WO2017143985A1 - Procédé de détection d'un marqueur de la tuberculose active - Google Patents

Procédé de détection d'un marqueur de la tuberculose active Download PDF

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WO2017143985A1
WO2017143985A1 PCT/CN2017/074390 CN2017074390W WO2017143985A1 WO 2017143985 A1 WO2017143985 A1 WO 2017143985A1 CN 2017074390 W CN2017074390 W CN 2017074390W WO 2017143985 A1 WO2017143985 A1 WO 2017143985A1
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sample
mycolic acid
exposing
antigen
substrate
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PCT/CN2017/074390
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English (en)
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Johannes Theodorus CASTROP
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Kei International Limited
Tomorrows Ip Limited
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Publication of WO2017143985A1 publication Critical patent/WO2017143985A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria

Definitions

  • the present invention relates to a method of detecting a marker for active tuberculosis, and a method for pre-treating a sample from a human or animal suspected of having active tuberculosis.
  • Mycobacterium tuberculosis is a pathogenic bacterial species in the family Mycobacteriaceae and the causative agent of most cases of tuberculosis (TB) .
  • TB skin test also called the Mantoux tuberculin skin test
  • TB blood tests also called interferon-gamma release assays or IGRAs
  • chest radiography X-ray
  • a definitive diagnosis of TB is made by identifying M. tuberculosis in a clinical sample (e.g. sputum, pus, or a tissue biopsy) .
  • a clinical sample e.g. sputum, pus, or a tissue biopsy
  • WO 2005/116654 and WO 2013/186679 describe methods based on this principle and disclose methods of detecting antigen specific biomarker antibodies for the diagnosis of active tuberculosis, by detecting binding of antibodies against mycolic acid antigens to immobilised mycolic acid antigens.
  • the inventor has observed that when a method of detecting a marker for tuberculosis, which is based on binding of antibodies against mycolic acid antigens in a sample to immobilised mycolic acid antigens, is applied to a sample derived from a healthy subject, there is also a high degree of binding of materials to immobilised mycolic acid antigens.
  • a sample from a healthy subject should not contain antibodies directed against Mycobacteria, such as antibodies against mycolic acid antigens. This high degree of binding to immobilised mycolic acid antigens of materials present in the samples of healthy subjects thus indicates that samples derived from healthy subjects contain materials which bind to mycolic acid antigens, but which are not indicative for tuberculosis.
  • the present invention aims to overcome the problems that derive from the binding of materials that are not indicative for active tuberculosis to immobilised mycolic acid antigens.
  • the invention in a first aspect relates to a method of detecting a marker for active tuberculosis, comprising the steps of: I) providing a sample from a human or animal suspected of having active tuberculosis; II) filtering the sample using a cut-off membrane filter with a nominal pore size of 10 nm or more and less than 100 nm; III) exposing at least part of the filtered sample to a substrate carrying an immobilised mycolic acid derived antigen; and IV) detecting binding of antibodies to said mycolic acid derived antigen.
  • the invention in a second aspect relates to a method for pre-treating a sample from a human or animal suspected of having active tuberculosis for detection, comprising the steps of: I) providing a sample from a human or animal suspected of having active tuberculosis; and II) filtering the sample using a cut-off membrane filter with a nominal pore size of 10 nm or more and less than 100 nm.
  • the inventor has surprisingly found that if a sample from a human or animal is filtered using a cut-off membrane filter with a nominal pore size of 10 nm or more and less than 100 nm before being subjected to a detection substrate carrying an immobilised mycolic acid derived antigen and detecting binding of antibodies to the antigen, the binding signal of materials that are not indicative for active tuberculosis to immobilised mycolic acid antigens is significantly reduced. This reduces the risk of that healthy subjects test positive for active tuberculosis.
  • Fig. 1 shows a diagram with results of tests with cut-off membrane filter with different cut-off sizes and samples derived from healthy persons (Control) and persons having active tuberculosis (TB+) .
  • the invention relates to a method of detecting a marker for active tuberculosis, comprising the steps of: I) providing a sample from a human or animal suspected of having active tuberculosis; II) filtering the sample using a cut-off membrane filter with a nominal pore size of 10 nm or more and less than 100 nm; III) exposing at least part of the filtered sample to a substrate carrying an immobilised mycolic acid derived antigen; and IV) detecting binding of antibodies to the antigen.
  • the invention further relates to a method for pre-treating a sample from a human or animal suspected of having active tuberculosis so that the sample can be used in a detection assay in which antibodies against mycolic acid derived antigens are detected, comprising the steps of: I) providing a sample from a human or animal suspected of having active tuberculosis; and II) filtering the sample using a cut-off membrane filter with a nominal pore size of 10 nm or more and less than 100 nm.
  • one or more samples from a human or animal suspected of having active tuberculosis may be compared to a sample from a human or animal which is confirmed to be healthy.
  • all samples in one analysis undergo the same treatment in accordance with the steps of the method of the invention.
