EP2909633A1 - Procédé et dispositif de détection de drogues illicites - Google Patents

Procédé et dispositif de détection de drogues illicites

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Publication number
EP2909633A1
EP2909633A1 EP13780125.4A EP13780125A EP2909633A1 EP 2909633 A1 EP2909633 A1 EP 2909633A1 EP 13780125 A EP13780125 A EP 13780125A EP 2909633 A1 EP2909633 A1 EP 2909633A1
Authority
EP
European Patent Office
Prior art keywords
analyte
receptor molecule
ligand
binding
test
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP13780125.4A
Other languages
German (de)
English (en)
Inventor
Sebastian Klaus
Frank Schwieger
Verena Zimmermann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Securetec Detektions Systeme AG
Original Assignee
Securetec Detektions Systeme AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Securetec Detektions Systeme AG filed Critical Securetec Detektions Systeme AG
Priority to EP13780125.4A priority Critical patent/EP2909633A1/fr
Publication of EP2909633A1 publication Critical patent/EP2909633A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9486—Analgesics, e.g. opiates, aspirine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/948—Sedatives, e.g. cannabinoids, barbiturates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention relates to a method for the determination of analytes, in particular illegal drugs, in a sample, as well as test elements, sampling elements and kits, which are suitable for carrying out the method.
  • Immunoassays based, for example, on "lateral flow technology" are widely used and accepted rapid test systems for the detection of analytes in body fluids or on surfaces
  • a sample containing the analyte is typically taken up and picked up from a surface with a suitable sampling element
  • the test element is then detected by means of an immunological detection reaction, which is based on the formation of immune complexes of antigens and antibodies.
  • Immunological detection systems generally provide high specificity and sensitivity due to the biophysical and biochemical properties of antigen-antibody binding. This is of particular importance in the detection of illicit drugs (e.g., amphetamines, metamphetamines, cannabis, cocaine, heroin). On the one hand, there is a need to quickly prove the use of illegal drugs. On the other hand, the test formats should also have high sensitivity and specificity to rule out false-positive as well as false-negative test results and provide a reliable indication of which drug was consumed.
  • illicit drugs e.g., amphetamines, metamphetamines, cannabis, cocaine, heroin.
  • the test formats should also have high sensitivity and specificity to rule out false-positive as well as false-negative test results and provide a reliable indication of which drug was consumed.
  • Synthetic cannabinoids are a group of more than 500 substances, of which In the 1980s and 1990s, most of them were produced as potential receptor agonists for the human cannabinoid receptors, with the aim of developing new analgesic drugs without the psychoactive component of cannabis.
  • EP 0699906 A2 discloses a rapid drug test, which as a surface, welding or saliva test under the name DrugWipe ® (company Securetec detection systems AG) is commercially available.
  • the test uses a substantially plastic wiper element ("wiper") with welded-on fleece, by means of which a sample of the analyte (for example, from a surface or from a solution) is taken and then directly transferred to an immunochromatographic test strip ("lateral flow").
  • lateral flow immunochromatographic test strip
  • the Chromatography is started, the result of the determination can be read by eye or using a suitable measuring device.
  • the detection of the analyte takes place in that bound to drug molecules (antigens) antibodies bind to a test line and form a colored line due to their gold marking, which can be optically detected in the readout window.
  • Antibodies which are not loaded with drug molecules are intercepted before reaching the test line by means of polyhaptens, which are present on the test strip in immobilized form, and are not visually visible in the readout window.
  • the DrugWipe ® is the only product that forms a colored line in the presence of a specific analyte in a sample and thus has a so-called positive indicator. All other commercial products have a negative display where non-appearance of a line is considered positive for the analyte.
  • Another advantage of the DrugWipe ® is the small sample volume needed to detect drugs and other substances. While in the case of DrugWipe ® a sample volume of less than 25 ⁇ is sufficient, other commercial tests require a sample volume of at least 100 ⁇ up to several milliliters.
  • the small sample volume that implementing the DrugWipe ® - is needed for rapid drug tests constitutes a decisive advantage over other commercially available test systems because drug users often have a very dry mouth due to the physiological effect of the drug and is available only little saliva for sampling , This is particularly beneficial for consumers of cannabis and synthetic cannabinoids, as the use of these drugs is always accompanied by a very dry, low-salivary oral cavity. Consequently, there are - especially in such cases - often only very small sample volumes are available which with the exception of DrugWipe ® - Generally speaking, rapid drug tests are not enough to obtain a trustworthy or error-free result. If it is even possible to perform a rapid drug test, sampling often takes several minutes, which is not reasonable for the subject or the sampler.
  • OraLab ® 6 developed by Varian, is used to detect drugs from saliva samples and is also based on the lateral flow technology. While the specificity of the test is about 90-100%, the sensitivity for amphetamines and opiates is only between 50 and 90%, and for A 9 -tetrahydrocannabinol and cocaine, sometimes even well below 50% (see DRUID study, published on the TIAFT 2009 in Geneva). A major drawback of this test is still the fact that relatively large sample volumes are needed, which are often not available in acute drug users.
  • Dräger's rapid drug test DrugTest ® 5000 which is also based on lateral flow technology, is a reliable test system for the detection of drugs, according to data from the DRUID study (TIAFT 2009, Geneva).
  • this test format has two significant disadvantages .
  • the results on the test strip can only be read out by means of a read-out device, which is very expensive to purchase and only partially suitable for hard field use outdoors.
