WO2003016903A2

WO2003016903A2 - Device and method for determining an analyte

Info

Publication number: WO2003016903A2
Application number: PCT/AT2002/000246
Authority: WO
Inventors: Walter Pils; Dietmar Pils
Original assignee: Walter Pils; Dietmar Pils
Priority date: 2001-08-20
Filing date: 2002-08-16
Publication date: 2003-02-27
Also published as: WO2003016903A3; AU2002332942A1; WO2003016903A8

Abstract

The invention relates to a method for determining at least one analyte from a sample by an immunochemical reaction with a device consisting of several zones. Said at least one analyte is applied on a starting zone in a reagent, especially an organic reagent, and flows into at least one other zone with one or several fields under the effect of capillary forces, whereby at least one specific binding partner, to which at least one substance is conjugated, is temporarily immobilized in a field.

Description

Device and method for the detection of an analyte

The invention relates to a method for the detection of at least one analyte from a sample by an immunochemical reaction with a device consisting of several zones. Furthermore, the invention relates to a device for the detection of at least one analyte from one or more capillary-capable materials, on which at least one starting zone and an adjacent zone consisting of one or more fields in which or at least one immobilized reagent is arranged. The invention further relates to a reagent for performing the method according to one of claims 1 to 17 and an analysis kit according to claim 57. The invention also relates to the use of the device, the reagent and the analysis kit according to claims 58 to 60.

Due to the increasing misuse of drugs and drugs, there is an increasing need to prove them in humans in order to impair them e.g. exclude their ability to react during working hours or on the road. The determination and quantification of analytes, in particular illegal substances, in biological samples, e.g. Blood and urine is a useful analytical method to verify drug abuse. It is currently common to detect drugs or drug derivatives in the blood, serum, plasma or urine. Due to the complex sampling, e.g. Blood, which can only be obtained from medical specialists, can only be detected in special laboratories and therefore involves a great deal of effort. Due to the complex analysis procedures, there are often large time delays until a final result is available.

The detection of illegal substances from the urine offers an alternative. However, the detected amounts of drugs or drug substitutes from positive urine tests do not clearly correspond to the concentrations that are detected in the blood, since these substances are sometimes still detectable in the urine days after their consumption, but are no longer present in the blood and therefore at the time there is no more drug-specific impairment of the test person when the urine is released.

To determine the current condition of a person, only analyzes that determine the drug concentrations from the blood are useful. However, the concentration of illegal substances in the blood correlates very well with that of saliva. To detect drugs or drug substitutes from saliva must be very sensitive Tests are used because there are sometimes only lower concentrations of these substances in the saliva than in the blood. It is therefore expedient to carry out an analysis which enables the detection of illegal substances from the saliva.

DE 19622503 AI describes a method for the detection of analytes on surfaces which have come into contact with body fluids. This document describes a test strip made of one or more capillary-active sheet-like materials capable of chromatography in fluid contact with one another with an eluent application zone at one end and a target zone at the other end. There is a capture zone between the eluent feed zone and the target zone, in which a capture reagent is immobilized. The capture reagent can either bind the analyte, a specific analyte binding partner or a labeled binding partner. Between the eluent application zone and the capture zone there is still a conjugate zone that contains a migratable labeled binding partner. The binding partner can specifically bind either the analyte or the specific analyte binding partner or the capture reagent, the binding of the labeled binding partner leading to a detectable signal in the capture zone or in the target zone. This signal indicates the presence of the analyte. Furthermore, a wiping element is separated from the test strip surface and a pressing device which eliminates contact between the wiping element and the test strip surface between the eluent application zone and the conjugate zone. With the wiping element, body fluids are absorbed from a body region. Direct markings, such as metal markings, in particular the gold marking, are preferred as markings for detection, because the test result can be read directly from the eye. Antibodies and antibody fragments can be considered as binding partners for the analyte. With this method, absolute amounts of up to 10 ng analyte, in particular drugs on surfaces, can be detected. A disadvantage of this disclosure is that low concentrations of analytes cannot be detected.

