NL2014085B1 - A method, portable device, kit and biosensor for detecting a marker for active tuberculosis. - Google Patents

Abstract

The present invention is based on the principle of detecting antibodies against mycolic acid derivatives, such as cord factors antibodies or mycolic acid (MA) antibodies as markers for active tuberculosis in a sample. The present invention relates to a method of detecting a marker for active tuberculosis, a portable device for carrying out a method of detecting a marker for active tuberculosis, a kit and biosensor for use in diagnosing tuberculosis.

Description

A method, portable device, kit and biosensor for detecting a marker for active tuberculosis

The present invention relates to a method of detecting a marker for active tuberculosis, a portable device for carrying out a method of detecting a marker for active tuberculosis, a kit and biosensor for use in diagnosing tuberculosis .

Introduction

Mycobacterium tuberculosis is a pathogenic bacterial species in the family Mycobacteriaceae and the causative agent of most cases of tuberculosis (TB).

Nine million people fell ill with TB in 2013, including 1.5 million cases among people with HIV. In 2013, 1.5 mil lion people died from TB, including 360000 among people who were HIV-positive. TB is one of the top three killers of women worldwide, 510000 women died from TB in 2013. Of the TB deaths among HIV-positive people, 50% were among women. At least 550000 children became ill with TB and an estimated 80 000 children who were HIV-negative died of TB in 2013. Globally in 2013, an estimated 480000 people developed multidrug-resistant TB (MDR-TB) and there were an estimated 210000 deaths from MDR-TB. At least one case of extensively drug-resistant TB (XDR-TB) has been reported by 100 countries by the end of 2013. On average, an estimated 9% of MDR-TB cases have XDR-TB. A reliable and fast way of diagnosing tuberculosis is therefore of utmost importance.

Several methods of diagnosing tuberculosis have been developed, but all methods have their disadvantages. Diagnosing active tuberculosis based merely on signs and symptoms is difficult, as is diagnosing the disease in those who are immunosuppressed. The TB skin test (also called the Mantoux tuberculin skin test), TB blood tests (also called interferon-gamma release assays or IGRAs), and chest radiography (X-ray), and tests on the presence of acid-fast-bacilli (AFB) on a sputum smear, indicate some of the infected individuals in days. A definitive diagnosis of TB is made by identifying M. tuberculosis in a clinical sample (e.g. sputum, pus, or a tissue biopsy). However, the difficult culture process for this slow-growing organism can take two to six weeks for blood or sputum culture.

Humans or animals infected with the M. tuberculosis normally produce antibodies directed against the Mycobacterium. Presence of these antibodies in a sample taken from infected individuals indicates the infection. WO 2005/116654 describes a method based on this principle and discloses detecting a marker for active tuberculosis. The method of WO 2005/116654 involves obtaining first, second and third samples from a subject suspected of having active tuberculosis, diluting the first sample and exposing part of it to an immobilized mycolic acid antigen in a test vessel and part of it to an immobilized mycolic antigen in a control vessel. The second sample is exposed to mycolic acid antigen-containing liposomes and the third sample is exposed to liposomes not containing mycolic acid antigens. The second sample is added to the test vessel and the third to the control vessel and binding of antibodies to the mycolic acid and antigen in both the test and control vessel is detected. The degree of binding between the test and control vessels is compared and lesser binding in the test vessel is an indicator of the presence of antibodies to the mycolic acid antigen. A further development was described in WO 2013/186679, which discloses a method of detecting antigen specific biomarker antibodies for the diagnosis of active tuberculosis, the method including the steps of: providing a lipid antigen-presenting liposomal composition comprising liposomes comprising a sterol-modified lipid and a purified mycobacterial lipid cell wall component or analogue or derivative thereof; immobilizing the liposomes to produce immobilized mycolic acid antigens comprising the purified mycobacterial lipid cell wall component or analogue or derivative thereof; obtaining a first, a second and a third sample from a human or animal suspected of having active tuberculosis, wherein each sample may contain antibodies to the antigen, the first sample having a lower concentration by dilution than the second and third samples; exposing part of the first sample to the immobilized mycolic acid antigens in a test vessel; exposing part of the first sample to the immobilized mycolic acid antigens in a control vessel; exposing the second sample to the lipid antigen-presenting liposomal composition provided in the first step ; exposing the third sample to liposomes not containing mycolic acid antigen; adding the second sample, after exposure to the mycolic acid antigen-containing liposomal composition provided in the first step, to the test vessel; adding the third sample, after exposure to the liposomes not containing mycolic acid, to the control vessel; detecting binding of antibodies to the mycolic acid antigen in both the test and control vessels in real time; and comparing the degree or extent of binding between the test and the control vessels, the weaker binding in the test vessel being an indicator of the presence of antibodies to the mycolic acid antigen in the sample that indicates active tuberculosis in the human or animal from which the sample originated.

The methods described in WO 2005/116654 and WO 2013/186679 provide reliable methods for diagnosis of tuberculosis, but have several disadvantages. First, the methods described in WO 2005/116654 and WO 2013/186679 require provision of several samples from the same subject. Second, these methods require several dilution steps and several transfer steps of samples to different unconnected compartments, requiring a complicated and large set of instrumental parts. Third, these methods involve separate incubation steps of the sample and dilutions thereof and consequently several separate measurements. For these reasons the methods of WO 2005/116654 and WO 2013/186679 are complicated and time consuming. In fact, the total time from obtaining a sample from a subject suspected of having active tuberculosis to determining whether or not an individual is infected with active tuberculosis using the methods disclosed in WO 2005/116654 and WO 2013/186679 takes an estimated time of at least 2 hours per sample. Furthermore, because the above methods require a complicated and large set of instrumental parts, said methods may be useful in a hospital environment, but not in areas which are deprived from hospitals and well developed healthcare, for instance in developing countries. It is in particular in these countries where tuberculosis is most prevalent and where reliable and fast diagnosis is most desired.