  • 300 ⁇ of saliva is needed as a sample volume for this test, which is why sampling takes several minutes, especially for drug users with a dry mouth. In this respect, there are considerable problems in terms of economy and ease of use for this test.
  • the test kit Rapid STAT ® offered by the company MAVAND represents another rapid drug test is commercially available, in which a dilute with buffer saliva sample to an immunochromatographic test strip is incubated with labeled binding partners and which, according to Manufacturer's information allows detection to a lower limit of 15 ng / ml of A 9 -tetrahydrocannabinol.
  • a significant disadvantage of this test is its extremely complex and cumbersome handling, which makes it unsuitable for use in traffic police, and the comparatively low specificity for ⁇ 9 - tetrahydrocannabinol of 80-90% (see DRUID study, TIAFT 2009, Geneva ).
  • EP 0 81 1 842 A2 and EP 0 699 906 A2 disclose methods for the determination of analytes in a body fluid or on a contaminated surface.
  • a test kit which comprises a test strip of one or more capillary-active chromatographic materials, a sampling element separate from the test strip surface, and a pressure device for contacting the test strip surface and sampling element.
  • at least part of the sample is transferred from the sampling element to the test element and the analyte, after the start of the chromatography and binding to an analyte-specific binding partner, analyzed.
  • WO 2005/121793 A2 discloses a method for detecting a methamphetamine in a liquid sample.
  • a kit is preferably used which comprises a chromatographic test strip and optionally a sampling element.
  • the test strip in turn comprises (a) a dry porous material on which a pseudoephedrine / carrier conjugate or antibody capable of binding to both the methamphetamine and the conjugate is immobilized in a detection zone, and (b) a separate one Marker release zone, which either the Antibodies in labeled form, provided that the detection zone contains immobilized pseudoephedrine / carrier conjugate, or detectable pseudoephedrine / carrier conjugate, if the detection zone contains immobilized antibody, in the fluid can release.
  • a disadvantage of the detection methods described in EP 0 699 906 A2, EP 0 81 1 842 A2, WO 2005/075982 A2 and WO 2005/121793 A2 is furthermore that no pretreatment of the sample is carried out before the beginning of the chromatography and the analyte-specific binding partner is not incubated directly with the analyte. Rather, the analyte and the analyte-specific binding partner come into contact with each other only during the course of the chromatography, whereby, in particular, the sensitivity of the method is adversely affected and at least partially the legally required minimum detection limits for drug molecules are not reached.
  • synthetic cannabinoids comprise a variety of substances, which in turn show a high structural variability (ie have only minor or no structural similarities with each other). Furthermore, there is usually no pronounced structural similarity to the natural cannabis active agent ⁇ 9 -tetrahydrocannabinol, as is apparent from FIG. 1, for example. Accordingly, the detection of synthetic cannabinoids usually requires a complex, device-based laboratory examination, which is neither time nor local feasible.
  • the object underlying the present invention was thus to provide a method for the determination of analytes, in particular for the determination of illegal drugs, in which the disadvantages of the prior art are at least partially eliminated.
  • the method should allow a simple, rapid and reliable on-site determination of a variety of cannabinoids and opioids, especially of 9- tetrahydrocannabinol and synthetic cannabinoids, which can be performed with a small sample volume while ensuring high sensitivity and specificity ,
  • This object is achieved according to the invention by a method for determining an analyte in a sample, comprising the steps:
  • receptor molecule is the ligand binding domain of a
  • Narcotic-binding receptor molecule in particular the
  • the ligand-binding domain of the receptor molecule is in native, truncated or mutated form and optionally conjugated to a heterologous molecule.
  • the first step of the method according to the invention requires the provision of a test element suitable for the determination of the analyte, which comprises at least one receptor molecule binding the analyte.
  • the test elements used for this purpose comprise any physical form familiar to the person skilled in the art, which is suitable for determining the presence or / and the amount of an analyte in a sample.
  • the test element is preferably designed such that it generates an optically detectable signal in the presence of the analyte to be determined, which enables a qualitative and / or quantitative determination of the analyte.
  • test elements in the context of the present invention include, in particular, test elements for carrying out a heterogeneous, homogeneous or chromatographic test, an enzyme-linked immunosorbent assay (ELISA) or a fluorescence resonance energy transfer (FRET) assay.
  • ELISA enzyme-linked immunosorbent assay
  • FRET fluorescence resonance energy transfer
  • test elements are known in the art and can be selected by the expert according to the particular requirements.
  • the test elements may be microfluidic structures such as microchannels, Stages, branches or / and chambers, which may optionally be achieved an improved transfer of the sample in the test element.
  • the test element is a chromatographic test strip onto which the analyte can be applied, for example, in the form of an aqueous or nonaqueous solution.
  • the chromatographic test strip is formed from a single chromatographically suitable, possibly strip-shaped material.
  • the chromatographic test strip preferably comprises a plurality of capillary active surfaces of the same or different chromatographic materials which are in fluid communication with each other and thus form a transport path along which a fluid, driven by capillary forces, passes through all regions of the test element can flow.
  • Any known liquid-absorbing, porous or capillary-active material may be used as the chromatographic material, e.g. Cellulose and derivatives thereof, glass fibers, as well as nonwovens and woven fabrics of artificial or natural materials. Chromatographic test strips which can be used in the context of the present invention are described, for example, in EP 0 699 906 A2, to the disclosure of which reference is hereby expressly made.