  • the strong decrease in background signal and the associated lower risk of false positives make it even possible to reliably diagnose whether a person has tuberculosis without the necessity of a reference sample obtained from a healthy subject.
  • the strong decrease in background signal and the associated lower risk of false positives also make it possible to reliably diagnose whether a person has tuberculosis without the necessity to divide a sample from a human or animal suspected of having active tuberculosis into two sample fractions of which one is exposed to mycolic acid antigens before the two fractions are exposed to a detection substrate with mycolic acid antigens such as for instance described in WO 2005/116654 or WO 2013/186679.
  • a sample from a human or animal suspected of having active tuberculosis is provided.
  • These samples may in particular be blood samples of a human or animal suspected of having active tuberculosis.
  • the sample is preferably a whole blood sample.
  • the sample may be obtained by any regular means of obtaining blood from a subject.
  • samples may be used that have been collected at an earlier stage, stored until use under suitable conditions and provided at a suitable moment.
  • a sample may be used in the detection method of the invention on the spot, i.e. as a point of care test.
  • the sample is preferably pre-filtered or separated to plasma or serum before the step of filtering the sample using a cut-off membrane filter with a nominal pore size of 10 nm or more and less than 100 nm.
  • a suitable filter for such a pre-filtering step is a 0, 2 micron filter. Pre-filtering the sample with a filter with larger pores than the cut- off membrane filter also prevents that aggregates of materials such as blood cells cause clogging of the cut-off membrane filter.
  • the sample is preferably a blood derived sample.
  • the sample may be a whole blood sample, a plasma sample or a serum sample.
  • Blood serum is blood plasma without clotting factors and is preferred as plasma.
  • the word plasma in this application may therefore as well refer to (blood) serum.
  • Serum is preferred because it contains less different materials than blood plasma which may lead to aspecific interactions or unwanted biological activity.
  • serum may have a lower viscosity than blood plasma. Using serum therefore may circumvent the need for diluting a sample, which saves time and materials.
  • Plasma or serum in this application may therefore also refer to diluted plasma or serum.
  • a dilution of the blood or plasma may therefore be implemented in the method of the invention, such as 5 to 10 x dilution, a 10 to 20 x dilution, a 20 to 50 x dilution, a 50 to 100 x dilution, a 250 to 5000 x dilution, a 750 to 1250 x dilution, such as for instance a 5 x, 10 x, 20 x, 50 x, 100 x, 200 x, 500 x, 4000 x, 2000 x or 1000 x dilution.
  • such dilution may take place before the step of separating the plasma from the blood step (pre-filter step or separating step) .
  • dilution may take place after the pre-filter step or separating step, and before the step of filtering the sample using a cut-off membrane filter with a nominal pore size of 10 nm or more and less than 100 nm.
  • the dilution may also be performed as an alternative to said pre-filter/separating step, and before the step of filtering the sample using a cut-off membrane filter with a nominal pore size of 10 nm or more and less than 100 nm.
  • Dilution may be performed with any suitable diluent, for example a PBS based buffer, such as a blocking buffer.
  • a PBS based buffer such as a blocking buffer.
  • Such buffer may for example be a PBS/AE buffer comprising NaCl, KCl, KH 2 PO 4 , Na 2 HPO 4 and EDTA in water at physiological pH.
  • Such buffer may be a PBS based buffer consisting of 8.0 g NaCl, 0.2 g KCl, 0.2 g KH 2 PO 4 , and 1.05 g Na 2 HPO 4 per liter of double distilled, deionized water containing 1 mM EDTA and 0.025% (m/v) sodium azide which is adjusted to pH 7.4.
  • the whole blood sample or plasma or serum may be further diluted with agents that prevent blood clotting, such as EDTA, heparine or citrate.
  • a detergent may be added in low concentration to the blood/plasma/serum to avoid sticking of components of the test system used.
  • a cut-off membrane filter with a nominal pore size of 10 nm or more and less than 100 nm is used.
  • the inventor has observed that it is this range of pore sizes that results in a significant reduction of materials that are not indicative for active tuberculosis in a sample, while antibodies that are indicative for tuberculosis are not reduced or reduced to a lesser extent so that the background signal derived from these materials is markedly reduced.
  • the signal derived from the actual markers for tuberculosis is therewith significantly less obscured by high background signal, so that the signal derived from the actual markers for tuberculosis becomes more pronounced.
  • nominal pore size is meant average pore size, in which more than about 90%of the pore sizes are within the range of about plus or minus 10%of the nominal pore size, e.g., such as plus or minus 1 nm in the case of 10 nm nominal pore size.
  • Nominal pore size as referred to in this application are sizes as determined by electron microscopy.
  • the cut-off membrane filter has a nominal pore size between 25 and 75 nm, such as between 25 and 70, 65, 60, 55 or 50 nm. Even more preferred is that the nominal pore size is between 25 and 45 nm, such as between 30 and 40 nm, between 33 and 38 nm or between 34 and 36 nm. It is even more preferred that the nominal pore size is about 35 nm. In these preferred ranges the amount of materials that are not indicative for active tuberculosis in a sample are highly reduced, whilst still maintaining a sufficiently high amount of actual markers for tuberculosis in the filtrate.