Therefore the invention aims to provide a method for determining whether or not an individual is infected with active tuberculosis (e.g. pulmonary or extra-pulmonary tuberculosis) which is fast and reliable, and which can be carried out outside of a professional medical environment, i.e. outside of a hospital, for instance on the streets. The invention also aims to provide a portable device which can be used in such method and a kit which can be used to assemble said device "on the spot". The invention also aims to provide a biosensor for use in said method, device or kit.

Summary of the invention

In a first aspect the invention relates to a method of detecting a marker for active tuberculosis as defined in claim 1.

In a second aspect the invention relates to a portable device defined in claim 13.

In a third aspect the invention relates to a kit for use in diagnosing tuberculosis, as defined in claim 22.

In a fourth aspect the invention relates to a biosensor as defined in claim 25.

Description of the figures

Fig. 1 describes an exemplary embodiment of the device of the invention.

Description of the invention

The present invention relates in one aspect to a method for detecting antibodies against mycolic acid derivatives, such as cord factors antibodies or mycolic acid (MA) antibodies in a sample.

The method of the invention comprises the steps of: i) providing a sample from a human or animal suspected of having active tuberculosis; ii) contacting said sample to a sterol lipid; iii) obtaining at least two fractions of said sample either before or after exposing it to said sterol lipid; iv) exposing the first of said fractions to a substrate carrying an immobilised mycolic acid derived antigen; v) exposing the second of said fractions to a substrate not carrying an immobilised mycolic acid derived antigen; vi) exposing the sample fraction exposed in step iv) to a test substrate carrying an immobilised mycolic acid derived antigen and exposing the sample fraction exposed in step v) to a control substrate carrying an immobilised mycolic acid derived antigen; vii) detecting binding of antibodies to the antigen of step vi) in real time; and viii) comparing the degree or extent of antibody binding between the test and control substrates, any observed lesser binding to the test substrate being an indicator of the presence of antibodies to the antigen in the sample that indicates active tuberculosis in the human or animal from which the sample originated.

The method enables samples, preferably blood samples to be analyzed in a reliable and fast way without the need to transfer samples from and into several vessels. The subsequent pre-incubation steps of the sample with a sterol lipid, followed by subjecting a fraction of the sample to a pre-incubation step with a mycolic acid derived antigen to obtain a test sample and another fraction of the sample to a pre-incubation without a mycolic acid derived antigen to obtain a control sample render the sample suitable for direct application in the eventual detection step which entails binding of mycolic acid antigens in the pretreated sample to a biosensor substrate carrying a mycolic acid. The biosensor substrate therefore does not require pre-incubation or pretreatment of the sensor with a dilution of the sample. Accordingly, the method of the invention does not require the several dilution and transfer steps required in methods of the prior art.

Furthermore the method of the invention is advantageously suitable for carrying out by means of a portable device, in particular by means of the device defined in claim 11. This makes it possible to determine whether or not an individual is infected with active tuberculosis (e.g. pulmonary or extra-pulmonary tuberculosis) in fast and reliable way, allowing detection of tuberculosis in less than 15 to 25 minutes. Furthermore the method can be carried out outside of a professional medical environment.

In particular the invention provides a quick method for determining whether or not an individual is infected with active tuberculosis as a point of care test i.e. as a test at or near the site of patient care. The test of the present invention provides simple medical tests which can be performed at the bedside.

Furthermore, the method only requires taking one sample from a subject, which sample can be used directly in the method of the invention.

The method of the invention will now be explained in more detail with reference to the steps of claim 1.

Step i

The method of the invention is based on diagnostic testing of samples, in particular blood samples of a human or animal suspected of having active tuberculosis. For this purpose in step i) of the method of the invention a sample from a human or animal suspected of having active tuberculosis is provided. The sample is preferably a whole blood sample. The sample may be obtained by any regular means of obtaining blood from a subject. In order to be used in the method of the invention samples may be used that have been collected at an earlier stage, stored until use under suitable conditions and provided at a suitable moment. Alternatively, a sample may be used in the method of the invention on the spot, i.e. as a point of care test. In the latter situation the portable device of the invention will be particularly suitable to use.

In case the sample is a whole blood sample, the sample may depending on the way of detection of binding of antibodies to the antigen be filtered or separated to plasma or serum before step ii) . In case the detection is carried out by means detecting mass differences, a filtering step may be preferred to filter out high mass components such as blood cells. In case binding is detected by fluorescence, one may choose not to filter the sample. This would even shorten the time required for diagnosis.

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. The choice of blood plasma or blood serum depends on whether the device of the invention is designed to separate the whole blood into plasma or serum. Serum is preferred because it contains less different materials than blood plasma which may lead to aspecific interactions or unwanted biological activity. In addition 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.

About 55% of whole blood consists of plasma/serum. If a whole blood sample is not filtered perfectly or if the patient's physical situation necessitates it, it may be desired to dilute the whole blood sample or plasma or serum. The words 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 a 250 to 5000 x dilution, a 750 to 1250 x dilution, such as for instance a 4000, 2000 or 1000 x dilution. Depending on the viscosity of the sample, such dilution may take place before the separating the plasma from the blood step or after the separating step or alternative to the separating step. For instance the dilution step may take place after the separating step but before step iv) or v) or after step v)/vi) and before step vii) . It is preferred to dilute the sample after step v)/vi) and before step vii) i.e. just before the samples enters the biosensor. This way the volume of the sample is kept as low as possible during most of the steps of the method, which is beneficial to the speed of the process and the compactness of the device used therein.