  • the analyte-binding receptor molecule which may be present in soluble form or in immobilized form on the test element, comprises by definition a ligand-binding domain, which is optionally conjugated to a heterologous molecule.
  • ligand-binding domain refers to a sequence of several, ie at least two consecutive amino acids of a receptor molecule via which an analyte to be determined binds to the receptor molecule Protein, which is preferably derived from a membrane protein, more preferably from a transmembrane protein, most preferably from a G protein-coupled receptor.
  • the heterologous molecule is preferably a heterologous polypeptide, more preferably an immunoglobulin domain.
  • the analyte-binding receptor molecule comprises the ligand-binding domain, especially the extracellular ligand-binding domain of a narcotic binding receptor molecule, such as a cannabinoid or opioid binding receptor molecule. More preferably, within the scope of the present invention, a receptor molecule is used which comprises the ligand-binding domain, in particular the extracellular ligand-binding domain of a receptor molecule selected from the group consisting of cannabinoid receptor 1, cannabinoid receptor 2, opioid receptor ⁇ , opioid receptor ⁇ , opioid receptor ⁇ 1 and opioid receptor ⁇ 2.
  • the ligand-binding domain may be a native ligand-binding domain, a truncated native ligand-binding domain or a mutated native ligand-binding domain, wherein the term "native ligand-binding domain” refers to the ligand-binding domain of a naturally occurring receptor molecule and preferably the naturally occurring receptor molecule
  • Naturally occurring receptor molecules which are particularly preferred in the context of the method of the invention include the human cannabinoid receptor 1 (see Figures 6 and 7), the human cannabinoid receptor 2 (see Figures 8 and 9), the human opioid receptor K (see Figures 10 and 11), the human opioid receptor ⁇ 1 and the human opioid receptor ⁇ 2.
  • an analyte-binding receptor molecule which has an amino acid sequence which is shorter than a naturally occurring receptor molecule and / or which comprises a truncated form of a native ligand-binding domain.
  • truncated form of a native ligand-binding domain refers to any fragment of a native ligand-binding domain capable of binding the analyte and an amino acid sequence truncated to the ligand-binding domain of a naturally occurring receptor molecule, e.g.
  • 5% and 75% truncated amino acid sequence wherein the truncation of the amino acid sequence at the N-terminus, at the C-terminus and / or realized between the N-terminus and the C-terminus of the naturally occurring receptor molecule or the native ligand-binding domain of the receptor molecule can be.
  • the amino acid sequence is shortened by up to 10%, 20%, 30%, 40% or 50% compared to the amino acid sequence of the native analyte-binding receptor molecule.
  • an analyte-binding receptor molecule in which the amino acid sequence of the ligand-binding domain by up to 10%, 20%, 30%, 40% or 50% compared to the amino acid sequence of the ligand-binding domain of a naturally occurring receptor molecule, such as the amino acid sequence of the ligand binding domain of human cannabinoid receptor 1, human cannabinoid receptor 2, human opioid receptor ⁇ , human opioid receptor ⁇ 1 or human opioid receptor ⁇ 2, is shortened.
  • an analyte-binding receptor molecule which comprises a has a mutated amino acid sequence towards a naturally occurring receptor molecule, and / or which comprises a mutated form of a native ligand-binding domain.
  • mutated form of a native ligand-binding domain refers to a genetically altered variant of a native ligand-binding domain capable of binding the analyte and an amino acid sequence modified from the ligand-binding domain of a naturally occurring receptor molecule, for example an amino acid sequence having a sequence identity between 50% and 99%, wherein the alteration of the amino acid sequence at the N-terminus, at the C-terminus and / or between the N-terminus and the C-terminus of the naturally occurring receptor molecule or the native ligand-binding Domain of the receptor molecule can be realized.
  • the mutations may be of natural origin (eg, point mutations or transcript or splice variants of a naturally occurring gene) or introduced using recombinant methods known in the art, such as site-specific removal, replacement or / and addition of DNA-level nucleotides of amino acids at the protein level.
  • at least one amino acid exchange results within the amino acid sequence of the naturally occurring receptor molecule, in particular within the amino acid sequence of the native ligand-binding domain, whereby, for example, an increase in the thermal and / or chemical stability of the receptor molecule can be achieved.
  • the aim is to maintain the binding properties of the native receptor molecule to the analyte and at the same time to increase its stability for use in a test element, in particular a stabilization in connection with the processes of drying (dehydration), storage over 1-2 years in the dry state and the rehydration of interest.
  • the analyte-binding receptor molecule has a mutated amino acid sequence, it is preferred that this amino acid sequence is at least 80%, in particular at least 95% identical to the amino acid sequence of the native analyte-binding receptor molecule.
  • an analyte-binding receptor molecule in which the amino acid sequence of the ligand-binding domain is at least 80%, in particular at least 95% identical to the amino acid sequence of the ligand-binding domain of a naturally occurring receptor molecule, such as the amino acid sequence of the ligand-binding domain of human cannabinoid receptor 1, human cannabinoid receptor 2, human opioid receptor ⁇ , human opioid receptor ⁇ 1 or human opioid receptor ⁇ 2.
  • receptor molecules or ligand-binding domains which can be used within the scope of the method according to the invention include, in addition to the cannabinoid and opioid receptors shown in FIGS. 6-13 of the present application, inter alia. the natural or described in WO 92/02640 A1, WO 95/07983 A1, WO 98/33937 A2, WO 00/04046 A2, WO 03/002718 A2, WO 2004/007551 A1, US 6,235,496 B1 and US 2002/0077285 A1 recombinantly produced DNA and amino acid sequences.