US Pat. No. 6,248,598 B1 describes a method which, on the one hand, for the production of saliva and, on the other hand, for the detection of at least one analyte therein without any additional external

Need to add reagents. By determining drugs from saliva, short-term consumption of these can also be demonstrated. The US B1 describes a method for the detection of an analyte with a device which fluidly connects a first part with absorbent material, which absorbs saliva in the mouth, to a second part and then enables a visual detection of analytes. light. In this second part there is a ligand that specifically binds an analyte, an indicator consisting of an analyte with a visually detectable label, an area on which the presence or absence of the analyte is indicated and possibly a control area. Furthermore, a holder in which a window for visual detection is located is described. One end of the device is for 10 to

120 sec. In the mouth to accumulate saliva, removed again and within 1 to 60 min. after saliva collection, the presence or absence of the analyte can be visually indicated.

WO 00/208 62 AI describes a method and an apparatus for the in vitro detection of many different analytes, from serum, plasma, blood, saliva or urine of human or animal origin. The detection system consists of a plastic device with an absorbent pad with an affinity membrane. The device has specific regions on the membrane in which antigens are immobilized. Each device has a quality control substance to check that the device is working properly. In the case of antibody detection, the control material is usually human IgG. In the case of antigen detection, the control material is a specific antigen. Any area can be used as a control area in a multi-analyte detection system. The device consists of a microporous membrane with a first and a second surface, a liquid flowing from one side to the other. The

Membrane has a multiplicity of readable regions which are arranged spaced apart from one another over the entire membrane, an antigen, antibody or hapten being immobilized on each region. Furthermore, the device has a cover for the first side of the membrane with a recess for receiving liquids, whereby a region of the membrane is visible. Finally, the device consists of an absorbent

Material that is in contact with the second side of the membrane for the absorption of the liquid that has diffused through the membrane. A method for detection with this device is also described.

A disadvantage of these methods and these devices is that the detection limits of

10 ng for the respective drug or for the drug substitute cannot be fallen below. It has been found to be particularly disadvantageous that there is often only a small concentration of illegal substances in the saliva, which is very easy to obtain.

The object of the invention is therefore possibilities for the detection of low concentrations to provide different analytes. It is also a sub-object of the invention to increase the specificity for the detection of drugs and drug substitutes.

The object of the invention is achieved by the method according to the features in the characterizing part of claim 1. The advantage of this method is that very low concentrations of an analyte are sufficient to be able to be detected using the method according to the invention. This method therefore offers a highly sensitive method to detect analytes from a sample. Simple detection methods for the determination of analytes allow them to be carried out outside of laboratories, e.g. on the street, in police stations, in prisons, in factories or other workplaces, at home, practically anywhere. This detection method eliminates the need for costly laboratory analysis and thus enables cost savings.

It also proves advantageous according to claim 2 that the sensitivity of this detection method can be increased considerably by the additional chemical reaction caused by the substance by the substance that is bound to the specific binding partner.

Furthermore, a further development of the method according to claim 3 proves to be advantageous because unphysiological substrates, for example for enzymes, such as. B. ß-galactosidase, horseradish peroxidase, etc. after the chemical reaction result in colored reaction products and thus allow visual and spectrophotometric detection. In the streptavidin, avidin and protein A proteins, the multiple binding sites for biotin and Fc parts of antibodies have proven to be advantageous. For example, an antibody can be labeled with biotin. Proteins such as streptavidin and avidin bind to one of their four binding sites in a highly specific manner to biotin. Free biotin binding sites are then saturated with a biotin-labeled enzyme, eg alkaline phosphatase. The addition of chromogenic substrates causes a color mixture to precipitate, which enables the binding to be detected. Since the biotin-binding proteins contain four biotin binding sites and the enzymes used also carry several biotin groups, complexes with many protein-enzyme-biotin molecules can form, which significantly increases the sensitivity of detection. Alkaline phosphatase is used as a marker enzyme, since a sensitive, histochemical color reagent and signal amplifier system develops in conjunction with suitable substrates. The enzyme ß-galactosidase cleaves natural and artificial ß-D-galactosides into galactose and the corresponding residues (mostly alcohol compounds). unphysiological Substrates for the enzyme, such as ONPG or X-gal, produce colored reaction products after hydrolysis and oxidation and thus allow visual and spectrophotometric detection.

Furthermore, an embodiment according to claim 4 is advantageous, wherein by separating the analyte in the many zones, a reaction with a dye component or its precursor as well as with a conjugated antibody can take place and these can then be detected in an analysis field. Another advantage is that, regardless of the presence of an analyte in the sample, a marking in the zone of the control field can be detected in order to control the functionality of the analysis method.

