. An in vitro method for detecting whether a patient suffers from a Th1 or Th2 mediated disease, which comprises contacting a sample of a body fluid of said patient with at least one redox indicator dye dissolved in a buffer solution with a pH-value between 5 and 1 1 , and determining whether said patient suffers from a Th1 or Th2 mediated disease based on the nature of the color change resulting from the direct reaction of said redox indicator dye with a metabolite in said sample of body fluid.

2. The in vitro method of claim 1 , which comprises:

(i) obtaining a color reagent by dissolving said at least one redox indicator dye compound in a buffer solution, wherein the pH-value of the buffer solution is between 5 and 1 1 ,

(ii) contacting the fluid sample with the color reagent obtained in step (i) in the absence of any additional reducing agents, and

(iii) determining the color of said color reagent in said fluid sample obtained in step (ii) after said contacting step, whereby the color is indicative of Th2 or Th1 mediated disease infection.

3. The in vitro method of claim 2, wherein step (iii) comprises determining the color of said color reagent in said fluid sample within 2 minutes after said contacting step, whereby an intense color reaction at this time point is indicative of Th2 mediated disease infection.

4. The in vitro method of claim 2 or 3, wherein step (iii) comprises or further comprises determining the color of said color reagent between 3 to 15 minutes after said contacting step, whereby the absence of a color reaction at this time point is indicative of Th1 mediated disease infection.

5. The in vitro method of claim 2, wherein step (iii) further comprises comparing the color of the color reagent with a reference.

6. The in vitro method of any one of claims 1 to 5, wherein the body fluid is urine.

7. The in vitro method of any one of claims 1 to 6, wherein the redox indicator dye is a tetrazolium-based compound.

8. The in vitro method of any of claims 1 to 7, wherein the at least one redox indicator dye is chosen from the group consisting of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide (XTT), 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium (MTS) and 3,3'-[3,3'-dimethoxy-(1 , 1 '-biphenyl)-4,4'-diyl]-bis[2-(4- nitrophenyl)-5-phenyl(-2H tetrazolium (NBT).

9. A kit for detecting and discriminating indications of Th1 and Th2 mediated diseases in a body fluid, comprising at least one redox indicator dye, a buffer with a pH between 5 and 1 1 , a reaction container, a vial for collecting a body fluid, and a pipetting device and optionally instructions for performing the method according to any one of claims 1 to 7, wherein said kit does not comprise any additional reducing agents.

10. The kit of claim 9, which further comprises a color reference corresponding to the envisaged color reaction for a Th1 and Th2 mediated disease.

1 1. The kit of claim 9 or 10, wherein said body fluid is urine.

12. The kit of any one of claims 9 to 11 , wherein the redox indicator dye is a tetrazolium- based compound.

13. The kit of any one of claims 9 to 12, wherein the reaction container is a fluid reaction container or a carrier.