Dilution may be performed with any suitable diluent, for example a PBS based buffer. Such buffer may for example be a PBS/AE buffer comprising NaCl, KC1, KH2P04, Na2HP04 and EDTA in water at physiological pH. Such buffer may be a PBS based buffer consisting of 8.0 g NaCl, 0.2 g KC1, 0.2 g KH2P04, and 1.05 g Na2HP04 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.

Optionally a detergent may be added in low concentration to the blood/plasma/serum to avoid sticking of components to walls of tubings, vessels or containers.

Step ii

In step ii, the sample is exposed to, i.e. contacted with a sterol lipid.

Although the inventors do not wish to be bound by any theory, it is assumed that the sterol lipid scavenges away the anti-cholesterol antibodies from the sample that would otherwise cross react with the mycolic acid antigens on the sensor substrate and lead to false positive diagnosis of tuberculosis.

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.

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 precoated with a sterol lipid, which is preferably cholesterol. At the end of the channel 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), i.e. 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.

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. In this context the substrate should be understood to be a support material.

Steps iv and v

In the next step a fraction of the sterol lipid exposed sample is exposed to a substrate carrying an immobilised mycolic acid derived antigen (step iv) ) and another fraction of the sterol lipid exposed sample to a substrate not carrying an immobilised mycolic acid derived antigen ( step v)) .

To obtain at least two fractions, at a certain moment in the process the blood/plasma stream has to be divided (step iii). For sake of convenience and to optimize reliability of the method, it is preferred that 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. It is preferred that 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.

Alternatively 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. Alternatively the blood stream is divided before filtering. This is less preferred because in this case two filters 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 iv) and v) are preferably of the same material.

In addition, since sterol lipids, (e.g. cholesterol) and mycolic acid derivatives are both hydrophobic, the substrates for exposure in steps iv) and v) will be of the same material as the substrate that may be used to in step ii). It will be understood that suitable substrate material is inert for non-specific binding of molecules of this s amp1e.

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 iv) and v) 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 iv) or without a mycolic acid derivative (step v) and pass it along the length of these channels. The distance of both channels to the biosensor 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 iv), and a second sample stream into a container comprising a second substrate not carrying an immobilised mycolic acid derived antigen (step v) and incubate the samples for less than 10 minutes, such as 2 to 8, 3 to 7, 4 to 6 or about 5 minutes.

Step vi

In this step the divided sterol lipid exposed sample streams, either exposed to mycolic acid derivatives in step iv(test stream) or not in step v (control stream) are passed to further substrates, test and control substrates respectively, carrying mycolic acid derivatives, preferably the same derivatives as in step iv.

These test and control substrates are contained in a biosensor comprising a first chamber (test chamber) with a test substrate for receiving the test stream exposed to mycolic acid derivatives in step iv) , and in a second chamber (control chamber) with a control substrate for receiving the control stream not exposed to mycolic acid derivatives in step v). The substrates in both chambers are preferably of the same material and carry the same mycolic acid derivative. A biosensor is in this context can be any means capable to generate a measurable signal when antibodies contact the mycolic acid derivatives on the test and control substrates.

Preferably the test and control substrates of step vi) are 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. Preferably 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 .

It is also well possible that the substrates of step vi) are 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.

Steps vii and viii

In step vii the binding of antibodies to the immobilised mycolic acid antigens is detected. Because detection takes place in real time, binding of antibodies on the substrates of step vi is directly detected during the binding process. It is therefore to be understood that steps vi and vii are not steps that need to take place at separate times. This also applies for step viii, i.e. comparison of binding to test and control substrates and detection may take place while the binding process of step vii is still going on. For detection in principle all real-time, label free analysis techniques may be used, such as surface plasmon resonance or electrochemical impedance spectroscopy isothermal titration calorimetry, bio-layer interferometry, optical gratings, photonic crystal, acoustic resonant profiling, quartz crystal microbalances.

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 make the comparison of step viii) of the degree or extent of binding between the test and control substrates, wherein any observed lesser binding to the test substrate is an indicator of the presence of antibodies to the antigen in the samples that relates to active tuberculosis in the human or animal from which the samples originated. In this context it should be understood that lesser can be interpreted qualitatively and quantitatively, i.e. lesser binding may be interpreted as having less binding events as well as having weaker bindings.

Device

In another aspect the invention relates a portable device for carrying out a method of detecting a marker for active tuberculosis, comprising at least one container comprising a sterol lipid arranged and configured to receive a sample stream from a human or animal suspected of having active tuberculosis; means for dividing said sample stream into at least a first and a second sample stream; said means either connected upstream or downstream of said at least one container comprising a sterol lipid; a further container for receiving said first sample stream in downstream connection with a said container comprising a sterol lipid, and comprising a first substrate carrying an immobilised mycolic acid derived antigen; a still further container for receiving the second sample stream in parallel arrangement to said further container and in down stream connection with a said container comprising a sterol lipid; and comprising a second substrate not carrying an immobilised mycolic acid derived antigen, and a biosensor comprising a first chamber for receiving the first sample stream in connection with said further container, comprising a substrate carrying an immobilised mycolic acid derived antigen and a second chamber for receiving the second sample stream in connection with said still further container comprising the same substrate carrying an immobilised mycolic acid derived antigen as the first chamber.