Furthermore, it proves to be advantageous that different analytes can be detected simultaneously by arranging several analysis fields. A further advantage is the temporary immobilization of the reagents in the dye field and in the conjugate field, because this eliminates the need to add these reagents during the analysis, thus simplifying the process and preventing confusion of the reagents.

The arrangement of running fields enables the analytes to be freed from particulate contaminants during the passage of the capillary-capable material.

An embodiment according to claim 5 proves to be advantageous, according to which a multiplicity of analytes can be detected by this method. It has also proven to be advantageous that such a large number of analytes can be detected using the same method without having to adapt the method. Another advantage is that, due to the diversity of this method, no special training of the personnel to be carried out is required for the various analytes, and thus a large number of analytes can be evaluated by only one person. It has also proven to be advantageous that several analytes can be evaluated simultaneously using one method and on one device.

The further developments according to claims 6, 7, 8 and 9 are advantageous, according to which in particular analytes which influence the physiological balance of humans can be detected. It proves to be advantageous that the method ensures simple handling and can therefore be carried out by anyone. This multiply applicable method minimizes the cost of analyzing a wide variety of analytes. The analytes can be determined on site and do not have to be transported to a special laboratory. Because of the quick test result Furthermore, it can be decided at an early stage whether the analyte is affecting the individual and thus further dangerous situations can be avoided. A rapid test result also prevents the possibility of manipulating biological samples, for example by taking doping maskers, such as sample oath, to prevent the detection of steroid hormones. The immediate availability of a result on site makes the argumentation much more difficult for the test person. This could prevent, for example, athletes from receiving an Olympic medal, although they are not entitled to it, but one has to wait a few days after the award ceremony until a final result of the doping control with conventional tests is available.

Both drugs and psychotropic drugs affect the respondents' ability to react at the workplace and their ability to drive in traffic. If a rapid test result is available using this method, the immediate danger of such situations can be recognized immediately.

A further development of the method according to claim 10 is also advantageous, according to which the risk of contamination for the personnel who carries out the analysis can be minimized by the rapid detection of antibodies against infections. On the one hand, it can be decided by the detection of a viral infection that special caution should prevail for the further examination measures. On the other hand, due to the rapid analysis results, intensive treatment of supposedly infected people or even their quarantine can be dispensed with.

According to claim 11, the method enables the detection of different hormones, e.g. of HCG, which is only detectable during pregnancy. This method also makes it easy to quickly prove pregnancy.

A further development of the method according to claim 12 has also proven to be advantageous, according to which, in the event of a conspicuous medical history, a first statement about the presence of tumor markers can be made quickly, and thus the time period until the result of the finding and thus the uncertainty for the patient is minimized.

Furthermore, the embodiment of the method according to claim 13 proves to be advantageous, wherein the correct treatment of the intoxication can be started immediately by detecting the toxin and, for example, the appropriate antidote can be administered immediately.

A further development according to claims 14, 15 and 16 is advantageous, according to which the analyte comes from a sample of any origin and can be used for the method. A particular advantage of this method is that, despite the variety of samples, regardless of their origin, a reliable, reproducible result is always achieved. It has proven to be particularly advantageous that the method has a high tolerance limit for contamination and that the samples therefore do not have to be cleaned before the analysis.

The development of the method according to claim 17, according to which very small amounts of an analyte can be detected, is also advantageous. The high sensitivity of the method thus enables the detection of analytes in the picogram range.

The object of the invention is independently achieved by a device according to the features in the characterizing part of claim 18. One advantage of this is that this device provides a very sensitive possibility for the detection of at least one analyte. On the other hand, it is advantageous that the simple structure of the device enables it to be operated and evaluated by chemically and medically untrained personnel, and the analyte can thus be detected independently of a specific location.

A further development according to claim 19 proves to be advantageous because it can prevent non-specific bindings and therefore only specific bindings can take place and can also be detected. It is also advantageous that during the detection no signals which result from unspecific binding influence the readability of the device.