  • the nominal pore size of the membrane is such, that at least a measurable extent of IgG antibodies against mycolic acid derivatives is able to pass to through the membrane. If a pore size of less than 10 nm is used the amount of materials that are not indicative for active tuberculosis in a sample would be significantly reduced, but the amount of actual markers for tuberculosis has also been scavenged away to such an extent that the filtrate becomes unsuitable for detection of markers for active tuberculosis.
  • the membrane of the cut-off membrane filter is preferably a hydrophilic membrane.
  • Suitable hydrophilic materials for the membrane of the cut-off membrane filter may be polyethersulphone, polyvinylidene difluoride and regenerated cellulose. These materials are low protein binders so that antibodies against mycolic acid antigens do not bind to the membrane. Polyethersulphone membranes are preferred because these provide higher flow rates and lower protein binding than competitive regenerated cellulose. This results in lower processing times and the highest possible recoveries of antibodies against mycolic acid antigens which are specific for active tuberculosis in the filtered sample.
  • Suitable cut-off filters can be obtained commercially. For instance cut-off filters with Molecular Weight Cut Off (MWCO) of 100 KDa, 300 KDa and 1000 KDa are available from Pall Corporation These MWCOs correspond to membrane nominal pore sizes of 10 nm, 35 nm and 100 nm respectively.
  • MWCO Molecular Weight Cut Off
  • Filtering the sample using abovementioned cut-off membrane filter can take place by means of any suitable filter method, using any suitable filter device. Filtering can take place by gravity or capillary force, but is preferred that filtering takes place using ultracentrifigation, such as centrifugation at 10.000 g. This way filtering takes place with few seconds, thus reducing the time of treatment of the sample, so that diagnosis of tuberculosis can take place in a fast way.
  • the filtered sample or part of it can be used directly for detection of antibodies against mycolic acid antigens or be stored until further use.
  • filtrate i.e. the part of the sample that flows through the membrane.
  • At least part of the sample is exposed to a sterol lipid, prior to exposing it to the detection substrate carrying an immobilised mycolic acid derived antigen.
  • at least part of the sample is exposed to said sterol lipid after filtering it with said cut-off membrane filter, and prior to exposing it to said substrate carrying an immobilised mycolic acid derived antigen.
  • the inventor has found that this results in even more reduction of materials that are not indicative for active tuberculosis in a sample, while the background signal derived from these materials is even more reduced, thus lowering the risk of false positive tuberculosis detection even more.
  • the exposure time of the sample to the sterol lipid is preferably less than 10 minutes, such as 2 to 8, 3 to 7, 4 to 6 or about 5 minutes.
  • the exposure time depends on the way the sample is brought into contact with the sterol lipid.
  • the sterol lipid used in the context of the invention preferably is cholesterol or a derivative thereof.
  • the sterol lipid may also be a sterol modified phospholipid.
  • Such sterol-modified lipid may a sterol-modified phospholipid, for instance a sterol-modified phosphatidylcholine lipid or glycerophospholipid.
  • the sterol is preferably cholesterol.
  • a good example of a sterol-modified lipid suitable for the purposes of the invention is 1-palmitoyl-2-cholesteryl carbonoyl-sn-glycero-3-phosphocholine.
  • the sterol lipid is preferably immobilized on a surface.
  • An example is a substrate having a coating containing cholesterol or cholesterol ester wherein the cholesterol ester is cholesterol linoleate, wherein a weight ratio of linoleic acid to cholesterol is in the range from 1: 3 to 1: 20.
  • One exemplary way of exposing the sample to the sterol lipid is to expose the sample to beads that are pre-coated with a sterol lipid, which is preferably cholesterol.
  • a substrate may also be coated with a sterol lipid, preferably cholesterol, in combination with other molecules.
  • said sterol lipid is cholesterol immobilized on a substrate together with phosphatidyl choline.
  • the sterol lipid scavenges away the anti-cholesterol antibodies from the blood/plasma/serum that would otherwise cross react with the mycolic acid antigens on the substrate and lead to false positive diagnosis of tuberculosis.
  • Phosphatidyl choline will bind to hydrophobic materials in the blood sample, rendering the sample more hydrophilic after exposure. The resulting hydrophilic sample will easier to be handled in the subsequent method steps and be less prone to clotting.
  • Sterol lipids preferably cholesterol may be immobilised together with pectin on a substrate, e.g. the inner wall of a tubing or on beads.
  • the sterol lipid scavenges away the anti-cholesterol antibodies from the blood/plasma/serum that would otherwise cross react with the mycolic acid antigens on the substrate and lead to false positive diagnosis of tuberculosis.
  • pectin scavenges away cholesterol in the sample and therewith also cholesterol antibodies. This results in even more reduction of materials that are not indicative for active tuberculosis in a sample, while the background signal derived from these materials is even more reduced, thus lowering the risk of false positive tuberculosis detection even more.