Preferably the device of the invention comprises a skin penetrating end connected to a first tubing, which is arranged and configured receive a blood sample from the skin penetrating end and to carry said blood sample away from the skin site to said container comprising a sterol lipid.

In another preferred embodiment the device comprises a filter unit in connection with said first tubing for receiving said blood sample and configured to separate plasma from said whole blood sample.

In a further preferred embodiment the portable device comprises a skin penetrating end connected to a first tubing, which is arranged and configured receive a blood sample from the skin penetrating end and to carry said blood sample away from the skin site; a filter unit in connection with said first tubing for receiving said blood sample and configured to separate plasma from said whole blood sample; a first container for receiving said sample, said first container comprising a sterol lipid; means for dividing said sample into at least a first and a second sample stream; a second container for receiving said first sample stream, said second container comprising a first substrate carrying an immobilised mycolic acid derived antigen; a third container for receiving the second sample stream, said third container comprising a second substrate not carrying an immobilised mycolic acid derived antigen; and a biosensor comprising a first chamber for receiving the first sample stream in connection with the second container, comprising a substrate carrying an immobilised my-colic acid derived antigen and a second chamber for receiving the second sample stream in connection with the third container comprising the same substrate carrying an immobilised mycolic acid derived antigen as the first chamber .

The stream through the apparatus is preferably driven by a pump, preferably a continuous flow pump, for example a peristaltic pump or a diaphragm pump. The pump is preferably located downstream of the biosensor, in order to be able to suck the sample through the apparatus and to prevent contamination of the sample before analysis.

The skin penetrating means may be any suitable means to obtain a blood sample from a human or animal, such as a needle syringe or the like.

The first tubing has dimensions that are suitable for purpose of the device, i.e. it needs to be well adaptable to the skin penetrating means and the first container.

The components of the device, i.e. the containers, means for diluting, filter unit and biosensor may be interconnected by suitable interlinking tubings. Alternatively the components of the device may be designed such that they can be connected directly to each other. The material of the tubings used in the invention can be any suitable material which is known to the person skilled in the field of testing blood samples. Suitable materials are inert to blood/plasma/serum components and include poly-tetrafluorethylene (e.g. Teflon®), polypropylene, poly-etherketone (PEEK) and polyethylene.

Further components may be connected between the components of the device. Such further components may be connected in tubings interlinking the components are directly attached to the components.

The device of the invention may also comprise a means for dilution of the blood or plasma. Such means may be implemented before the sterol lipid containing container, before the filter unit, between the filter unit and the sterol lipid containing container, between the sterol containing container and the other (further and still further or (second and third) containers or between the fur-ther/still further or second /third containers and the respective chambers of the biosensor. Preferably the means for diluting the sample are connected between the further or second container and the biosensor and between the still further or third container and the biosensor. For instance a 10 ml container may be implemented at the outlet of the further or second and still further or third containers. A suitable buffer may already be present in the container or be added into this container to provide the desired dilution. This way the volume of the sample is kept as low as possible during most of the steps of the method of the invention, which is beneficial to the speed of the process and the compactness of the device of the invention .

The containers may be vessels or a channel or tubing, such as a spiral channel or spiral tube. A spiral channel or tube is preferred because such structure takes little space whilst maintaining a long flow path. A spiral channel or tube may be advantageously implemented on a microchip. This contributes to the compactness of the device of the invention.

The filter unit may comprise a filter matrix. Preferably the filter is implemented on a filter microchip for the sake of compactness.

The material of the containers is preferably the same and suitable materials may be bk-7 glass, polytetrafluo-rethyleen, polypropylene, polyether ketone or polyethylene .

The substrates of the containers have to be material that is inert for non-specific binding of molecules of the sample, for instance bk-7 glass, polytetrafluorethyleen, polypropylene, polyether ketone or polyethylene.

The device comprises a container comprising a substrate carrying an immobilised mycolic acid derived antigen (second container) and a container comprising a substrate not carrying an immobilised mycolic acid derived antigen. Instead of an immobilised mycolic acid derived antigen, the latter container may an inert coating or no coating, as long as no aspecific binding takes place.

Preferably the test and control substrates of the chambers of the biosensor are 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 biosensor comprises a first and a second chamber. It is to be understood that the two chambers do not need to be in one compartiment or housing. For instance the biosensor can comprise two separate sensor units each comprising a chamber with a substrate carrying mycolic acid derivative antigens, one sensor unit being in connection via a tubing with a container comprising a substrate carrying an immobilised mycolic acid derived antigen, the other sensor unit being in connection via a tubing with a container comprising a substrate not carrying an immobilised mycolic acid derived antigen.

Preferably, the biosensor comprises a Si-based ring resonator. This enables the device of the invention to be very compact.

In this respect the invention also relates to a biosensor, comprising at least two chambers comprising a silica based of substrate with an immobilised mycolic acid derived antigen and a Si ring resonator.

It is also well possible that the substrates of the chambers of the biosensor are 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 device of the invention may be connected to any suitable automated analysis means, such as a computer with suitable software programs to carry out the comparison of binding of mycolic acid antibodies to the immobilised antigens in the chambers of the biosensor.

An exemplary embodiment of the device of the invention is shown in Fig. 1.