  • the disclosure of the above-mentioned documents is hereby incorporated by reference.
  • a significant advantage of the method according to the invention is that the analytes can be detected with high specificity and sensitivity.
  • the specific and sensitive detection of structurally diverse analytes from a particular class of substances can be performed.
  • using the ligand-binding domains of human cannabinoid receptor 1 and human cannabinoid receptor 2 it is possible to have both ⁇ 9 -tetrahydrocannabinol and all currently known as well as future ones to detect synthetic cannabinoids.
  • the analyte-binding receptor molecule may comprise a detectable label, such as an enzyme label, dye label, fluorescent label, or particle label.
  • a detectable label such as an enzyme label, dye label, fluorescent label, or particle label.
  • particle marking in which the analyte-binding receptor molecule is covalently or non-covalently bound to the surface of suitable nanoparticles e.g., gold or platinum particles
  • the marking is particularly preferably a gold marking, which has the advantage that the test result can be recorded and evaluated by the user directly visually and visually.
  • a sample containing the analyte is applied to the test element in a further step of the method of the invention using suitable means.
  • the sample is picked up from a surface by means of a sampling element, which can have one or more sampling surfaces, and the sample sampling element is subsequently brought into contact with the test element, at least a portion of the sample being transferred from the sampling element to the test element.
  • sampling can be carried out in any desired manner, for example by scraping off, wiping off or aspirating the sample from a suitable surface, in particular a body surface such as tongue or skin.
  • the sampling element is preferably a wiping element which makes it possible to wipe off a sample from a surface to be examined and to subsequently transfer the sample to the test element, for example by utilizing capillary effects.
  • the wiping element has one or more wiping surfaces (which are independent of one another), wiping elements having 2, 3 or 4 wiping surfaces being preferred in the case of several wiping surfaces. If a wiping element with a plurality of wiping surfaces is used, usually also several (independent) test elements are used, each of these test elements being brought into contact with a wiping surface of the wiping element.
  • the at least one wiping surface which is preferably welded to a surface of the wiping element, may in principle be made of any material which appears to be of use to the person skilled in the art for the purposes of the present invention and does not adversely affect subsequent transfer of the analyte to the test element.
  • specially absorbent materials in particular fabrics, nonwovens and / or porous matrices (eg membranes and sponges), have proven to be expedient.
  • nonwovens in particular nonwovens based on cellulose, polyester or / and glass fibers, are particularly preferably used, the fibers optionally being able to be held together with the aid of an organic binder.
  • suitable Nonwovens or fibers are described, for example, in DE 38 02 366 A1 and EP 0 699 906 A2, to the disclosure of which reference is expressly made.
  • the thickness of the wiping surface (s) or the material used for this purpose is for the purposes of the present invention of minor importance and is usually in a range of 0.1 to 3 mm.
  • the dimension of the wiping surface (s) is advantageously adapted to the dimension of the test element, i. the width of the wiping surface (s) should neither exceed nor undercut the width of the test element.
  • Preferred dimensions of the wiping surface (s) are in the range of 0.3 to 2 cm in length and in the range of 0.3 to 1 cm in width.
  • the geometry of the wiping surface (s) can be adapted to the particular requirements of the surface to be examined, with a triangular, quadrangular (for example, square, rectangular, diamond-shaped) or roller-shaped configuration of the wiping surface (s) being considered particularly advantageous.
  • a roll-shaped configuration of the wiping surface (s) has the advantage that a particularly good contact between the wiper element and the test element is realized by the large surface of the wiping surface (s) and, accordingly, an increase in the sensitivity of the method can be achieved.
  • the test element or / and the sampling element comprises an analyte-transfer reagent.
  • the analyte transfer reagent which comprises the transfer of the analyte from the surface to be examined to the sampling element and / or the subsequent transfer of the analyte from the sampling element to the test element, in particular by blocking free binding sites on the sampling element or / and influencing the analyte properties favors, for this purpose, for example, on the test element and / or the sampling element, in particular in the area of any existing sample surface (s), be impregnated.
  • the test element used for determining the analyte comprises the analyte transfer reagent described above, while in another embodiment the sample receiving element comprises the analyte transfer reagent.
  • both the test element and the sampling element comprise an analyte-transfer reagent which contains at least one analyte-unspecific substance selected from the group consisting of a protein, a protein mixture, a carbohydrate and a sugar alcohol.
  • the concentration of analyte-specific substance in the analyte-transfer reagent may be adjusted according to the particular requirements of the test element by a person skilled in the art, but is usually about 0.01 to about 15 wt .-%, based on the total weight of the analyte-transfer reagent.
  • the test element comprises the analyte transfer reagent
  • the analyte transfer reagent contains an analyte-unspecific protein and / or an analyte-unspecific protein mixture, in particular an analyte-unspecific protein.
  • the protein used according to the invention is preferably a substance selected from the group consisting of gelatin, ovalbumin and bovine serum albumin, while, for example, skim milk powder can be used as the protein mixture.
  • the sampling element preferably comprises an analyte-transfer reagent which contains a carbohydrate or / and a sugar alcohol, in particular a sugar alcohol.
  • carbohydrate refers to monosaccharides and oligosaccharides of the general empirical formula C n H 2 nOn, which are each natural or synthetic Origin.
  • monosaccharides are naturally occurring tetroses, pentoses and hexoses, such as, for example, erythrose, threose, ribose, arabinose, lyxose, xylose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose and fructose, which in each case are Form or in the L-form can be used.
