WO2010089098A1

WO2010089098A1 - Method and means for diagnosing tuberculosis

Info

Publication number: WO2010089098A1
Application number: PCT/EP2010/000658
Authority: WO
Inventors: Detlef Müller-Schulte
Original assignee: Deklatec Gmbh
Priority date: 2009-02-05
Filing date: 2010-02-03
Publication date: 2010-08-12
Also published as: WO2010089098A4

Abstract

The invention relates to a simple rapid diagnosis for tuberculosis, without cumbersome laboratory equipment, by means of novel magnetic and/or colored polymer particles able to deposit themselves complementarily on the bacteria or infectious agents by means of immobilized bacteria-specific ligands, thus forming a visually detectable magnetic and/or colored cluster.

Description

Methods and means for tuberculosis diagnostics

description

Tuberculosis (TBC) is one of the three most common infectious diseases in the world caused by mycobacteria, in particular Mycobacterium tuberculosis (M.L), by droplet transmission. One-third of the world's population is considered infected, meaning that around 2 billion people worldwide carry the bacterium. The route of infection is via inhaled bacteria, which are transmitted by coughing, sneezing or speaking. According to the WHO, the number of new infections per year is 8-9 million cases with 2-3 million deaths. Even in the most advanced countries, the cure for adequate therapy is below 80%. However, this information is not available for non-industrialized countries, which are much lower due to lack of sanitary measures and access to appropriate medical care. Due to the increasing

Traveling and globalization is also increasingly posing a major problem for Western TB. In addition, the TBC issue of AIDS infection, in which TBC is one of the most opportunistic diseases, has become a major challenge for the medical community. To treat TBC adequately, the

Diagnostics, especially in relation to an early detection a crucial role.

TBC diagnostics has long been the standard in medicine, with different test procedures being used, depending on the situation and location. Until recently, the tuberculin skin test with purified tuberculin was performed to diagnose latent TBC. However, since this test proved to be not always meaningful, he was replaced in 2004 by the Mendel Mantoux skin test, which in turn has proved to be not always safe. In addition to these rather simple tests, a number of molecular biological and immunological tests have been developed in recent years, the aim of which was to improve and secure diagnostics. In U.S. Patents 7,122,196 and No. 7,087,713 describes antibodies capable of detecting TBC polypeptides and DNA sequences. U.S. Patent 7,115,361 discloses a method for detecting a mycobacterial CD8 T cell II response using antigenic TBC peptides. Detection of a M. tuberculosis specific rpoB sequence is described by amplification technique in US Patent 7,094,542. Oligonucleotides for amplifying M. twόercw / os / s-specific nucleic acid sequences and their use for the detection of M. tuberculosis are described in US Patent 7,009,041. ^'Iüscher also on a amplification method for detecting M. tuberculosis-specific nucleic acid sequences is based in U.S. Patent 5,908,744 described method.

The detection of the pathogen DNA via the amplification of the nucleic acids by means of the PCR technique {polymerase chain reactiori) is often severely limited by PCR inhibitors, which are present in the clinical samples.

In order to avoid the disadvantages of the previous nucleic acid detection, a method is disclosed by the company. Genpoint AS, Oslo, Norway, (DE 698 02 191 T3, corresponding to WO 1998/051693, as reference here attached), which the binding and lysis of cells, binding thereof and detection of cell nucleic acid on particulate magnetic carriers.

Another detection technique for the detection of pathogens is based on gold colloids, as disclosed in WO 1998/004740 and in US Pat. No. 7,323,309. Here, the colouration based on the plasmon resonance inherent coloration is used, which changes due to particle size conditionally. By binding of such antibodies or nucleic acid sequences to the colloids, which specifically recognize corresponding target structures (nucleic acids, epitopes) of the pathogen, particle agglomeration occurs, which manifests itself in a color change. The disadvantage of the methods based on this principle is the low color depth of the colloids, which is usually masked by the intrinsic staining of the clinical samples (blood, sputum, faeces, urine), as well as in the complicated detection procedure (see J.-Nam et al., J.Amer.Chem.Soc, Vol. 126, 5932, 2004).

In addition to these molecular biological test procedures, which invariably require state-of-the-art laboratory equipment, the QuantiFERON TB Gold Test from Cellestis (Australia) and the T-Spot TB Test (Oxford-Immunotec, GB) have recently become more popular Proven practice. This test is based on the detection of interferon-gamma (IFN-γ) secretion by stimulation of sensitized T-effector cells using specific mycobacterial antigens (eg ESAT-6: early secretory antigen target, CFP-10: culture filtrate protein). comes about. Although the detection has a number of advantages such as sensitivity, rapid detection (24-48 hours), unaffected by BCG vaccination (bacille-Calmette-Guerin) as well as detection of false negative test results, the test method in any case relies on a correspondingly high-tech laboratory infrastructure ,

An alternative to the methods described above is a membrane-based rapid test for the detection of active TBC from serum or whole blood of Fa. Bio-Vita is (EP 0966544), which is based on a complex formation of a gold-labeled antibody-binding protein with a TBC antigen. In case of infection, this complex is detected by a colored band. However, because of lack of sensitivity and lack of specificity, the procedure is not recommended as an alternative to the tuberculin skin test (Pulmonology, Vol. 59, 681, 2005). All methods known from the prior art have in common that they depend either on a highly technical laboratory infrastructure or on reagents which do not allow simple application without adequate laboratory equipment.

The object of the present invention is to provide, by providing easy-to-use methods, the possibility of carrying out rapid and sensitive diagnostics also in non-high-tech laboratories or nursing stations, as are widely used in third-world countries.