Fig. 1 shows a skin penetrating needle 1 connected to a first tubing 2, which is arranged and configured receive blood from the needle 1 and to carry the amount of blood away from the skin site. Tubing 1 is connected to a filter unit 3. This filter unit serves to separating plasma from said whole blood sample. Filter unit 3 is connected via tubing 13 to a first container 4, which is in the form of a spiral channel of which the inner wall is coated with a sterol lipid. The spiral channel is connected via tubing 14 with a means for dividing plasma 5 in the form of a passive valved branch point. One branch of the branch point is connected via tubing 16 with a second container 6 for receiving a first plasma stream, which comprises a first substrate carrying an immobilised mycolic acid derived antigen. The other branch of the branch point is connected via tubing 15 with a third container 7 for receiving a second plasma stream, which comprises a second substrate not carrying an immobilised mycolic acid derived antigen. Containers 6 and 7 in this embodiment are in the of a spiral channel of which the inner wall is either coated with a mycolic acid derived antigen (container 6) or not coated with a mycolic acid derived antigen (container 7) . Containers 6 and 7 are connecter via respectively tubings 17 and 18 with dilution containers 11 and 12 respectively. These containers may be prefilled with a suitable dilution buffer of comprise inlets 21, 22 for in troducing a dilution buffer. The dilution containers 21 and 22 are connected via respective tubings 19 and 20 to a test chamber 9 of a biosensor 8 and to a control chamber 10 of biosensor 8 respectively. The test and con- trol chambers 9 and 10 both comprise the same substrate carrying an immobilised mycolic acid derived antigen.

Kit

In another aspect the invention relates to a kit for use in diagnosing tuberculosis, comprising one or more skin penetrating means; one or more tubings; one or more containers coated with a sterol lipid and optionally phosphatidyl choline, pectin or β-cyclodextrin; one or more containers comprising a substrate with an immobilised mycolic acid derived antigen; and one or more biosensors comprising at least two chambers comprising a substrate with an immobilised mycolic acid derived antigen; and optionally means for diluting blood or plasma and/or a filter unit.

The kit may also comprise tools to assemble the components, such as screws, clamps, glue, tape, screwdrivers etc .

The kit may also comprise means to dilute the blood or plasma during blood analysis. Such as container (for instance of 10 ml) to be implemented at the outlet of the second and third containers. A suitable buffer may already be present in the container or be added into this container to provide the desired dilution.

The components of the kit enable the person that is to perform a tuberculosis diagnosis test to assemble the device of the invention on the spot, i.e. at the point of care. It is therefore to be understood that the components of the kit have the same characteristics and preferred properties as explained above for the device of the invention. The device of the invention is easy to be assembled by means of the components of the kit of the invention, i.e. no specialist technical background is required. The kit may be provided conveniently with instructions for assembly and use.

Sterol lipids

The sterol lipid used in the method, device or kit 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. In such sterol modified lipid the sterol is preferably cholesterol. A good example of a sterol-modified lipid suitable for the purposes of the invention is l-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. A substrate may also be coated with a sterol lipid, preferably cholesterol, in combination with other molecules .

Preferably 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 sensor 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.

In one embodiment a sterol lipid, preferably cholesterol is immobilised in together with pectin on a substrate, e.g. the inner wall of a tubing. In this embodiment 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 sensor substrate and lead to false positive diagnosis of tuberculosis. In addition pectin scavenges away cholesterol in the sample and therewith also cholesterol antibod ies. This leads to an even higher reliability of the method of the invention.

Alternatively a sterol lipid, preferably cholesterol is immobilised in together with a compound binding to cholesterol in the blood derivedsample, such as cholesterol binding heteropolysaccharide, such as β-cyclodextrin, pectin or dextrin. Such molecules scavenge away cholesterol in the sample and therewith also cholesterol antibodies. This leads to an even higher reliability of the method of the invention.

For instance a sterol lipid, preferably cholesterol is immobilised with β-cyclodextrin or pectin on a substrate, such as hollow fiber polypropylene membranes or glass .

Many methods are known to immobilise a sterol lipid, in particularly cholesterol on a substrate. The skilled person will be able to select the protocol suitable for his particular coating. For example a coating may be applied by initially dissolving cholesterol in an organic solvent and further diluting the dissolved cholesterol in an ethanol solution, permitting the solution to evaporate in place within a container, rinsing the coated container with buffered saline, air-drying the container, and sealing the container in vapor-proof pouches with desiccant.

Mycolic acid derived antigens

The mycolic acid derived antigen may be derived from mycobacteria selected from virulent and pathogenic mycobacteria. Preferably, 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.

Natural sources of mycolic acid derivatives include the cell walls of mycobacteria such as Mycobacterium tuberculosis include mixtures of different classes of com pounds and different mycolic acid homologues, often as derivatives in which they are bonded to the wall of the cell.

In addition to the mycolic acids themselves, the cells of mycobacteria also contain compounds derived from the acid, such as sugar esters of mycolic acids. Naturally occurring sugar esters comprise for instance trehalose-6, 6 ' -dimycolate, commonly referred to as TDM, also known as "cord factors"; and trehalose monomycolates (often referred to as TMM) . These sugar esters occur in nature as complex mixtures of different classes of mycolic acids and of different homologues within each class.

Because it is difficult to establish the identity of cord factors present in natural products and to separate individual molecular species it is preferred to use semisynthetic or more preferably synthetic mycolic acid derivatives for the purposes of the invention. Further, it is known that the structure of the mycolic acid unit affects the biological activity of the cord factor. Therefore when natural mycolic acid derivatives would be used in the present invention the difference in biological activity between these derivatives and thus the detection of binding of antibodies of the antigen in the sample may impart a factor of unpredictability and uncertainty to the outcome of the detection. In addition, deviations in the preparation of the natural mycolic acid derivatives may result in problems regarding reproducibility of different test batches .