According to the embodiment according to claim 20, the sensitivity of the device for detecting the analyte is increased. The dye components and their precursors act as part of an amplifier system. Furthermore, it has proven to be advantageous that the attachment of a dye field enables only the analyte to be applied to the device and not the dye components to be added to the reagent first. This again simplifies the handling of the device. Furthermore, it has been shown to be advantageous in that the entire detection method can be checked by arranging a control field. Regardless of the presence of an analyte in the sample, a signal is detected, thus enabling a functional check for the analysis. It is also advantageous that not only the analyte itself, but also a specific binding partner for it can be immobilized in the analysis zone and thus a large number of binding partners are available for binding the analyte from the sample. It also proves advantageous that a large number of different analytes can be analyzed with one device. The arrangement of a conjugate and dye field makes it unnecessary to add substances that are temporarily immobilized in these fields, thus minimizing the susceptibility to errors in the test. Furthermore, it proves advantageous that either the specific binding partner, to which the same substance is conjugated, or the same substance itself, which produces the same reaction with the dye or the dye precursors, are located both in the control zone and in the analysis zone. This enables precise control of the functionality of the device.

According to the development of the device according to claim 22, it is advantageous that specific binding sites of the at least one analyte are not blocked by unspecific reactions of the dye components or their precursors in advance of the analysis and would thus lead to a false negative result.

Embodiments of the device according to one of claims 23 and 24 are also advantageous, the dye component or its precursors serving as an enhancer system and the sensitivity of the analysis thus being increased. X-gal has the advantage over ONPG that the blue oxidigo dye formed as a reaction product after hydrolysis and oxidation is sparingly soluble and therefore does not diffuse.

Embodiments according to claims 25 and 26 also prove to be advantageous, according to which the addition of these substances results in better solubility of the dye components and their precursors and thus in turn improves the sensitivity of the analysis.

The further development according to claim 27 proves to be advantageous, according to which a control of the analysis is made possible, because in any case the substances bind to the specific binding partners present in the control field and thus the functionality of the analysis is checked on each device or on its own device can. An embodiment according to claim 28 is also advantageous, wherein even small amounts of specific binding partners for the substances are sufficient to obtain a detectable signal and thus the manufacturing costs of the device can be kept very low.

According to the embodiment according to claim 29, it proves to be advantageous that during the analysis in the analysis field there are in any case sufficient substance conjugated specific binding partners because the control field is arranged downstream and therefore too many substance-specific binding partners cannot already be intercepted in the control field and therefore for the analysis field would no longer have enough specific binding partners that are conjugated to a substance.

The refinements according to claim 30 also prove to be advantageous, the same analyte being arranged in the analysis field or a specific binding partner for the analyte to be detected and thus cross-reactions and false positive results can be excluded by unspecific binding.

An embodiment according to claim 31 is also advantageous in that a variety of analytes can be detected and the device can not only be used for the detection of a single analyte and production costs for the different designs of the devices can thereby be minimized. It also proves advantageous that the same method can be used for the detection of all analytes.

Furthermore, an embodiment according to claim 32 proves to be advantageous, according to which only a small amount of the analyte has to be applied to the device and thereby the costs for the material expenditure of the device are minimized.

Also of advantage are the developments of the device according to claims 33 and 34, with which the intensity of the signals resulting from the evaluation is available in various gradations, since too high a concentration would cause the signals to be superimposed, thereby jeopardizing the clarity of the analysis results. The device can thus be used universally regardless of the expected concentration of the analyte. Another advantage is that the different concentrations of the analytes enable quantification. The object of the invention is also achieved independently by a reagent according to the features in the characterizing part of claim 35. It proves to be advantageous that simple chemical compounds can be used for the production of the reagent, which cause very low costs in the production and procurement and thus consequently enable the reagent to be produced cost-effectively. Another advantage of this is that an intensification of the signal can be brought about and thus the sensitivity of the method and also of the device increases.

According to the development of the reagent according to claim 36, it proves advantageous that the analyte can already be bound in the reagent and is detected in the analysis method by a specific binding partner for the binding partner of the analyte. This enables indirect detection of the analyte.

According to the refinements of the reagent according to claims 37 to 39, components of the dye system or their precursors can already be added to the reagent and, for example, this can shorten the running distance of the analyte on the device and thus minimize the size of the device overall.

The developments according to claims 40 and 41 also prove to be advantageous, according to which the addition of these components results in a faster solubility of the dye components in the reagent and / or the adsorption of the dye components on the capillary-capable material of the device is reduced.

Also advantageous are the developments according to claims 42 to 47, according to which standard products from the chemical industry can be used for the production of the reagent, and thus the production costs for the reagent are kept low.