The starting point of the invention are magnetic and non-magnetic nanoparticles and microparticles, which undergo specific binding (complexing) with the mycobacteria (pathogen) due to special coatings. The complexation of the bacteria leads to a change in the optical and magnetic properties, which is used for sensitive detection by simple methods.

Magnetic (Fe ₃ O ₄ ), iron oxide (Fe ₂ O ₃ ) or iron oxyhydroxide (FeOOH) are preferably used as magnetic nanoparticles. The presentation of such substances are well known and can either by precipitation - usually for this iron (III) and iron (II) saline solutions with varying molar ratios (2: 1, 0.5: 1 to 4: 1) by adding from bases or by addition of heat to correspondingly colloidal magnetic dispersions (hereinafter also referred to as "magnetic colloid"), decomposition of corresponding metal carbonyls or sintering processes .The particle sizes are generally in the range from 10 to 300 nm. As is known to the person skilled in the art, the particle sizes of the magnetic colloids prepared with the aid of the aforementioned preparation method are known to depend on the concentration of the base, the type of base, the temperature, the stirring method and the salt ratio and the ion concentration. In order to prevent agglomeration of the magnetic colloids, which is caused especially by van der Waals forces, surface-active substances, such as surfactants or emulsifiers, are added to the magnetic nanoparticles, which prevents settling of the colloid in the aqueous phase. Such stabilized colloidal dispersions or magnetic colloids are also known under the name "ferrofluids." They are also commercially available today (Ferrofluidics Corp., USA, Advanced Magnetics, USA, Taibo Co, Japan, Liquids Research Ltd, Wales, Schering AG, Germany ).

As magnetic particles in the context of the present invention are in principle such materials in question, which have a spontaneous magnetization in a static magnetic field. This category includes ferromagnetic and superparamagnetic substances. This class of compounds includes, for example, all iron (II) and iron (III) oxides of the formula FeO, Fe ₂ Ch and iron mixed oxides of the formula Fe ₃ θ ₄ and ferrites of the general formula Mei _-x Zn _x Fe ₂ θ ₄ , where Me is cobalt or Nickel can be. Other compounds which are also ferromagnetic in nature and basically meet the conditions of the invention are nickel, cobalt, gadolinium and the rare earth metals dysprosium, holmium and terbium and ferromagnetic alloys of non-ferromagnetic elements. Compounds of this type, which are also generally known as "Heusler alloys", have, for example, the composition Cu ₂ AlMn or Cu ₂ SnMn, as well as alloys of iron, nickel, cobalt, aluminum, copper, neodymium and boron partially very high coercive force as hard magnetic materials are used, meet basically also the prerequisites required above. In order to be able to use the abovementioned materials in the compositions and processes according to the invention, they have a particle size of from 10 to 300 nm, preferably from 50 to 200. This ensures that the magnetic colloids are present in the polymer matrix in the subsequent encapsulation by a functional polymer in finely disperse form.

In addition to the magnetic properties, another essential aspect of the agents according to the invention is encapsulating the nanoparticles in a functional polymer shell (matrix). The polymer matrix must fulfill the following conditions: (i) chemical functionality and (ii) biocompatibility. The chemical functionality enables the chemical coupling of such bioligands in the form of antibodies, for example, capable of complementary binding with the surface structures of the bacterium. Biocompatibility meets the requirements for the use of magnetic particles in the diagnostic field. As a guideline for the biocompatibility in the selection of the relevant polymer matrices at least 90% maintenance of the cell integrity and cell morphology of HeLa cells after five hours of incubation was used. Polymers that meet these requirements usually belong to the class of hydrogels whose biocompatibility is primarily composed of the high molecular weight

Water content (> 80%) derived. Polymers meeting the above criteria are polysaccharide and oligosaccharides, polysaccharide derivatives such as hyaluronates, alginates, dextran, agarose, dextrin, starch, heparin, hydroxyethylcellulose, furthermore synthetic polymers such as polyvinyl alcohol, polyacrylic acid, polyurethanes, silica gels, polyoxyethylenes, polylactides, Polyglycolides, polycyanoacrylates,

Polyhydroxyethylmethacrylate and copolymers of these substances. Furthermore, proteins such as gelatin, casein, collagen, albumin, fibrinogen or polyamino acids have proven useful for this purpose. For the encapsulation of the magnetic colloids with the polymers, in principle, those methods are applicable which are able to completely encapsulate the magnetic colloids and give correspondingly isolated nano- or microparticulate magnetic particles. Advantageously, the well-known methods such as suspension crosslinking (suspension crosslinking), suspension polymerization or precipitation process ("solvent evaporation process") of organic phase are used for this purpose. These methods are known in the art and may be used by a person skilled in the art at any time.