Therefore in order to be able to provide a method, device kit or sensor with high reliability and reproducible results it is preferred to use semi-synthetic or even more preferred synthetic mycolic acid derivatives which are identical or closely analogous to single compounds found in natural mixtures.

Suitable semi-synthetic derivatives include semisynthetic cord-factors which may be prepared by attaching mycolic acids to the sugar group. These semi-synthetic factors however still contain mixtures of different homo-logue.

Therefore particular suitable mycolic acid derivatives for use in the method, device, kit and sensor of the present invention are synthetic cord factors, for example the synthetic cord factors described in WO 2010/08667, i.e. compounds of formula (M)x (S)y (M')z, wherein x is from 1 to 6, y is from 1 to 12, z is from 0 to 10, each M and each M' is independently a mycolic acid residue including a β-hydroxy acid moiety and each S is a monosaccharide unit.

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 substatrates in various ways that are known to the skilled person. Synthetic mycolic acid derived antigens may be synthetised with particular active groups that enable immobilisation to a substrate material. For instance, for immobilisation on silica, silane coupling chemistry may be applied.

Claims (25)

1. Werkwijze voor het detecteren van een marker voor actieve tuberculose, omvattende de stappen van: i) het verschaffen van een monster afkomstig van een mens of dier, welke vermoed wordt actieve tuberculose te hebben; ii) het blootstellen van het monster aan een sterol-lipide; iii) het verkrijgen van ten minste twee fracties van het monster ofwel voorafgaand ofwel na het blootstellen daarvan aan het sterol-lipide; iv) het blootstellen van de eerste van de fracties aan een substraat dat een geïmmobiliseerd van mycol-zuur afgeleid antigeen draagt; v) het blootstellen van de tweede van de fracties aan een substraat dat geen geïmmobiliseerd van mycol-zuur afgeleid antigeen draagt; vi) het blootstellen van de in stap iv) blootgestelde monsterfractie aan een testsubstraat dat een geïmmobiliseerd van mycolzuur afgeleid antigeen draagt en het blootstellen van de in stap v) blootgestelde monster-fractie aan een controlesubstraat dat een geïmmobiliseerd van mycolzuur afgeleid antigeen draagt; vii) het detecteren van de binding van antili-chamen aan het antigeen van stap vi) in ware tijd (realtime) ; en viii) het vergelijken van de graad of uitgebreidheid van de binding tussen de test- en controlesubstraten, waarbij enige waargenomen mindere binding aan het testsubstraat een indicator van de aanwezigheid van antilichamen tegen het antigeen in het monster is die actieve tubercu- lose in de mens of het dier waarvan de monsters afkomstig zijn indiceert.A method for detecting an active tuberculosis marker, comprising the steps of: i) providing a sample from a human or animal suspected of having active tuberculosis; ii) exposing the sample to a sterol lipid; iii) obtaining at least two fractions of the sample either before or after exposure thereof to the sterol lipid; iv) exposing the first of the fractions to a substrate that carries an immobilized mycolic acid-derived antigen; v) exposing the second of the fractions to a substrate that does not carry an immobilized mycolic acid derived antigen; vi) exposing the sample fraction exposed in step iv) to a test substrate that carries an immobilized mycolic acid-derived antigen and exposing the sample fraction exposed in step v) to a control substrate that carries an immobilized mycolic acid-derived antigen; vii) detecting the binding of antibodies to the antigen of step vi) in real time; and viii) comparing the degree or extent of binding between the test and control substrates, any observed less binding to the test substrate being an indicator of the presence of antibodies to the antigen in the sample that are active tuberculosis in humans or indicates the animal from which the samples originated. 2. Werkwijze volgens conclusie 1, waarbij het in stap i) verschafte monster een volbloedmonster is.The method of claim 1, wherein the sample provided in step i) is a whole blood sample. 3. Werkwijze volgens conclusie 2, waarbij na stap i) plasma gescheiden wordt van het volbloedmonster.The method of claim 2, wherein after step i) plasma is separated from the whole blood sample. 4. Werkwijze volgens één van de conclusies 1 tot 3, omvattende een aanvullende stap van het verdunnen van het volbloedmonster of het plasma.The method of any one of claims 1 to 3, comprising an additional step of diluting the whole blood sample or the plasma. 5. Werkwijze volgens één van de conclusies 1 tot 4, waarbij het van mycolzuur afgeleide antigeen ten minste één gekozen uit de groep van mycolzuur, cord-factor, chemisch gemodificeerd mycolzuur, chemisch gemodificeerde cord-factor, een synthetisch mycolzuurderivaat, een synthetisch cord-factorderivaat is.The method of any one of claims 1 to 4, wherein the 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 order derivative. 6. Werkwijze volgens één van de conclusies 1 tot 5, waarbij het sterol-lipide op een oppervlak geïmmobiliseerd is .The method of any one of claims 1 to 5, wherein the sterol lipid is immobilized on a surface. 7. Werkwijze volgens conclusie 6, waarbij het sterol-lipide geïmmobiliseerd is op een substraat, omvattende het sterol-lipide en een verbinding die aan cholesterol in het monster bindt, zoals pectine of β-cyclodextrine.The method of claim 6, wherein the sterol lipid is immobilized on a substrate comprising the sterol lipid and a compound that binds to cholesterol in the sample, such as pectin or β-cyclodextrin. 