  • the oligosaccharides such as lactose, maltose, sucrose, trehalose, gentianose, kestose and raffinose.
  • the carbohydrate is a substance selected from the group consisting of glucose, lactose, maltose, mannose and sucrose.
  • sugar alcohol denotes monosaccharide-sugar alcohols of the general empirical formula ⁇ ⁇ ⁇ 2 ⁇ + 2 ⁇ and disaccharide alcohols of the general empirical formula C n H 2n On-i, each of natural or synthetic origin
  • Preferred monosaccharide sugar alcohols include glycerol, erythritol, threitol, ribitol, arabinitol, xylitol, allitol, altritol, galactitol, glucitol, iditol, and mannitol, each of which may be in the D-form or the L-form
  • isomalt, lactitol and maltitol can be used for sugar alcohols
  • the sugar alcohol is a substance selected from the group consisting of glucitol, glycerol, lacitol, mannitol and xylitol.
  • the sample-taking element can be brought into contact with a region of the test element, preferably by slight mechanical pressing of its sampling surface (s), which is designed to apply the sample containing the analyte, at least a portion of the sample from the sampling element the test element is transmitted.
  • the pressure with which the sampling element is pressed onto the test element should be at least as great that a surface contact between the surface of the test element and the sampling surface / the sampling surfaces of
  • Sampling element exists and insofar as a fluid communication between the two elements is made possible.
  • the contacting of sample-taking element and test element leads to a direct contact between the sample (or the analyte) and the analyte-binding receptor molecule present on the test element.
  • a rapid contact between the analyte and the analyte-binding receptor molecule is realized, whereby a rapid formation of the complex of analyte and analyte-binding receptor molecule can take place and the sensitivity and specificity of the detection method is significantly improved.
  • the present invention also contemplates the possibility that contact between the analyte and the analyte-binding receptor molecule will occur only after transfer of the sample from the sampling element to the test element and the start of the chromatography.
  • the test element in a preferred variant additionally comprises at least one agent which, for example, by blocking or destroying unspecific binding sites in the sample or / and by changing the sample consistency chemical or / and mechanical treatment of causes the sample containing the analyte.
  • the analyte is optimally available for binding to the analyte-binding receptor molecule, thus improving the accessibility of the analyte for the analyte-binding receptor molecule as well as the transport over the individual regions of the test element.
  • a chemical sample conditioning agent it may be impregnated, for example, on the test element, wherein for impregnation preferably an aqueous or nonaqueous solution, which contains the chemical sample conditioning agent in a concentration of about 0.01 to about 5 wt .-%, is used.
  • an aqueous or nonaqueous solution which contains the chemical sample conditioning agent in a concentration of about 0.01 to about 5 wt .-%, is used.
  • at least one chemical sample preparation means and at least one mechanical sample preparation means are used in parallel in the test element.
  • Chemical sample conditioning agents which can be used in the process according to the invention include, in particular, acids, bases, buffers, organic solvents and detergents, with bases being considered to be particularly preferred.
  • acids include inorganic acids (eg, hydrochloric acid) and organic acids (eg, acetic acid and citric acid).
  • exemplary bases include, in particular, alkali and alkaline earth metal hydroxides, such as sodium hydroxide and calcium hydroxide, while calcium carbonate, Tris, PBS, phosphate buffers, borate buffers, BICINE buffers and HEPES may be used as buffers, among others.
  • detergents include, inter alia, octylglucoside, Cholamidopropansulfonat, polidocanol, polyalkylene glycol ethers (eg, Brij ®, ® Synperonic) and polysorbates (eg Tween ® 20, Tween ® 80).
  • exemplary organic solvents include, in particular, dimethylsulfoxide, ethanol, glycerol, isopropanol, methanol, and mixtures thereof.
  • Mechanical sample conditioning agents which can be used in the process of the invention include, for example, tissue or / and nonwovens, in particular nonwovens, by means of which the sample is filtered or separated before incubation of the analyte with the analyte-binding receptor molecule, so that especially solid and viscous sample components (for example, solid and viscous salivary components), which can adversely affect the detection method, are retained.
  • nonwovens which can effect mechanical conditioning of the sample include, but are not limited to, for example, the commercially available Ahlstrom 8964, Whatman Rapid 24Q and Freudenberg FS 2216 products.
  • one or more test elements can be accommodated in a housing.
  • the housing preferably has at least one recess, via which the sampling element can be brought into contact with the test element. If the sampling element has a plurality of sampling surfaces, then it is to be regarded as preferred that the housing contains only a single recess for receiving the sampling element. Alternatively, however, it is also possible that the housing contains a number of recesses corresponding to the number of sampling surfaces.
  • the recess (s) may be arranged in any shape (s) and have any dimension and geometry deemed appropriate to the skilled person, but are usually adapted to the location, dimension and geometry of the sample surface (s) of the sampling element. This makes it possible, for example, to obtain a predefined combination of sampling element and test element in order to avoid in this way improper use of the test system.
  • the housing may further comprise a holding device, which allows a reversible attachment of the sampling element to the housing, so that the latter can be removed, for example, for sampling and reassembled after sampling on the housing.
  • a holding device which allows a reversible attachment of the sampling element to the housing, so that the latter can be removed, for example, for sampling and reassembled after sampling on the housing.
  • the sampling element can be placed on the housing in such a way that intensive contact between sampling element or its sampling surface (s) and the test element is ensured, which results in good transfer of the analyte from the sampling element to the analyte-binding Ensures receptor molecule comprehensive test element.