In the suspension crosslinking process, an aqueous polymer solution in which the magnetic colloid is dispersed is suspended in a water-immiscible phase (organic phase) and then crosslinked with a suitable bi- or trifunctional crosslinker (U.S. Patents 6,204,033 and 4,345,588). Suitable organic phases are oils with a viscosity of 50 to 200 cp (20 ^° C.) or organic solvents into which the polymer is generally dispersed as a 1-20%, preferably as a 2 to 10%, solution together with the magnetic colloid , During the suspension process, the polymer phase is then covalently solidified by addition of crosslinkers to spherical particles, the magnetic colloid being physically encapsulated in the polymer matrix. Paraffin oils, vegetable oils, silicone oils or organic solvents such as xylene, chloroform, butanol, heptanol, toluene, benzene, ethyl acetate, hexane, heptane, octane and mixtures thereof have proven particularly suitable as the organic phase. In order to realize the optimum particle sizes for the various applications or methods, various process-technical measures can be used which enable the synthesis of both nanoparticulate and microparticulate particles. For the synthesis of very small particles in the range from 300 nm to 5 μm, two measures are particularly important: (i) Addition of 0.3-15% (w / w) / (v / v) emulsion stabilizers and (ii) mechanical comminution with the aid of a dispersing tool that works on the rotor-stator principle (eg Ultra-Turrax, IKA Werke, FRG ). The rotor speeds are in the range of 5000 rev / min to 24,000 rev / min., In general, with increasing speed, the particle sizes are shifted downwards (<1 micron). Examples of suitable emulsifiers are: polyoxyethylene adducts, polyoxyethylene sorbitol esters, polyethylene-propylene oxide block copolymers, alkylphenoxypolyethoxyethanols, fatty alcohol glycol ether phosphoric esters, sorbitan fatty acid esters, polyoxyethylene alcohols, polyoxyethylene sorbitan fatty acid esters or polyoxyethylene acids. The

Volume ratios of organic phase to polymer / magnetic colloid phase vary between 10: 1 and 60: 1, that of the polymer phase to the colloid phase usually between 1: 0.5 and 1: 4, preferably between 1: 1 and 1: 3, wherein the polymer-magnetic colloid ratio is determined by the respective amount of the solid content in the colloid. The solids content in the polymer is consistently 20 to 60% (w / w). This variable magnetic colloid content is important for the adjustment of the magnetic response or the magnetic attractive behavior of the particles in a magnetic field, which is crucial for the differential detection of the complexes and represents one of the inventive unique selling points.

Suitable crosslinkers for solidifying the polymer droplets into spherical particles are difunctional and trifunctional substances which can react with the functional groups (hydroxyl, carboxyl, amino groups, for example) of the various polymer matrices. Examples of these are: alkyl dihalides, glutaraldehyde, di- and triisocyanates, divinylsulfone, cyanogen bromide, triallyl isocyanurate, divinylethyleneurea, epichlorohydrin, 2-chloro-1-methylpyridinium iodide, 1,4-butanediol divinyl ether, divinylsuccinate, divinyl glutarate, divinyl adipate. The Concentration of the crosslinker is usually between 0.1-5 mol%, based on the polymer content.

In a further embodiment, as an alternative to the suspension crosslinking process, the suspension polymerization can also be used in order to encapsulate the magnetic colloids in a defined manner. For this purpose, water-soluble vinyl monomer are suspended individually or in a mixture in an organic phase which is immiscible with the monomers, and then free-radically polymerized with the addition of suitable stabilizers. Methods of this type are known from the literature (see Paine et al., Macromolecules, 23j 3104, 1990). As monomers, water-soluble monomers such as e.g. Vinylpyrrolidone, acrylamide, 2-hydroxyethyl methacrylate, hydroxyethyl acrylate, acrylic acid, methacrylic acid, N-isopropylacrylamide, acrolein and mixtures thereof in question. The suspension phases consistently correspond to the water-immiscible organic phases used in the suspension crosslinking. Also come here the same suspension stabilizers used as in the aforementioned crosslinking process. The proportions of polymer to magnetic colloid and organic phase to the aqueous phase are analogous to those in the aforementioned crosslinking process.

Another object of the invention relates to colored or fluorescent particles. These consist basically of the same polymer sheaths as those of the abovementioned magnetic particles, the difference being that the dyes or fluorescent dyes ("markers") are encapsulated in the polymer sheath instead of the magnetic colloids The term "colored" also includes fluorescent and quantum dot-containing particles in the following text Virtually all water-soluble substances can be used which bring about intensive coloring of the particles. Since the diagnosis is partly carried out in whole blood, preferably those dyes or fluorescent dyes are used which have a good contrast to the blood such as methylene blue, Oregon Green, malachite green, Eriochrom black T, Indian yellow, saffron, crystal violet, fuchsin, Erioglaucin A. Acridine orange, RiboGreen.

Another class of substances which are suitable for marking the particles and have extremely strong fluorescence are the so-called quantum dots. These are semiconductor nanocrystals, predominantly with a size in the range of 2 to 10 nm, which are formed from elements of groups II-VI, HI-V or IV-VI. Nonlimiting examples of this are: CdS, CdSe, ZnS, ZnSe, CdTe, MgS, ZnTe. The peculiarity of these materials is their very high fluorescence intensity and the precise setting of the emissions due to the variable band gaps between valence and conductor band, the latter being determined by the size of the particles. Thus, with decreasing crystallite size, the emissions are shifted towards the shorter-wave visible spectrum (blue shift) and vice versa. These precisely adjustable emission frequencies in conjunction with high fluorescence intensity has led to an interesting range of applications in physics and biotechnology in recent years. The syntheses of this class of substance are variously described in the literature (X. Gao, S. Nie, J. Phys. Chem. B Vol. 107, 1 1575, 2003) and, therefore, the general state of the art. For the encapsulation of these particles basically the same procedures are used as in the production of the magnetic particles. The nanocrystals are usually in a concentration of 0.1 to and 3% (w / w), based on the polymer, before. The basis for the use of the agents according to the invention is the specific complexation of the mycobacteria by the magnetic and / or colored particles, wherein the magnetic particles act as catcher and the colored particles as marker probes. In the following, the term "complexation" defines the specific or complementary, noncovalent binding of the magnetic and / or the colored particles to the cell wall structures of the bacteria or bacterial antigens. In order to be able to specifically complex the pathogens, the polymer matrix of the particles is primarily covalently coupled with antibodies or antibody fragments, also referred to below as "catcher structures", "scavengers" or "bio-ligands", which are capable of the cell wall structures (epitopes) or