8. Werkwijze volgens conclusie 6, waarbij het sterol-lipide geïmmobiliseerd is op een substraat dat het sterol-lipide en fosfatidylcholine omvat.The method of claim 6, wherein the sterol lipid is immobilized on a substrate comprising the sterol lipid and phosphatidylcholine. 9. Werkwijze volgens één van de conclusies 1 tot 8, waarbij het sterol-lipide cholesterol is.The method of any one of claims 1 to 8, wherein the sterol lipid is cholesterol. 10. Werkwijze volgens één van de conclusies 1 tot 9, waarbij de test- en controlesubstraten op silica gebaseerd zijn.The method of any one of claims 1 to 9, wherein the test and control substrates are silica-based. 11. Werkwijze volgens één van de conclusies 1 tot 10, waarbij detectie uitgevoerd wordt bij gebruik van een biosensorchip bij gebruik van een Si-ringresonator.The method of any one of claims 1 to 10, wherein detection is performed when using a biosensor chip when using an Si ring resonator. 12. Werkwijze volgens één van conclusies 1 tot 11, waarbij de detectie uitgevoerd wordt bij gebruik van op-pervlakteplasmonresonantie of elektrochemische impedantie-spectroscopie, isotherme titratiecalorimetrie, biolaagin-terferometrie, optische rasters, fotonisch kristal, akoestische resonantieprofilering, kwartskristalmicrobalansen.The method of any one of claims 1 to 11, wherein the detection is performed using surface plasmon resonance or electrochemical impedance spectroscopy, isothermal titration calorimetry, biolayer interferometry, optical grids, photonic crystal, acoustic resonance profiling, quartz crystal microbalances. 13. Draagbare inrichting voor het uitvoeren van een werkwijze voor het detecteren van een marker voor actieve tuberculose, omvattende: ten minste één houder (4) die een sterol-lipide omvat, die gerangschikt en geconfigureerd is om een mon-sterstroom afkomstig van een mens of dier, welke vermoed wordt actieve tuberculose te hebben te ontvangen; middelen (5) voor het verdelen van de monster-stroom in ten minste een eerste en een tweede monster-stroom; waarbij de middelen ofwel stroomopwaarts ofwel stroomafwaarts verbonden zijn van de ten minste ene houder (4) die een sterol-lipide omvat; een verdere houder (6) voor het ontvangen van de eerste monsterstroom in stroomafwaartse verbinding met een genoemde houder (4) die een sterol-lipide omvat; en die een eerste substraat omvat dat een geïmmobiliseerd van my-colzuur afgeleid antigeen draagt; een nog verdere houder (7) voor het ontvangen van de tweede monsterstroom in stroomafwaartse verbinding met een genoemde houder (4) die een sterol-lipide omvat; en die een tweede substraat omvat dat geen geïmmobiliseerd van mycolzuur afgeleid antigeen draagt; een biosensor (8), omvattende een eerste kamer (9) voor het ontvangen van de eerste monsterstroom in verbinding met de verdere houder (6) die een substraat dat een geïmmobiliseerd van mycolzuur afgeleid antigeen omvat, en een tweede kamer (10) voor het ontvangen van de tweede monsterstroom in verbinding met de nog verdere houder (7) en die hetzelfde substraat dat een geïmmobiliseerd van mycolzuur afgeleid antigeen draagt als de eerste kamer (9) omvat.A portable device for performing a method for detecting an active tuberculosis marker, comprising: at least one container (4) comprising a sterol lipid arranged and configured to receive a sample stream from a human or animal suspected of having received active tuberculosis; means (5) for dividing the sample stream into at least a first and a second sample stream; the means being connected either upstream or downstream of the at least one container (4) comprising a sterol lipid; a further holder (6) for receiving the first sample stream in downstream connection with said holder (4) comprising a sterol lipid; and comprising a first substrate carrying an immobilized mycolic acid derived antigen; still further container (7) for receiving the second sample stream in downstream connection with said container (4) comprising a sterol lipid; and comprising a second substrate that does not carry an immobilized mycolic acid derived antigen; a biosensor (8), comprising a first chamber (9) for receiving the first sample stream in connection with the further container (6) comprising a substrate comprising an immobilized mycolic acid derived antigen, and a second chamber (10) for receiving the second sample stream in connection with the still further container (7) and which comprises the same substrate that carries an immobilized mycolic acid derived antigen as the first chamber (9). 14. Draagbare inrichting volgens conclusie 13, verder omvattende een filtereenheid (3) in verbinding met de eerste buis (2) en de eerste houder (4) en geconfigureerd om plasma van een volbloedmonster te scheiden.The portable device of claim 13, further comprising a filter unit (3) in connection with the first tube (2) and the first container (4) and configured to separate plasma from a whole blood sample. 15. Draagbare inrichting volgens conclusie 13 of 14, waarbij de filtereenheid (3) een filtermatrix omvat.A portable device according to claim 13 or 14, wherein the filter unit (3) comprises a filter matrix. 