  • sample-receiving element wetted with the analyte is preferably brought into contact with each other for a period of at least 10 seconds, wherein the analyte-binding receptor molecule present on the test element can be incubated with the analyte to be determined, and optionally additionally a chemical or / and mechanical processing of the sample containing the analyte takes place.
  • the analyte on the one hand as completely as possible dissolved out of the sample matrix of the sampling element and on the other hand can react almost quantitatively with the analyte-binding receptor molecule.
  • an incubation time of about 10 seconds to about 600 seconds, more preferably from about 20 seconds to about 180 seconds, most preferably from about 30 seconds to about 90 seconds, has proved to be advantageous.
  • the sample volume required for the determination of the analyte can usually be reduced to less than 10 ⁇ and the sensitivity of the test system can be significantly improved.
  • the test element is contacted with an eluent.
  • the test element preferably comprises a (terminal) region, which is designed to receive eluent and usually comprises an absorbent material, such as tissue and / or non-woven.
  • the eluent migrates through the various areas of the test element, with analyte, analyte-binding receptor molecule and complexes thereof being carried along accordingly.
  • the capillary action of the individual components of the test element can be utilized, which are arranged or connected to one another in such a way that an uninterrupted flow of eluent is ensured.
  • eluent can in the context of the present invention in principle any eluent which appears to be suitable to a person skilled in the art can be used.
  • water and aqueous buffer solutions which optionally contain further substances, such as, for example, a carbohydrate, a sugar alcohol, a detergent, a salt or / and an organic solvent, each as described above, in a concentration of usually approximately, are used in the process described herein 0.05 to about 1 .5 wt .-% may include.
  • an aqueous eluent which comprises magnesium sulfate and a combination of borates (for example magnesium chloro borate, sodium tetraborate, calcium borate and calcium sodium borate) as constituents, the use of which increases the sensitivity and / or specificity of the analyte determination can be achieved.
  • borates for example magnesium chloro borate, sodium tetraborate, calcium borate and calcium sodium borate
  • test element is introduced into a housing, depending on the configuration of the housing there are various possibilities for obtaining wetting of the test element with eluent.
  • the eluent can be applied to the region of the test element designed for receiving eluent, for example by pressing on an ampoule containing the eluent, which is preferably stored inside the housing. If, however, the area of the test element designed to receive eluent projects out of the housing, it is possible to immerse the area in the eluent.
  • the determination of the analyte is initiated, which may include, for example, a competitive test format and / or a non-competitive test format (sandwich test format). Particularly preferred according to the invention is a combination of a competitive test format and a non-competitive test format.
  • the detection method according to the invention first comprises the formation of a Complex of analyte molecules and analyte-binding, optionally labeled receptor molecule, which is transported by means of the eluent, for example together with unbound receptor molecules and / or other substances present in the sample, to a region of the test element which is designed to detect the analyte.
  • the area formed for the detection, in particular for the optical detection of the analyte usually comprises a plurality of defined sections in which different reagents can be immobilized.
  • this region comprises a portion which is designed to bind unbound analyte-binding receptor molecules, a portion which is designed to bind the complex of analyte and analyte-binding receptor molecule, and optionally a portion in which a control signal is generated independently of analyte.
  • the region formed for the detection of the analyte can be formed from one or more materials which are suitable for the purposes of the invention, for example membranes made of Nylon® , nitrocellulose or polyvinylidene fluoride.
  • the section provided for binding unbound analyte-binding receptor molecules may comprise, for example, immobilized analyte analogs, in particular polyhaptens, which intercept the analyte-binding, optionally labeled receptor molecules as a result of formation of a complex at a defined position on the test element (capture line) and thus generation Avoid false-positive results.
  • the complex of analyte and analyte-binding receptor molecule is usually not immobilized on the capture line since the analyte blocks the binding sites required on the analyte-binding receptor molecule.
  • the binding of the complex of analyte and analybindendem Receptor molecule formed section preferably comprises a specific for the analyte binding receptor molecule binding partner (eg an antibody), which causes immobilization of the complex of analyte and analyte binding receptor molecule at a predetermined position on the test element (test line) and allows an optical determination of the analyte, if the analyzer binding Receptor molecule carries an optical label.
  • the immobilization of the complex is usually carried out via a free binding site of the analyte-binding receptor molecule, wherein a staining of the test line is accompanied by a positive detection of the analyte.
  • the interception line can optionally be covered appropriately.
  • the test element comprises a control section
  • a control line additionally appears, which functions as an indicator for perfect functionality of the test element. Excess eluent which leaves the area of the test element formed for the detection of the analyte can optionally be taken up with the aid of a liquid-absorbing material in a region of the test element specially designed for this purpose.
  • the region designed to detect the analyte does not comprise a portion designed to bind unbound analyte-binding receptor molecule.
  • a specific analyte-specific binding partner is preferably used to generate the complex of analyte and analyte-binding receptor molecule, as described, for example, in EP 1 579 222 B1.
  • Such complexes can subsequently be immobilized by means of a complex-specific binding partner, which ultimately leads to a simplified detection of the analyte, since the generation of false-positive signals, which are caused inter alia by not bound to the capture line analyte-binding receptor molecules can be avoided.