Antigens that are secreted in the course of infection by the pathogen to make a complementary bond. The invention in no way limiting examples thereof are antibodies against lipoarabinomannan, mannosylated lipoarabinomannan, mycolic acid, arabinogalactan, peptidoglycolipid, trehalose-6-phosphate phosphatase, cord factor, glycolipid, 19 kDa antigen, 38 kDa antigen, 14 kDa (TB68 epitope) Antigen, heat shock protein HSP 65, 14 kDa antigen, 27 kDa antigen and 25 kDa antigen or the 40 kDa antigen.

The recovery of such antibodies as monoclonal, polyclonal or recombinant species are well known in the art and may be utilized at any time by one of ordinary skill in the art (see PJ Cummings et al., Hybridoma, Vol. 17, 151, 1998 ). In addition to the antibodies, antibody fragments such as Fab or F (ab ') ₂ , single-chain molecules (scFv), artificial constructs such as "diabodies", "triabodies" or "minibodies" can also be used.

In addition to the antibodies directed against the surface epitope of the pathogen, it has surprisingly been found that a targeted binding between target pathogen (Mt) and particles is achieved by binding (immobilizing) receptor-analogous structures to the marker and magnetic probes. Receptor-analogous structures in the context of the invention are receptors on the surfaces of such cells, to which the Mt. binds and triggers the phagocytosis and thus the primary infection in the target cell. These include the complement receptors CR1, CR3 and CR4 of the macrophages, the macrophage mannose receptor (MR) and the surface protein A receptor (SPA-R). The receptors can be bound to the probes individually as well as in combination with the other capture structures. The combinatorial immobilization of the receptors and / or antibodies improves the number of possible complementary interactions between catcher / marker probes and pathogens and thus increases the probability of the binding relevant to the detection. The

Provision of the corresponding receptor-analogous structures is generally genetic engineering and is apparent from the prior art.

The coupling of the ligands takes place according to the known methods for the immobilization of bioligands to polymeric carriers (compare "Methods in

Enzymology ", Mosbach Hrsg, VoI 135 Part B, Academic Press, 1987, pp. 3-169) Suitable coupling agents are, for example: cyanogen bromide, carbodiimides, hexamethylene diisocyanate, tosyl chloride, tresyl chloride, 2-fluoro-1-methylpyridinium-toluene 4-sulfonate, epichlorohydrin, N-hydroxysuccinimide, chlorocarbonate, isonitrile, hydrazide, glutaraldehyde, l, r, -carbonyl-diimidazole, 1,4-butanediol diglycidyl ether Furthermore, the couplings of the bioligands via so-called spacer molecules are particularly advantageous , heterobifunctional, reactive compounds that can chemically bind both with the functional groups of the matrix (carboxyl, hydroxyl, sulfhydryl, amino groups) and with the bioligand, examples of which are: succinimidyl-4- (N-maleimido) methyl) -cyclohexane-1-carboxylate, 4-succinimidyloxycarbonyl-α- (2-pyridyldithio) toluene, succinimidyl-4- (p-maleimidophenyl) butyrate, N-γ- Maleimidobutyryloxysuccinimide ester, 3- (2-pyridyldithio) propionylhydrazide, sulfosuccinimidyl-2- (p-azidosalicylamido) ethyl-1,3'-dithiopropionate.

The combinatorial use of the magnetic and the marker particles surprisingly opens up a series of novel diagnostic applications whose unique feature is the simple visual detection of a complex formed without the aid of special analyzers. The extremely high sensitivity of the diagnostic methods according to the invention results from the fact that per test a more than ten thousand-fold excess of particles can be used with a variety of immobilized Bioliganden that can react with the corresponding antigens of the pathogen. As a result, the reaction equilibrium is shifted entirely to the side of the pathogen-magnetic particle complex, resulting in the enormous detection sensitivity of the process. In other words, only a few pathogens or antigens are required to be able to detect it. It has been shown that fewer than 1000 pathogens in the affected infected samples are sufficient to make a clear diagnosis statement. In addition to the reaction kinetics enabling a sensitive diagnosis, a further increase in the detection sensitivity results from the fact that the pathogens are not only "two-dimensionally complexed" but due to the statistical distribution of immobilized capture on the particle surface a three-dimensional pathogen particle cluster consisting of a variety of particles and bacteria form, which can be detected visually due to the size and color in a simple manner.

The selection of different particle sizes opens up three detection possibilities: (i) By using preferably 350 to 700 nm nanoparticles, a pathogen magnetic particle cluster is formed, which is due to the significantly increased magnetic moment in comparison to the base particles using a conventional hand magnets within a few seconds in the form of a clearly visible particle spot (patch) manifested on the test tube wall. This behavior of the magnetic particles, referred to as the differential magnetic attraction, means that the magnetic particle-pathogen cluster formed has a markedly increased magnetic moment with respect to the base particles due to the presence of several magnetic particles and thus with the help of a magnet placed on the sample tube within a few seconds to the tube wall migrates to form a clearly visible particle speckle. In contrast, the starting particles are not attracted by the magnet within this period. The attraction times vary analogously to the viscosity of the sample and are in the range of 30 (gastric juice, low viscosity) up to 240 seconds (sputum, blood, tissue sample). This purely visual proof does not require any optical or other technical-analytical aids.