  • a sterol lipid preferably cholesterol is immobilised together with a compound binding to cholesterol in the blood derived sample, such as cholesterol binding heteropolysaccharide, such as ⁇ -cyclodextrin, pectin, amphotericin B or dextrin.
  • a compound binding to cholesterol in the blood derived sample such as cholesterol binding heteropolysaccharide, such as ⁇ -cyclodextrin, pectin, amphotericin B or dextrin.
  • cholesterol binding heteropolysaccharide such as ⁇ -cyclodextrin, pectin, amphotericin B or dextrin.
  • a sterol lipid preferably cholesterol is immobilised with ⁇ -cyclodextrin or pectin, amphotericin B on a substrate, such as hollow fiber polypropylene membranes or glass or beads.
  • At least part of the filtered sample is exposed to a substrate carrying an immobilised mycolic acid derived antigen, i.e. to a detection substrate, and binding of antibodies to the antigen is detected.
  • Detection of binding of antibodies to the antigen on the detection substrate may take place in real time or by means of an end-point assay.
  • Suitable real time detection assays include surface plasmon resonance or electrochemical impedance spectroscopy, isothermal titration calorimetry, bio-layer interferometry, optical gratings, photonic crystal, acoustic resonant profiling, quartz crystal microbalances.
  • the substrate carrying an immobilised mycolic acid derived antigen which is used for detecting binding of antibodies to the antigen is silica based, such as substrates based on silicium dioxide.
  • Silica based substrates are particularly useful when ring resonance technology is used to detect binding of antibodies to the immobilised mycolic acid antigens.
  • the detection is carried out using a biosensor chip using Si-based ring resonator. This enables the method of the invention to be carried out with a very compact device.
  • the substrate carrying an immobilised mycolic acid derived antigen which is used for detecting binding of antibodies to the antigen is gold based.
  • Gold based substrates are particularly useful when surface plasmon resonance or electrochemical impedance spectroscopy are used to detect binding of antibodies to the immobilised mycolic acid antigens.
  • the detection of binding of antibodies and/or other material to the mycolic acid antigen on the detection substrate may be carried out in an automated device.
  • Various automated devices will be known to the person skilled in the art and the skilled person will be able to select suitable software means to determine the degree or extent of binding to the detection substrate.
  • the method of the invention when using a real time detection method, comprises i) providing a sample from a human or animal suspected of having active tuberculosis; ii) filtering the sample using a cut-off membrane filter with a nominal pore size of about 10 nm or more and less than 100 nm; iii) exposing the filtered sample to a sterol lipid; iv) obtaining at least two fractions of said sample either before or after exposing it to said sterol lipid; v) exposing the first of said fractions to a substrate carrying an immobilised mycolic acid derived antigen; vi) exposing the second of said fractions to a substrate not carrying an immobilised mycolic acid derived antigen; vii) exposing the sample fraction exposed in step v) to a test substrate carrying an immobilised mycolic acid derived antigen and exposing the sample fraction exposed in step vi) to a control substrate carrying an immobilised mycolic acid derived antigen; vii
  • test detection substrate and the control detection substrate in this embodiment may be separate entities. Alternatively the test substrate and the control substrate may be realized as different positions (spots) on one substrate entity.
  • the sample from a human or animal suspected of having active tuberculosis of step i) of this embodiment is as described above.
  • One exemplary way of exposing the sample to the sterol lipid in this embodiment is to lead the sample into a long spiral channel and pass it along the length of the channel, which is pre-coated with a sterol lipid, which is preferably cholesterol.
  • the lipid sterol exposed sample may pass a means for dividing the sample stream such as a passive valved branch point that leads the divided sample streams to the next substrate carrying an immobilised mycolic acid derived antigen of step vi) and the substrate not carrying an immobilised mycolic acid derived antigen of step vii) , e.g.
  • a container comprising a substrate carrying an immobilised mycolic acid derived antigen
  • a container comprising a substrate not carrying an immobilised mycolic acid derived antigen respectively.
  • An alternative exemplary way of exposing the sample to the sterol lipid is to inject it into a container coated with cholesterol, followed by incubation therein for less than 10 minutes.
  • Whole blood/plasma/serum is rich in hundreds different kind of molecules with hydrophilic to hydrophobic properties. Therefore substrate material will be used that is inert for non-specific binding of molecules of this sample.
  • the substrate should be understood to be a support material.
  • a fraction of the sterol lipid exposed sample is exposed to a substrate carrying an immobilised mycolic acid derived antigen (step v) ) and another fraction of the sterol lipid exposed sample to a substrate not carrying an immobilised mycolic acid derived antigen (step vi) ) .
  • the blood/plasma stream has to be divided (step iv of this embodiment) .
  • the stream is divided after exposure to the sterol lipid, i.e. that the sterol lipid exposed sample is divided into at least two fractions to provide a test stream and a control stream.
  • the sample is divided in two equal or substantially equal fractions, because this enables a simple comparison of both fractions without the need for correction calculations.
  • Dividing the sample stream may be carried out by any means for dividing the sample stream such as a passive valved branch point.