16. Draagbare inrichting volgens één van de conclusies 13 tot 15, omvattende: een huiddoordringend uiteinde (1) dat verbonden is met een eerste buis (2) die gerangschikt en geconfigureerd is om een bloedmonster uit het huiddoordringende uiteinde (1) te ontvangen en om het bloedmonster weg van de huidpositie te dragen; een f iltereenheid (3) in verbinding met de eerste buis (2) voor het ontvangen van het bloedmonster en geconfigureerd is om plasma van het volbloedmonster te scheiden; een eerste houder (4) voor het ontvangen van het monster, waarbij de eerste houder (4) een sterol-lipide omvat; middelen (5) voor het verdelen van het monster in ten minste een eerste en een tweede monsterstroom; een tweede houder (6) voor het ontvangen van de eerste monsterstroom, waarbij de tweede houder een eerste substraat omvat dat een geïmmobiliseerd van mycolzuur afgeleid antigeen draagt; een derde houder (7) voor het ontvangen van de tweede monsterstroom, waarbij de derde houder een tweede substraat omvat dat geen geïmmobiliseerd van mycolzuur afgeleid antigeen draagt; en een biosensor (8), omvattende een eerste kamer (9) voor het ontvangen van de eerste monsterstroom in verbinding met de tweede houder (6), die een substraat dat een geïmmobiliseerd van mycolzuur afgeleid antigeen omvat, en een tweede kamer (10) voor het ontvangen van de tweede monsterstroom in verbinding met de derde houder (7) en die hetzelfde substraat dat een geïmmobiliseerd van mycolzuur afgeleid antigeen draagt als de eerste kamer (9) omvat.A portable device according to any of claims 13 to 15, comprising: a skin penetrating end (1) connected to a first tube (2) arranged and configured to receive a blood sample from the skin penetrating end (1) and to carry the blood sample away from the skin position; a filter unit (3) in connection with the first tube (2) for receiving the blood sample and configured to separate plasma from the whole blood sample; a first container (4) for receiving the sample, the first container (4) comprising a sterol lipid; means (5) for dividing the sample into at least a first and a second sample stream; a second holder (6) for receiving the first sample stream, the second holder comprising a first substrate carrying an immobilized mycolic acid derived antigen; a third container (7) for receiving the second sample stream, the third container comprising a second substrate that does not carry an immobilized mycolic acid derived antigen; and a biosensor (8) comprising a first chamber (9) for receiving the first sample stream in connection with the second container (6), which comprises a substrate comprising an immobilized mycolic acid derived antigen, and a second chamber (10) for receiving the second sample stream in connection with the third container (7) and comprising the same substrate that carries an immobilized mycolic acid derived antigen as the first chamber (9). 17. Draagbare inrichting volgens één van de conclusies 13 tot 16, waarbij de houder (4) die een sterol-lipide omvat een buis is waarvan de binnenwand een coating heeft die een sterol-lipide in combinatie met fosfatidyl-choline omvat.The portable device of any one of claims 13 to 16, wherein the container (4) comprising a sterol lipid is a tube whose inner wall has a coating comprising a sterol lipid in combination with phosphatidylcholine. 18. Draagbare inrichting volgens één van de conclusies 13 tot 16, waarbij de houder (4) die een sterol-lipide omvat een buis is waarvan de binnenwand een coating heeft die een sterol-lipide en een verbinding die cholesterol in het monster bindt, zoals pectine of β-cyclodextrine, omvat.The portable device of any one of claims 13 to 16, wherein the container (4) comprising a sterol lipid is a tube whose inner wall has a coating that contains a sterol lipid and a compound that binds cholesterol in the sample, such as pectin or β-cyclodextrin. 19. Draagbare inrichting volgens één van de conclusies 13 tot 18, waarbij het substraat van de eerste en tweede kamers (9, 10) op silica gebaseerd is.The portable device of any one of claims 13 to 18, wherein the substrate of the first and second chambers (9, 10) is silica-based. 20. Draagbare inrichting volgens één van de conclusies 13 tot 19, waarbij de biosensor een Si-ringresonator omvat.The portable device of any one of claims 13 to 19, wherein the biosensor comprises an Si ring resonator. 21. Draagbare inrichting volgens één van de conclusies 13 tot 20, verder omvattende middelen voor het verdunnen van het monster verbonden tussen de verdere houder en de biosensor en tussen de nog verdere houder en de biosensor .The portable device of any one of claims 13 to 20, further comprising means for diluting the sample connected between the further container and the biosensor and between the still further container and the biosensor. 22. Kit voor gebruik bij het diagnosticeren van tuberculose, omvattende één of meer huiddoordringende middelen; één of meer buizen; één of meer houders met een sterol-lipide en eventueel fosfatidylcholine, pectine of β- cyclodextrine; één of meer houders, omvattende een substraat met een geïmmobiliseerd van mycolzuur afgeleid anti-geen; en één of meer biosensoren, omvattende ten minste twee kamers die een substraat met een geïmmobiliseerd van mycolzuur afgeleid antigeen omvatten; eventueel middelen voor het verdunnen van bloed of plasma en/of een filtereenheid.A kit for use in diagnosing tuberculosis, comprising one or more skin penetrants; one or more tubes; one or more containers with a sterol lipid and optionally phosphatidylcholine, pectin or β-cyclodextrin; one or more containers comprising a substrate with an immobilized mycolic acid derived antigen; and one or more biosensors, comprising at least two chambers comprising a substrate with an immobilized mycolic acid derived antigen; optionally means for diluting blood or plasma and / or a filter unit. 23. Kit volgens conclusie 22, waarbij de biosensor een Si-resonator omvat.The kit of claim 22, wherein the biosensor comprises an Si resonator. 24. Kit volgens conclusie 22 of 23, waarbij het sterol-lipide cholesterol is.The kit of claim 22 or 23, wherein the sterol lipid is cholesterol. 25. Biosensor (8), omvattende ten minste twee kamers (9, 10) omvattende siliciumsubstraat met een geïmmobili seerd van mycolzuur afgeleid antigeen en een Si-ringresonator. -o-o-o-A biosensor (8) comprising at least two chambers (9, 10) comprising a silicon substrate with an immobilized mycolic acid derived antigen and an Si ring resonator. -o-o-o-