(ii) Magnetic microparticles (particle size 5 to 30 μm) form in the sample (eg blood, urine, serum or sputum) a pathogen-magnetic particle cluster which, after appropriate separation by a conventional hand magnet, can be detected either visually or with the aid of a conventional microscope is.

(iii) Combinational application of magnetic and stained microparticles (probes) both coated with the same pathogen-specific scavengers. The cluster formed can be separated magnetically and detected by its color without special optical aids. With the help of this as well The methods set out under (i) and (ii) can detect bacterial counts of less than 1000.

The salient features of these procedures are: (a) ease of use - only a few pipetting steps are needed, (b) detection without optical aids, and (c) extreme detection sensitivity.

In addition to the complexation of the pathogens with the aid of specific ligand-bearing particles, a further embodiment of the diagnostic method according to the invention consists of using magnetic and colored / fluorescent polymer particles, on whose surface a respective antibody directed against a different epitope of interferon-γ (IFN-γ) are statistically coupled. The basis of the test procedure is the detection of IFN-γ produced by stimulation of T-effector cells due to incubation with mycobacterial antigens, e.g. ESAT-6 and / or CFP-10 is distributed. This process is the basis of the T SPOT-TB process (Oxford Immunotec, Oxford, UK), which has been on the market for some time. Disadvantage of the method, however, is its time-consuming and complicated handling, which can only be performed in a correspondingly state-of-the-art laboratory. The application of the agents according to the invention makes it surprisingly possible to apply this detection principle within a significantly simplified test procedure and thereby make this diagnostic principle accessible for simply equipped laboratories or medical facilities.

After addition of the antibody-carrying polymer particles, the IFN-γ formed can be formed by forming a particle cluster composed of several crosslinked particles, which can be detected either by settling in the sample tube or by differential magnetic attraction. Different combinations are suitable for this test procedure, depending on whether the detection in the sputum, Urine, whole blood or serum is performed. Possible combinations are: (i) magnetic particles and colored particles, especially suitable for whole blood, (ii) nanoparticles and microparticles, (iii) two magnetic particles suitable for high viscosity sputum. The particle sizes which are particularly suitable for these embodiments are in the range from 0.8 to 10 μm, the particle sizes being used in accordance with the viscosities of the respective samples. Particle sizes of 3-10 microns and larger are preferably used in higher viscosity media because the magnetic attraction of these particles results in faster detection due to the higher magnetic moment to nanoparticles.

Another embodiment is the use of only one type of particle to which the two anti-IFN-γ antibodies are immobilized simultaneously in a random manner. It is crucial in this embodiment that the immobilization density of the antibodies remains low in order to (a) obtain the steric accessibility for the IFN-γ and (b) to suppress intraparticular crosslinking by the cytokine. Particularly suitable for this embodiment are microparticles with a size> 3 microns, whose concentration is chosen so that the reaction is largely shifted in the direction of clustering. The advantage of the aforementioned methods compared to the methods known from the prior art is that

(i) the test can be carried out in a tube, (ii) only a few pipetting steps are necessary, and

(iii) no technical aids such as microscope, nephelometer, photometer, etc. are necessary for pathogen detection.

The agents and methods according to the invention are explained in more detail below with reference to some examples. example 1

A 10% (w / w) gelatin solution in 2 ml of 0.05 M Na phosphate buffer, pH 8.2, is prepared by heating the mixture to 80 ^° C. Thereafter, the solution is brought to 45 ⁰ C. To this solution, first 0.5 ml of one according to a protocol of Shinkai et al. (Biocatalysis, Vol. 5, 61, 1991) added magnetic colloid added. This is followed by the addition of 0.5% (v / v) 12% glutaraldehyde solution. The suspension is then sonicated for 2 minutes at 45 ° C in an ultrasonic bath. Thereafter, the resulting mixture is dissolved in 60 ml vegetable oil preheated to 45 ° C (viscosity 84 cp) in which 0.8% (v / v) Pluronic L61, 0.8% (v / v) Tetronic 1 101 and 2.5% (v / v ) Dehymuls FCE are dissolved and homogenized at 6,000 rpm with the aid of a dispersing tool (T25 Ultra-Turrax, IKA Werke, FRG) under nitrogen atmosphere for 2 minutes. Thereafter, the dispersion is cooled down to <10 ° C by means of ice cooling. The suspension is allowed to react with gentle stirring for 3 hours at room temperature. To

After addition of 50 ml of petroleum ether, the magnetic fraction is separated by means of a neodymium-boron-iron hand magnet and washed ten times alternately with petroleum ether and ethanol. This is followed by washing five times with PBS buffer. There are obtained polymer particles with a mean size of 0.943 microns. The recovered particle fraction is then suspended in 3 ml of 0.1 M Na phosphate buffer, pH 8.2, and activated with the addition of 0.5 ml 12% glutaraldehyde solution over a period of 2 hours at room temperature. The fraction is washed several times with redistilled water with the aid of magnetic separation. The washed fraction is dissolved in a mixture consisting of 2 ml of 0.05 M phosphate buffer, pH 8.2, in which 0.85 mg of anti-cord factor (trehalose-6,6'-dimycolate) IgG and 2 mg of cyanoborohydride are dissolved, over a period of 3 hours with gentle shaking reacted. It is washed several times with redistilled water and PBS buffer, pH 7.2. Incubation of the recovered fraction with a TBC infected sputum sample leads within 30 min. To bacteria-magnetic particle clusters, which can be detected by means of a hand magnet within 250 sec. As a brown-black particle agglomerate on the tube wall.