  • the section designed to bind the complex of analyte and analyte-binding receptor molecule then preferably comprises a complex-specific binding partner (eg an antibody), which immobilizes the complex of analyte and analyte-binding receptor molecule at a predetermined position on the test element (test line) and in this way allows optical determination of the analyte, if the analyte-binding receptor molecule carries an optical label.
  • the immobilization of the complex is usually carried out via a free binding site of the complex-specific binding partner, wherein a staining of the test line is accompanied by a positive detection of the analyte.
  • the qualitative or / and quantitative determination of the analyte carried out in the last step of the method according to the invention can be carried out in any desired manner.
  • Optical detection methods in particular photometric or fluorimetric detection methods, are preferably used in the context of the present invention.
  • Particularly preferred according to the invention is an optical-visual detection of the analyte.
  • analytes in particular from 5 to 50 different analytes, are determined simultaneously by means of the method according to the invention.
  • the simultaneous detection of a plurality of different analytes from a class of substances such as the simultaneous detection of various cannabinoids, occurs via a single analyte-binding receptor molecule.
  • the test element For the application of the sample formed area in particular one of the number of different substance classes corresponding number of different analyte-binding receptor molecules and, if the test element does not contain a section for capturing unbound analyte-binding receptor molecules, optionally a number of different substance classes corresponding number of complex-specific binding partners, as described above are defined. In this way, it can be ensured that the parallel determination of analytes from different substance classes takes place essentially independently of one another and no interference occurs.
  • the method according to the invention makes it possible to determine one or more analytes with high sensitivity and specificity.
  • SAMHSA Department of Health and Human Services: Proposed Revision to mandatory guidelines for federal workplace drug testing programs, Federal Register (2004), 69, 19673-19732
  • Bosker et al. the lower limit of detection for A s -tetrahydrocannabinol at 4 ng / ml saliva, the lower detection limit for synthetic cannabinoids at 10 ng / ml saliva.
  • the method of the invention can be used to determine any biological or chemical substance that is detectable, for example, using immunological techniques or receptor-ligand techniques.
  • the method described herein is used to detect natural, semisynthetic or fully synthetic anesthetics, as listed in the German Narcotic Law and which bind in vivo to a receptor molecule used in the invention used.
  • anesthetics include, but are not limited to, dissociatives, delirants, empathogens, entactogens, hypnotics, narcotics, psychedelics, sedatives, and stimulants.
  • At least one analyte selected from the group consisting of natural, semisynthetic or fully synthetic amphetamines, benzodiazepines, cannabinoids, ketamines, methamphetamines, opioids and tropane alkaloids is determined, with cannabinoids and opioids being preferred as analytes.
  • cannabinoids A 9 -tetrahydrocannabinol and synthetic cannabinoids are preferred as analytes, while preferred examples of opioids include in particular codeine, desomorphine, fentanyl, heroin, methadone and morphine.
  • Particularly preferred according to the invention is a detection of A 9 -Tetrahydrocannabinol and synthetic cannabinoids.
  • the analyte may be from any source, such as an object wetted with the analyte, particularly from the surface of an object wetted by the analyte, or from a bodily fluid, particularly blood, urine, saliva, or sweat.
  • a bodily fluid particularly blood, urine, saliva, or sweat.
  • the presence or / and amount of an analyte in a sample of saliva or sweat is determined.
  • the amount of sample required to carry out the process is usually from about 0.1 ⁇ to about 200 ⁇ , preferably from about 0.5 ⁇ to about 40 ⁇ , more preferably from about 1 ⁇ to 15 ⁇ , and most preferably from about 2 ⁇ to about 10 ⁇ ,
  • test element for determining an analyte, comprising:
  • test element comprises at least one analyte-binding receptor molecule, wherein the ligand-binding domain of the receptor molecule is in native, truncated or mutated form and is optionally conjugated to a heterologous molecule.
  • the invention relates to a sampling element for receiving an analyte from an object and for transferring the analyte to a test element, wherein the sampling element comprises at least one analyte-binding receptor molecule, wherein the ligand-binding domain of the receptor molecule in native, truncated or mutated form is present and optionally conjugated with a heterologous molecule.
  • the invention relates to a kit for the determination of an analyte, which is preferably used for carrying out the method described above and comprises the following constituents:
  • test element (a) a test element comprising
  • test element or / and the sampling element comprises at least one analyte-binding receptor molecule, wherein the ligand-binding domain of the receptor molecule is in native, truncated or mutated form and is optionally conjugated to a heterologous molecule.
  • test element according to the invention the sampling element according to the invention and the test element or sampling element contained in the kit according to the invention, reference is made to the statements in connection with the description of the method according to the invention.
  • Figure 1 Chemical structure of A 9 -Tetrahydrocannabinol and various selected substances from the group of synthetic cannabinoids.
  • Figure 3 cross section of an embodiment of a test element for
  • test element comprises:
  • a fourth region which is designed for optical detection of the analyte and comprises a test line, if a complex of analyte and analyte-binding receptor molecule has been formed
  • FIG. 4 shows a detailed view of the regions 3 and 4 of the test element according to FIG.
  • FIG. 3 in which the chromatography course of a sample without
  • Analyte is shown.
  • the analyte-binding receptor molecule which is bound to the surface of suitable nanoparticles, is immobilized in region 3 of the test element at the polyhapten line, since it interacts with the polyhaptens via its free binding sites.
  • FIG. 5 detailed view of the regions 3 and 4 of the test element according to FIG.
  • FIG. 3 in which the chromatography course of a sample with analyte is shown.