  • the sample stream (which is pre-filtered to plasma or serum using a filter with larger pore size than 100 nm as described above, such as a microfilter of 0, 2 micron) is divided into at least two fractions after the pre-filtering step, but before exposure to the sterol lipid.
  • the blood stream is divided before pre-filtering. This is less preferred because in this case two microfilters would be required.
  • the substrate carrying an immobilised mycolic acid derived antigen and the substrate not carrying an immobilised mycolic acid derived antigen of steps v) and vi) are preferably of the same material.
  • sterol lipids, (e.g. cholesterol) and mycolic acid derivatives are both hydrophobic, the substrates for exposure in steps v) and vi) will be of the same material as the substrate that may be used in step iii) . It will be understood that suitable substrate material is inert for non-specific binding of molecules of this sample.
  • One exemplary way of exposing the sample to the substrates of step v) and vi) of this embodiment is to lead the divided sample streams into long spiral channels, preferably implemented in micro-chips, pre-coated either with a mycolic acid derivative (step v) or without a mycolic acid derivative (step vi) and pass it along the length of these channels.
  • the distance of both channels to the biosensor with the detection substrates will need to be of the same length. It is important that there is as less as possible non-specific binding the substrate material.
  • An alternative exemplary way of exposing the divided lipid sterol exposed samples to a substrate coated either with a mycolic acid derivative or without a mycolic acid derivative is to inject the a first stream into a container comprising a substrate carrying an immobilised mycolic acid derived antigen (step v) , and a second sample stream into a container comprising a second substrate not carrying an immobilised mycolic acid derived antigen (step vi) and incubate the samples for less than 10 minutes, such as 2 to 8, 3 to 7, 4 to 6 or about 5 minutes.
  • Suitable end point assays include enzyme-linked immunosorbent assay (ELISA) , Western blotting, radioactive labelling assay, photospectrometric assay, immunofluorescence, immunoprecipitation, immunocytochemistry, immunohistochemistry, electrochemical impedance spectroscopy.
  • ELISA enzyme-linked immunosorbent assay
  • Western blotting radioactive labelling assay
  • photospectrometric assay immunofluorescence
  • immunoprecipitation immunocytochemistry
  • immunohistochemistry immunohistochemistry
  • electrochemical impedance spectroscopy electrochemical impedance spectroscopy.
  • an end point assay detection takes place by means of an immunogold filtration assay.
  • the detection substrate is a microporous membrane, preferably a nitrocellulose membrane or a PVDF membrane, which is coated with an immobilised mycolic acid derived antigen.
  • an end-point assay interaction of antibodies with the mycolic acid derived antigens may be carried out using secondary antibodies that bind the heavy chain of the antibodies against the mycolic acid derivatives.
  • secondary antibodies are commercially available.
  • the secondary antibody may be coupled to beads, for instance gold beads, or associated with liposomes.
  • Examples of secondary antibodies may be protein A or G, possibly conjugated with an enzyme that enables detection.
  • a particular suitable technique or detecting the binding of antibodies to the immobilised mycolic acid antigens on the detection substrate in an end-point assay is the so-called immunogold filtration assay (IGFA) , and in particular the dot immunogold filtration assay (DIGFA) .
  • IGFA immunogold filtration assay
  • DIGFA dot immunogold filtration assay
  • Immunogold filtration assays are methods combining ELISA and immunogold technique and are methods in which a sample to be assayed is allowed to filtrate through a microporous membrane, preferably a nitrocellulose membrane, and is captured by a capture probe coated on the membrane.
  • a colloidal gold labelled probe is allowed to filtrate through the microporous membrane in the same manner.
  • a microporous membrane as the carrier for the capture probe and employing the capillary action and permeability of the membrane antigens and antibodies can easily react and may conveniently be subjected to optional washing and/or blocking steps.
  • the colloidal gold labelled probe binds to the capture probe the colloidal gold particles aggregate and a red dot appears which is visible with the naked eye.
  • Immunogold filtration assays are simple and rapid detection methods because no instruments are required except a membrane and the reagents and the results can be observed by the naked eye within a few minutes.
  • the microporous membrane may be for example a nitrocellulose membrane, a cellulose acetate membrane or a PVDF membrane with a suitable pore diameter.
  • nitrocellulose is used.
  • a suitable pore diameter is 0, 2 to 5 ⁇ m.
  • the detection substrate is a microporous membrane, preferably a nitrocellulose membrane, which is coated with a mycolic acid derived antigen.
  • the pretreated samples can be applied to the membrane.
  • colloidal gold-labeled second antibodies can be added onto the membrane to have gold particle aggregation in the antigen-antibody reaction place.
  • aggregation visible red or brown spots are formed.
  • the intensity of the spot is proportional to the amount of reactions between antigen and antibody, i.e. to the amount of antibodies in the pre-treated sample. In other words a sample from a person suffering from tuberculosis will result in a more intense spot than a sample from a person which is healthy.
  • the membrane may be washed with a suitable buffer, for example a PBS based buffer.