Example 2

To a 4 ml of 25% (w / w) aqueous dextran solution (average molecular weight 40,000) are added 1.2 ml of 5 M NaOH and 3 ml of MnZn ferrite suspension (BASF, Ludwigshafen, mean particle size 12 nm). The mixture is sonicated for 10 minutes under ice-cooling and then injected by syringe into 80 ml of conventional cottonseed oil containing 0.3 g of polyoxyethylene sorbitan monooleate. It is followed by repeated sonication for 3 min. At 0 ⁰ C. While stirring (2000 rpm, KPG stirrer) 0.5 g of cyanogen bromide, dissolved in 20 ml of ether was added. After 15 minutes, the magnetic particles are isolated by magnetic separation (neodymium-boron-iron magnet) and washed several times with ether and ethanol alternately. The recovered particles have a mean size of 4.8 microns and a magnetic content of 28%. The magnetic particle fraction is then lyophilized and then activated with a solution consisting of 3 ml of absolute dimethylformamide (DMF) and 0.4 ml of hexamethylene diisocyanate over a period of 30 min. This is followed by repeated washing with absolute ethanol and DMF. The washed fraction is then reacted with 2 ml of DMF in which 1 mM 6-aminocaproic acid is dissolved over a period of 60 min. At room temperature. This is followed by repeated washing with bidistilled water. The repeatedly washed with water, carboxylated carrier is incubated twice with 0.005 M HCl, washed twice with bidistilled water and then with 2 ml of 20 mg of N-cyclohexyl-N '- (2-morpholinoethyl) - carbodiimide-methyl-p-toluenesulfonate containing 0 , 1 MES buffer, pH 4.5, for 30 min. At room temperature activated. It is washed five times with ice water, followed by a όhour incubation with 0.45 mg of anti-lipoarabinomannan IgG (GeneTex Inc. San Antonio, USA) dissolved in 3 ml of 0.05 M MES buffer, pH 6.0. The reacted product is incubated at room temperature over a period of 2 hours with 5 ml of 0.05 M Tris / HCl buffer / 1% ethanolamine, pH 8.5 and then washed several times with bidistilled water and sterilized PBS buffer. Incubation of the recovered magnetic particle fraction with a M. tuberculosis- containing sample leads within a period of 30 to 60 min. To a bacterial magnetic particle cluster, which is visually detectable analogously to Example 2.

Example 3

4 ml of an aqueous 10% (w / w) hydroxypropyl cellulose solution are mixed with 1.2 ml of magnetic colloid prepared according to the protocol of Shinkai et al. (Biocatalysis, Vol. 5, 61, 1991), mixed and sonicated for 2 min in an ultrasonic bath (60 W) with ice cooling. The solution is adjusted to pH 10.5 with 3 M NaOH. Then, 120 μl of divinyl sulfone are added. The suspension is then added under nitrogen to 150 ml of olive oil (viscosity 95 cp) containing 0.6% (w / w) Tween 80, 0.1% (w / w) Prisorine 3800 and 1.2% (v / v ) Span 85 are dissolved using a dispersing tool (Ultra-Turrax; 12,000 rpm). The dispersion is continued for 10 min under nitrogen (5 ml / min). Thereafter, the reaction mixture is allowed to react for a further 30 min. With gentle stirring at 4 ° C. The magnetic phase is separated by placing the magnetic fraction on a glass column densely packed with steel wool (filling volume: about 5 ml, inner diameter: 1.2 cm), which is surrounded by an annular neodymium-boron-iron magnet. The retentate is then washed three times with 20 ml PBS buffer, pH 7.2. It is followed by removal of the magnet and elution with 5 ml of PBS buffer. There are magnetic particles with a mean particle size of 0.82 microns. The product is lyophilized over a period of 5 days. thereupon 3 ml of molecular sieve-dried dimethylsulfoxide in which 0.6 mM 4-dimethylaminopyridine and 0.3 mM 2-fluoro-1-methyl-pyridinium-toluene-4-sulfonate are dissolved are added. Reaction time: 45 minutes at room temperature. The product is washed several times alternately with absolute ethanol and tetrahydrofuran and finally twice with 0.05 M K phosphate buffer, pH 8.0, with the aid of the magnetic separation column. It is then eluted with 3 ml of PBS buffer, pH 7.2. To the eluate is added 0.5 ml of the same buffer in which 35 μg of anti-38 kDa IgG (Santa Cruz Biotechnology Inc., CA, USA) are dissolved. Reaction time: 12 hours at 4 ° C with gentle shaking. For deactivation, the support for 2 hours at 4 ⁰ C with 5 ml of 0.1 M Tris-HCl buffer / 1% glycine, pH 8.5 is dissolved, incubated. After washing with bidistilled water, the magnetic fraction is then stored for 2 hours in 4 ml of 0.1 M Tris-HCl buffer / 5% glycerol / 0.1% serum albumin, pH 7.5. After washing the magnetic particles repeatedly with bidistilled water, the magnetic particles are dispersed in 2 ml of sterile physiological saline. 30 to 60 minutes incubation of the magnetic particle fraction obtained with a sample infected with mycobacteria results in a magnetic particle-pathogen cluster which can be detected analogously to Example 2.