  • the analyte-binding receptor molecule which is bound to the surface of suitable nanoparticles, is not immobilized on the polyhapten line in region 3 of the test element, since its binding sites are occupied by the analyte and no interaction with the polyhaptens is possible. Instead, the complex of analyte and analyte-binding receptor molecule migrates into the region 4 of the
  • Test element where it is immobilized on the test line.
  • FIG. 6 Nucleotide sequence of human cannabinoid receptor 1 (SEQ ID NO: 1).
  • FIG. 7 Amino acid sequence of human cannabinoid receptor 1
  • FIG. 8 Nucleotide sequence of human cannabinoid receptor 2
  • FIG. 9 Amino acid sequence of human cannabinoid receptor 2
  • FIG. 10 Nucleotide sequence of human opioid receptor 1
  • FIG. 11 Amino acid sequence of human opioid receptor 1
  • FIG. 13 Amino acid sequence of human opioid receptor ⁇ 1,
  • sequence-optimized cDNA sequences of the human cannabinoid receptors 1 and 2 are cloned into a plasmid vector of the pET (pET21 a (+) or pET1007D) type.
  • the expression of the receptors in cell-free environment and the purification of the receptors takes place as in Klammt et al. 2004 (High level cell-free expression and specific labeling of integral membrane proteins, Eur. J. Biochem., 271 (3): 568-80) and Klammt et al. 2005 (Evaluation of detergents for the soluble expression of alpha-helical and beta-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system, FEBS J. 272 (23): 6024-38).
  • the two purified cannabinoid receptors 1 and 2 are conjointly or individually conjugated to the surface of 40 nm gold particles.
  • a di-sodium tetraborate buffer is used to achieve optimal binding to the gold surface.
  • the gold conjugate is placed on the web of a lateral flow test strip and then dried.
  • the storage of the gold conjugate impregnated nonwoven takes place at a relative humidity of ⁇ 5%.
  • the dried-on gold conjugate dissolves and migrates over the test strip.
  • an eluent eg a running buffer
  • the dried-on gold conjugate dissolves and migrates over the test strip.
  • On the test strip is a zone containing proteins on the surface of which A 9 -THC molecules are applied.
  • the gold conjugate binds to these immobilized A 9 -THC molecules when there is no A 9 -THC or any other cannabinoid in a sample to be tested.
  • ⁇ 9 - THC or another cannabinoid in the sample binds to the cannabinoid receptors on the surface of the gold conjugate. This will prevent the cannabinoid receptors from binding to the immobilized ⁇ 9 . Bind THC molecules on the test strip.
  • this lateral flow rapid test can provide a yes / no statement about the presence or absence of cannabinoids in a sample,
  • a water-soluble variant of the coupled opioid receptor ⁇ can be used as a computer-aided design of the protein (Perez-Aguilar et al., 2013: A Computationally Designed Water-Soluble Variant of a G-Protein Coupled Receptor: The Human Mu Opioid Receptor, PLoS ONE 8 (6) e66009).
  • the sequence-optimized cDNA sequence of the human opioid receptor ⁇ is cloned into the expression plasmid pET-28b (+) (EMD / Novagen), which is transformed into the bacterial strain E. coli BL21 (DE3) (EMD / Novagen) for the expression of the receptor , Expression and purification are carried out as in Perez-Aguilar et al. 2013 (A Computationally Designed Water-Soluble Variant of a G-Protein Coupled Receptor: The Human Mu Opioid Receptor, PLoS ONE 8 (6) e66009).
  • the purified opioid receptor ⁇ is conjugated to the surface of 40 nm gold particles. For conjugation, a di-sodium tetraborate buffer is used to achieve optimal binding.
  • the gold conjugate is placed on the web of a lateral flow test strip and then dried.
  • the storage of the gold conjugate impregnated nonwovens takes place at a relative humidity of ⁇ 5%.
  • the dried gold conjugate dissolves and travels across the test strip.
  • eluent e.g., a running buffer
  • the gold conjugate binds to these immobilized morphine molecules when there is no morphine in a sample to be examined.
  • an opioid is present in the sample, it binds to the opioid receptors on the surface of the gold conjugate.
  • the opioid receptors can no longer bind to the immobilized morphine molecules on the test strip.
  • this lateral flow rapid test can provide a yes / no statement about the presence or absence of opioids in a sample.

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Abstract

La présente invention concerne un procédé de détermination d'analytes, en particulier de drogues illicites, dans un échantillon, ainsi que des éléments d'essai, des éléments de prélèvement d'échantillon et des trousses qui sont appropriés pour mettre en œuvre le procédé.
EP13780125.4A 2012-10-22 2013-10-21 Procédé et dispositif de détection de drogues illicites Withdrawn EP2909633A1 (fr)

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EP13780125.4A EP2909633A1 (fr) 2012-10-22 2013-10-21 Procédé et dispositif de détection de drogues illicites

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EP12189463.8A EP2722669A1 (fr) 2012-10-22 2012-10-22 Procédé et dispositif destinés à la vérification de drogues illégales
EP13780125.4A EP2909633A1 (fr) 2012-10-22 2013-10-21 Procédé et dispositif de détection de drogues illicites
PCT/EP2013/071961 WO2014064046A1 (fr) 2012-10-22 2013-10-21 Procédé et dispositif de détection de drogues illicites

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EP13780125.4A Withdrawn EP2909633A1 (fr) 2012-10-22 2013-10-21 Procédé et dispositif de détection de drogues illicites

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WO2014064046A1 (fr) 2014-05-01

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