  • a PBS based buffer may for example be a PBS/AE buffer comprising NaCl, KCl, KH 2 PO 4 , Na 2 HPO 4 and EDTA in water at physiological pH.
  • Such buffer may be a PBS based buffer consisting of 8.0 g NaCl, 0.2 g KCl, 0.2 g KH 2 PO 4 , and 1.05 g Na 2 HPO 4 per liter of double distilled, deionized water containing 1 mM EDTA and 0.025%(m/v) sodium azide which is adjusted to pH 7.4.
  • the microporous membrane may be coated with said mycolic acid derived antigen in a dot wise manner.
  • a DIGFA assay the pre-treated samples are applied to the membrane in the form of dots.
  • the colloidal gold-labelled second antibodies are added in the form of dots.
  • a DIGFA assay is particularly preferred because at different spots on several membranes various antigens deriving from various mycobacterial strains may be immobilized. This way it becomes possible to provide information on which mycobacterial strain a patient is infected with.
  • DIGFA Another advantage of using DIGFA is that samples derived from different persons suspected of having active tuberculosis can be compared in one test, because DIGFA enables fast and reliable detection of antibody-antigen interaction in an unlimited amount of spots, depending on the size of the membrane.
  • the detection of binding of antibodies and/or other material to the mycolic acid antigen may be carried out in an automated device.
  • Various automated devices will be known to the person skilled in the art and the skilled person will be able to select suitable software means to quantify the degree or extent of binding on the detection substrates.
  • the detection of binding of antibodies and/or other material to the mycolic acid antigen may be performed by a visual detection technique or any other suitable detection technique.
  • detection by means of the end-point assay takes place visually, preferably with the naked eye. This has the advantage of easy detection without the need for expensive and complicated detection technology.
  • binding of antibody antibodies and/or other material to the mycolic acid antigen may be assessed by means of the naked eye.
  • a visual signal e.g. the red staining in case a DIGFA assay is applied as end-point assay, may also be detected with help of a mobile app, i.e. a computer program designed to run on mobile devices such as tablet computers or smart phones.
  • a mobile app i.e. a computer program designed to run on mobile devices such as tablet computers or smart phones.
  • an app can be used that is designed to compare the binding signal between different samples or sample fractions and which indicates whether the human or animal from which the sample originated has active tuberculosis.
  • the method of the invention when using an end-point detection method, comprises i) providing a sample from a human or animal suspected of having active tuberculosis; ii) filtering the sample using a cut-off membrane filter with a nominal pore size of about 10 nm or more and less than 100 nm; iii) exposing at least part of the filtered sample to a sterol lipid; iv) obtaining at least two fractions of said sample either before or after exposing it to said sterol lipid; v) exposing the first of said fractions to a substrate carrying an immobilised mycolic acid derived antigen; vi) exposing the second of said fractions to a substrate not carrying an immobilised mycolic acid derived antigen; or storing at least part of the second of said fractions until step vii) , skipping the step of exposing the second of said fractions to a substrate not carrying an immobilised mycolic acid derived antigen; vii) exposing at least part
  • step i) of this embodiment is as described above.
  • One exemplary way of exposing the sample to the sterol lipid is to lead the sample into a long spiral channel and pass it along the length of the channel, which is pre-coated with a sterol lipid, which is preferably cholesterol.
  • the lipid sterol exposed sample may pass a means for dividing the sample stream such as a passive valved branch point that leads the divided sample streams to the next substrate carrying an immobilised mycolic acid derived antigen of step v) and the substrate not carrying an immobilised mycolic acid derived antigen of step vi) , e.g. a container comprising a substrate carrying an immobilised mycolic acid derived antigen, and a container comprising a substrate not carrying an immobilised mycolic acid derived antigen respectively.
  • An alternative exemplary way of exposing the sample to the sterol lipid is to inject it into a container coated with cholesterol, followed by incubation therein for less than 10 minutes.
  • Another alternative exemplary way is to expose the sample to the sterol lipid, wherein the sterol lipid is contained in a compartment of a column. In such a compartment the sterol lipid is preferably coated on beads.
  • Whole blood/plasma/serum is rich in hundreds different kind of molecules with hydrophilic to hydrophobic properties. Therefore substrate material will be used that is inert for non-specific binding of molecules of this sample.
  • the substrate should be understood to be a support material.
  • a fraction of the sterol lipid exposed sample is exposed to a substrate carrying an immobilised mycolic acid derived antigen (step v) ) and another fraction of the sterol lipid exposed sample to a substrate not carrying an immobilised mycolic acid derived antigen (step vi) ) .
  • the blood/plasma stream has to be divided (step iv) .
  • the stream may be divided after exposure to the sterol lipid, i.e. that the sterol lipid exposed sample is divided into at least two fractions to provide a test stream and a control stream.
  • the sample is divided in two equal or substantially equal fractions, because this enables a simple comparison of both fractions without the need for correction calculations.
  • Dividing the sample stream may be carried out by any means for dividing the sample stream such as a passive valved branch point.