Example 4

A mixture consisting of 7 ml of 10% (w / w) aqueous acrylamide solution, 0.5 ml of 30% N, N'-methylenebisacrylamide and 2 ml of vacuum distilled acrylic acid are flushed with argon for 15 min. Then 3.5 ml of magnetic colloid EMG 507 (from FerroTec, USA) and 0.4 ml of 5 N NaOH solution are added to the solution. The mixture is sonicated for 5 min. With ice cooling in an ultrasonic bath (60 W). After adding 1.2 ml of 35% ammonium peroxodisulfate solution to the aqueous phase, it is dissolved in 300 ml of xylene in the 0.8% (v / v) Prisorine 3800 and 0.1% (v / v) Igepal 520 are, with stirring (1200 rpm) and ice cooling and a continuous stream of argon dispersed. After 20 sec. Are inflicted 0.4 ml of N, N, N ', N'-Tetrarnethyl-ethylenediamine, and the mixture 8 min. At 10 ⁰ C stirred further. Thereafter, the mixture is allowed to react for a further 20 min. At 15 ° C. The magnetic phase is separated by placing the magnetic fraction on a glass column densely packed with steel wool (filling volume: about 5 ml, inner diameter: 1.2 cm), which is surrounded by an annular neodymium-boron-iron magnet. The retained fraction is then washed with a total of 50 ml of PBS buffer, pH 7.2, (flow rate: 1 ml / min). The magnetic particles are washed twice with 10 ml of 0.005 M HCl and, after removal of the magnet, by charging 5 ml of 0.1 M morpholinoethanesulfonic acid buffer (MES), pH 4.7, (flow rate: 0.5 ml / min) from the separation column eluted. Magnetic particles with a mean particle size of 645 nm are produced. To the eluate is added 0.5 mM N-cyclohexyl-N '- (2-morpholinoethyl) carbodiimide-methyl-p-toluenesulfonate and 1 mM N-hydroxysuccinimide; Reaction time: 30 min. At room temperature. After activation, the support is applied to the magnetic separation column and washed with a total of 10 ml of ice water. The washed magnetic fraction is eluted after removal of the magnet with 4 ml of MES buffer, pH 5.5, and divided into two equal particle fractions. The two fractions are washed with 1 ml of bidistilled water in which 450 μg of anti-INF-γ-IgG (MAI-23837, ABR-Affinity BioReagent Inc., USA) and 450 μg of anti-INF-γ-IgG (MA 1- 37933) were incubated for 4 hours at room temperature 4 ° C. It is then washed with 20 ml of PBS buffer / 0.5% Tween 20, pH 7.2, and the magnetic fraction with 5 ml of 0.05 M Tris / HCl buffer / 1% ethanolamine, pH 8.5, eluted. The eluate is left in this elution solution for 2 hours. After washing the support with 40 ml of PBS buffer, pH 7.2, with the aid of the magnetic separation column is eluted with 4 ml of sterile physiological saline. Both eluates can now be probed with a cell sample stimulated with ESAT-6 or CFP-10, which according to a protocol of T. Meier et al. (Eur. J. Clin. Microbiol. Infect. Dis., Vol. 24, 529, 2005) were used to detect IFN-γ. For this purpose, 10 × 10 ⁶ particles are incubated for 2 hours at room temperature. The presence of IFN-γ manifests itself in a magnetic particle cluster, which becomes visible on the wall by applying a conventional hand magnet (neodymium-boron-iron) to the sample tube after 30 to 45 seconds.

Example 5

In 5 ml of ethylene glycol 0.8% polyvinyl alcohol (molecular weight 84 kDa) are dissolved at 16O ⁰ C. After the solution has cooled down to 80 ^° C., 1.5 ml of ferrofluid 507 (from Ferro Tee, USA) are added to the solution. This is followed by a two-minute treatment in an ultrasonic bath at 8O ⁰ C. Then allowed to cool the dispersion to 70 ⁰ C. The solution is then dissolved in 60 ml of rapeseed oil preheated to 70 ^° C. (viscosity 140 cp), in which 1.2% (w / w) Pluronic 6200 and 0.5% Prisorine 3800 are dissolved, with stirring (1000 rpm) suspended for two minutes. Thereafter, the stirring vessel is cooled down with ice water. After about 4 minutes periförmige particles fall out. It is stirred for 15 minutes under ice cooling. Thereafter, 50 ml of petroleum ether are added and the magnetic particle fraction with the aid of a neodymium-boron-iron hand magnets separated. It is with several times

Petroleum ether, methanol and ice water washed. There are particles with a mean particle size of 8.6 microns.

In a manner analogous to that described above, a second particle fraction is prepared which contains 2.5 mg methylene blue instead of the magnetic colloid. The purification of the non-magnetic fraction is analogous to the magnetic fraction with the difference that each one centrifugation step is applied. The fractions obtained are then each with 2.5 ml of epichlorohydrin and 5 ml 3 M NaOH solution for two hours at 60 ⁰ C reacted. This is followed by intensive vigilance with ethanol and redistilled water using a magnetic separation step or by centrifugation.

Both fractions are immobilized separately with an antibody directed against Mycobacterium tuberculosis 38 kDa (HTM83) protein (Santa Cruz Biotechnology Inc, CA, USA) as follows: 2 ml of 0.5 M K phosphate buffer / 2.5 M ammonium sulfate, pH 8.5, in which 70 μg of IgG are dissolved, is reacted with the particle fractions over a period of 24 hours at room temperature. Followed by a β-hour incubation with 4 ml of 0.1 M Tris-HCl buffer / 1% ethanolamine, pH 8.5. This is followed by repeated washing with sterilized PBS buffer, pH 7.2. 30 to 10 minutes incubation of the recovered magnetic particle and color particle fraction with a mycobacteria-infected sample gives a colored magnetic particle-bacteria cluster, which can be detected analogously to Example 2.