  • the sample stream (which is filtered to plasma or serum) is divided into at least two fractions after the filtering step, but before exposure to the sterol lipid.
  • the blood stream is divided before filtering. This is less preferred because in this case two filters would be required.
  • the stream is in this case divided before exposure to the sterol lipid in step iii) , so that steps iii) and v) can take place in the same column and step iii) and vi) can take place in another column.
  • the substrate carrying an immobilised mycolic acid derived antigen and the substrate not carrying an immobilised mycolic acid derived antigen of steps v) and vi) are preferably of the same material.
  • sterol lipids, (e.g. cholesterol) and mycolic acid derivatives are both hydrophobic
  • the substrates for exposure in steps v) and vi) may be of the same material as the substrate that may be used to in step iii) .
  • suitable substrate material is inert for non-specific binding of molecules of this sample.
  • the exposure time of the sample to the sterol lipid is preferably less than 10 minutes, such as 2 to 8, 3 to 7, 4 to 6 or about 5 minutes.
  • One exemplary way of exposing the sample to the substrates of step v) and vi) is to lead the divided sample streams into long spiral channels, preferably implemented in micro-chips, pre-coated either with a mycolic acid derivative (step v) or without a mycolic acid derivative (step vi) and pass it along the length of these channels.
  • An alternative exemplary way of exposing the divided lipid sterol exposed samples to a substrate coated either with a mycolic acid derivative or without a mycolic acid derivative is to inject the a first stream into a container comprising a substrate carrying an immobilised mycolic acid derived antigen (step v) , and a second sample stream into a container comprising a second substrate not carrying an immobilised mycolic acid derived antigen (step vi) and incubate the samples for less than 10 minutes, such as 2 to 8, 3 to 7, 4 to 6 or about 5 minutes.
  • steps v) and vi) take place in a column.
  • step vi) said another fraction of the sterol lipid exposed sample is exposed to a substrate not carrying an immobilised mycolic acid derived antigen, it may also be possible that the step of exposing said fraction to a substrate not carrying an immobilised mycolic acid derived antigen is skipped and that said fraction is stored until further use, such as in step vii) .
  • step vii) of this embodiment the divided sterol lipid exposed sample streams, either exposed to mycolic acid derivatives in step v (test stream) or not in step vi (control stream) are passed to detection substrates, test and control substrates respectively, carrying mycolic acid derivatives, preferably the same derivatives as in step v) .
  • the test and control substrates may be any of the detection substrate as described above for end-point methods.
  • the mycolic acid derived antigens as referred to in the present application may be derived from mycobacteria selected from virulent and pathogenic mycobacteria.
  • the term mycolic acid derived antigen is to be understood as a compound comprising a mycolic acid residue which is capable of binding to anti-mycolic acid antibodies.
  • the mycolic acid antigen is derived from Mycobacterium tuberculosis.
  • Said mycolic acid derived antigen is at least one selected from the group of mycolic acid, cord factor, chemically modified mycolic acid, chemically modified cord factor, a synthetic mycolic acid derivative, a synthetic cord factor derivative.
  • the mycolic acid derived antigen may suitably be selected from one or more of mycolic acids obtained from natural sources, synthetically prepared mycolic acids, sulfur-containing mycolic acids, structural analogues of mycolic acids, and mycolic acid wax esters.
  • the mycolic acid derived antigen also includes salts and/or esters of these derivatives.
  • Natural sources of mycolic acid derivatives include the cell walls of mycobacteria such as Mycobacterium tuberculosis include mixtures of different classes of compounds and different mycolic acid homologues, often as derivatives in which they are bonded to the wall of the cell.
  • Esters of mycolic acid derivatives can also be used such as esters of alcohols (e.g. monohydric alcohols and polyhydric alcohols) and sugar esters.
  • Sugar esters are particularly preferred.
  • Sugar esters include esters with a monosaccharide, disaccharide or an oligosaccharide. Said saccharides may conveniently include sugar units based on hexoses and/or pentoses. Glucose esters are suitable examples of these esters.
  • Further suitable sugar esters are trehalose esters, including trehalose monomycolates and trehalose dimycolates. For instance cord factors, which are trehalose monomycolates or trehalose dimycolates are well known examples of sugar esters that are suitable. These compounds occur in nature as complex mixtures of different classes of mycolic acids and of different homologues within each class.
  • semi-synthetic or more preferably synthetic mycolic acid derivatives for the purposes of the invention.
  • the structure of the mycolic acid unit affects the biological activity of the cord factor.
  • Suitable semi-synthetic derivatives include semi-synthetic cord-factors which may be prepared by attaching mycolic acids to the sugar group. These semi-synthetic factors however still contain mixtures of different homologue. Therefore particular suitable mycolic acid derivatives for use in the context of the present invention are synthetic cord factors, for example the synthetic cord factors described in WO 2010/08667, i.e.
  • Salts of mycolic acid derivatives can also be used, for instance ammonium salts, or alkali metal and alkaline earth metal salts.
  • Sulfur-containing mycolic acids and/or esters or salts thereof may also be used. These compounds are analogues of natural mycolic acid compounds containing sulfur.