Claims

claims

1. Polymers, magnetic colloid and / or visual marker-containing nano- and micro-particles for tuberculosis diagnostics, characterized in that the polymeric particles are coated with one or more bioligands, the

Cell wall structures, antigens of Mycobacterium tuberculosis or infection-related secreted antigens bind complementary.

2. Polymer nanoparticles and microparticles, according to claim 1, characterized in that the visual markers are water-soluble dyes, water-soluble fluorescent substances or quantum dots.

3. Polymeric nanoparticles and microparticles, according to claim 1, characterized in that the polymer particles having bioligands coupling reactive groups.

4. Polymeric nanoparticles and microparticles, according to claim 3, characterized in that the coupling groups with antibodies, antibody fragments (Fab, F (ab ') ₂ ), artificially produced antibodies ("diabodies", "triabodies" or "minibodies") , Single chain molecules (scFv), modified antibodies,

Antibody conjugates, antigens, proteins, glycoproteins or oligosaccharides are implemented.

5. Polymeric nanoparticles and microparticles, according to claim 4, characterized in that the antibodies, antibody fragments, antigens, artificially produced

Antibodies, modified antibodies, antibody conjugates, proteins, glycoproteins or oligosaccharides specific to lipoarabinomannans, mycolic acids, arabinogalactans, peptidoglycolipids, trehalose-6-phosphate Phosphatase, cord factor, glycolipids, the 19 kDa antigen, the 38 kDa antigen, the 14 kDa antigen (TB68 epitope), the heal shock protein HSP 65, the 14 kDa antigen, the 27 kDa antigen, the 25 kDa antigen or the 40 kDa antigen.

6. Polymeric nanoparticles and microparticles, according to claim 3 or 4, characterized in that the coupling groups are reacted with two antibodies, antibody fragments or artificial antibodies which are directed against two different epitopes of IFN-γ.

7. Polymeric nanoparticles and microparticles, according to one of claims 1 to 6, characterized in that the polymer from the group of polysaccharides, polycyanoacrylates, polyacrylates, dextran-grafted polyamino acids (polyamino acid-g-dextran), starch, heparin, dextran sulfate, Agarose, alginates, hyaluronic acids, chitosans, cellulose derivatives, pullulans, hydroxypropylcelluloses, dextran-g-polyethylene glycol alkyl ethers, pectins, dextranes, polylactides, polyglycolides, polyurethanes, polyethyleneimines, gelatin, caseins, collagens, albumins, fibrinogens or polyamino acids, silica gels, Polyvinyl alcohols, polyether esters, polyacroleins, polyesters, polyacrylic acids, poly-N-isopropylacrylamides, poly-N-substituted acrylamides, poly-N-substituted methacrylamides, poly (ε-caprolactones), polyoxyethylene-oxypropylenes, linear copolymers thereof, graft copolymers or block copolymers thereof is.

Polymeric nanoparticles and microparticles, according to claim 7, characterized in that the polymer or copolymers or graft copolymers has cationic groups in the form of secondary, tertiary or quaternary amino functions.

9. polymer nanoparticles and microparticles, according to any one of claims 1 to 8, characterized in that the particles have a size of 100 nm to 600 nm.

10. Polymeric nanoparticles and microparticles, according to one of claims 1 to 8, characterized in that the particles have a size of 0.8 to 2000 microns.

1 1. Polymeric nanoparticles and microparticles, according to any one of claims 1 to 10, characterized in that the polymer particles are crosslinked with a bi- or trifunctional substance.

12. Polymeric nanoparticles and microparticles, according to one of claims 1 to 1 1, characterized in that the magnetic colloids ferro-, ferri- or superparamagnetic or ferrites or magnetic substances with a Curie temperature in the range of 40 ° C to 500 ° C are.

13. Polymeric nanoparticles and microparticles, according to claim 12, characterized in that the magnetic content in the polymer particles is 10 to 60% (w / w).

14. Polymeric nanoparticles and microparticles according to one of claims 1 to 13, characterized in that the visual markers are present in a concentration of 0.1 to 5% (w / w) in the polymer particle.

15. Polymeric nanoparticles and microparticles, according to any one of claims 1 to 14, characterized in that the particles by means of emulsion polymerization,

Suspension polymerization, suspension crosslinking or precipitation.

16. A method for the diagnosis of tuberculosis with the aid of magnetic and / or visual marker-containing nano- and / or microparticles, according to one of claims 1 to 15, characterized in that the polymeric nano- and / or microparticles with a sputum, gastric juice -, urine, ejaculate, punctate or tissue sample are incubated and by specific binding to the

Mycobacteria (M. tuberculosis) or antigens released by mycobacterial infection form a visually detectable magnetic and / or colored particle cluster.

17. A method for the diagnosis of tuberculosis, according to claim 16, characterized in that the polymeric nano- or microparticles are used alone or in combination with the marker particles.

18. A method for the diagnosis of tuberculosis, according to claim 16 or 17, characterized in that two polymeric nano- or microparticle fractions coated with one each directed against a different epitope of IFN-γ antibody and with an infected and sensitized with M. tuberculosis antigens sample be incubated.

19. A method for the diagnosis of tuberculosis, according to claim 18, characterized in that the IFN-γ release by incubation of a M. tuberculosis infected sample with the M. tuberculosis antigens ESAT-6 and / or CFP-10 is triggered.

20. Use of magnetic and / or colored nano- and / or microparticulate particles alone or in combination according to one of claims 1 to 15 as an agent for tuberculosis diagnosis.