  • Mycolic acid wax esters comprise a cyclopropyl group or an alkene and an internal ester group and can be isolated from natural sources or prepared synthetically.
  • Suitable compounds for use in the detection method of the invention are described inter alia in WO 2016/024116.
  • the mycolic acid antigen may be in a form selected from homogenous and heterogenous compound mixtures.
  • the mycolic acid derived antigen may for instance be used in combination with a phospholipid such as phosphatidylcholine.
  • the mycolic acid antigen may be immobilised on the substrates in various ways that are known to the skilled person. Synthetic mycolic acid derived antigens may be synthesised with particular active groups that enable immobilisation to a substrate material.
  • silane coupling chemistry may be applied for immobilisation on silica.
  • the mycolic acid derived antigen may be suitably immobilised as follows.
  • Mycolic acid derived antigens may be obtained in lyophilized form and be reconstituted in a solvent mixture, for instance a chloroform: methanol: water mixture, and diluted to a concentration in the order of several nanomolars, for instance 1 nM. This dilution can then be spotted on a nitrocellulose membrane, wherein each spot is separated at a predetermined distance, e.g. 1 cm. After drying of the spots the mycolic acid derived antigens are immobilized on the membrane.
  • Alternative immobilisation methods may for instance involve dissolving the antigens in hexane or hot PBS to form an antigen coating solution before spotting the solution on a membrane.
  • cholesterol was coupled to beads.
  • Toyopearl AF-Amino-650M beads Tosoh Bioscience
  • Beads were prepared in accordance with the manufacturer’s instructions.
  • cholesterol 7-keto cholesterol was conjugated to the beads basically as described in Abdel-Magid AF et al. (Reductive Amination of Aldehydes and Ketones with Sodium Triacetoxyborohydride. Studies on Direct and Indirect Reductive Amination Procedures. J. Org. Chem.
  • Serum derived from humans that were known to be suffering of tuberculosis was diluted 1: 5 in blocking buffer (TB+ samples) .
  • the TB+ samples used were derived from both smear positive and smear negative patients. Also samples were used derived from healthy humans (Control samples) . Two fractions of 0, 5 ml were obtained and transferred each to a 0, 2 micron spin filter and centrifuged at 10000g. The flow-through was pooled from 1: 5 to 1: 20 in blocking buffer.
  • the total pre-treatment of the samples in this particular set up may take place in less than 10 minutes, after which analysis can directly follow.
  • the ELISA signal of the control samples can be considered as background signal as these samples are derived from healthy humans that should not test positive for tuberculosis. As expected the TB+ samples give a higher signal than the control samples derived from healthy humans, thus indicating the presence of antibodies against mycolic acid (derivatives) .
  • Cut-off filters Prior to any cut-off membrane filter treatment samples were filtered with 0, 2 micron spin filter. Cut-off filters were used with Molecular Weight Cut Off (MWCO) of 10 kDa, 100 KDa, 300 KDa and 1000 KDa. This corresponds to membrane nominal pore sizes of 5 nm, 10 nm, 35 nm and 100 nm respectively. As a control, the cut-off membrane filter step was skipped. Again for each treatment 6 samples from different individuals (in case of TB+ samples SMEAR + and SMEAR -) were subjected to treatment as described above. For each treatment the samples were from the same 6 individuals.
  • MWCO Molecular Weight Cut Off
  • the average ELISA signal obtained from 6 samples subjected to the same treatment was determined and standardised to the signal obtained from the treatment in which only a 0, 2 micron filter was used.
  • the ELISA signal of the samples in which only a 0, 2 micron filter was used was set at 100%.
  • the average ELISA signal is shown in Table 3 below. Standard deviations are also indicated.
  • the ELISA signal derived from mycolic acid specific antibodies in the sample from tuberculosis patients becomes more pronounced, because the background signal caused by unspecific binding to the test substrate has been reduced. From this it follows that filtering a sample from a human or animal suspected of having active tuberculosis using a cut-off membrane filter with a nominal pore size of 10 nm or more and less than 100 nm, before exposing the sample to a substrate carrying an immobilised mycolic acid derived antigen and detecting binding of antibodies to the antigen results in strong reduction of background signal. This way the risk of testing false positive for tuberculosis is reduced. Accordingly the method of the invention herewith provides a method of detecting a marker for active tuberculosis which is more sensitive than the methods known in the art.

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Abstract

La présente invention concerne un procédé de détection d'un marqueur de tuberculose active, et un procédé permettant de prétraiter un échantillon d'un humain ou d'un animal suspecté d'être atteint de tuberculose active.
PCT/CN2017/074390 2016-02-22 2017-02-22 Procédé de détection d'un marqueur de la tuberculose active WO2017143985A1 (fr)

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WO2013186679A1 (fr) * 2012-06-11 2013-12-19 University Of Pretoria Composition de liposomes comprenant un lipide à modification stérol et composant de paroi cellulaire lipidique mycobactérien purifié et son utilisation dans le diagnostic de la tuberculose
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