Furthermore, the further developments according to claims 48 to 50 prove to be advantageous, according to which constant conditions in the reagent and thus a reproducible result are guaranteed throughout the analysis. Maintaining constant conditions also improves the functionality of the device. It has proven to be particularly advantageous that the addition of a mixture of the buffers mentioned makes it possible to buffer the reagent above a larger pH value and the detection reaction of the analyte is not disturbed, so that a reproducible analysis result is also available when examining contaminated samples , According to the developments according to one of claims 51 and 52, it has proven to be advantageous that a detergent brings about an improvement in the solubility for the dissolution of substances with complicated molecular structures and also large molecules.

Furthermore, the configurations according to claims 53 and 54 prove to be advantageous, according to which both the osmolarity of the reagent and the solubility are increased by the addition of these substances.

Further developments according to claims 55 and 56, according to which the shelf life of the reagent is extended, are also advantageous.

The object of the invention is also achieved independently by an analysis kit according to the features in the characterizing part of claim 57. The advantage of this is that all the necessary devices and reagents for carrying out the analysis are made available. Another advantage is that the simple way of storing the analysis kit at room temperature makes it possible to store it regardless of the location.

The object of the invention is also achieved independently by using the device according to the features in the characterizing part of claim 58. It has been shown to be advantageous that the device provides a reliable means for analyzing at least one analyte in a sample.

The object of the invention is also achieved independently by the use of the reagent according to the features in the characterizing part of claim 59. It has proven to be advantageous that the use of the reagent provides an inexpensive means with enhancement potential.

The object of the invention is also achieved independently by using the analysis kit in accordance with the features in the characterizing part of claim 60. It proves to be advantageous that the analysis kit enables rapid and uncomplicated determination of the analyte regardless of the location.

For a better understanding of the invention, this will be explained in more detail with reference to the following figures, which show a device for detecting at least one analyte in a schematically simplified representation. The invention is illustrated below in the Drawing illustrated embodiments explained in more detail.

Show it:

Figure 1 shows a square device from the start zone and analysis field.

Figure 2 shows a square device from the start zone, zone and target zone.

3 shows a quadrangular device consisting of the start zone, zone and target zone;

4 shows a round device comprising the starting zone and the analysis field;

5 shows a round device comprising the start zone, zone and target zone;

6 shows a star-shaped device consisting of a starting zone and an analysis field;

7 shows a star-shaped device comprising the start zone, zone and target zone;

8 shows a hexagonal device consisting of a start zone and an analysis field;

Fig. 9 shows a hexagonal device from the start zone, zone and target zone.

As an introduction, it should be noted that in the differently described embodiments, the same parts are provided with the same reference numerals or the same component names, and the disclosures contained in the entire description can be applied analogously to the same parts with the same reference numerals or the same component names. The location information selected in the description, e.g. above, below, laterally etc. refer to the figure described and illustrated immediately and are to be transferred to the new position in the event of a change of position. Furthermore, individual features or combinations of features from the different exemplary embodiments shown and described can also represent independent, inventive or inventive solutions.

Fig. 1 shows a side view of a device 1 on a carrier material 2, wherein the at least one capillary material 3 by means of a connecting layer 4, such as. B. one Double adhesive film, is connected to the carrier material 2. It is also possible to apply the capillary-capable material to a one-sided adhesive carrier material (laminating). A starting zone 5 and a further zone 6, which is formed from at least one capillary-capable material 3, are arranged on the device 1.

FIG. 2 shows a further embodiment of the device 1, it being noted at this point that the invention is not restricted to the specially shown embodiment variants of the device, but rather that various combinations of the individual embodiment variants with one another are possible and this variation possibility is due to this the teaching of technical action through objective invention lies in the ability of the specialist working in this technical field. The scope of protection also includes all conceivable design variants which are possible by combining individual details of the design variant illustrated and described.

2 shows a side view of a device 1 on a carrier material 2, the

Carrier material 2 is connected to at least one capillary-capable material 3 via a connecting layer 4. A starting zone 5, a zone 6 and a target zone 7 are arranged on the capillary-capable material 3. Zone 6 consists of several fields, e.g. a conjugate field 8 with specific binding partners to which at least one substance is conjugated, at least one running field 9, an analysis field 10 and / or control field 11 and a dye id 12.

FIG. 3 shows a square device 1, with several analysis fields 10 being arranged in zone 6. A different analyte 13 can be bound to each of these analysis fields 10 and thus the device 1 for the detection of several different analytes

13 can be used. In an alternative embodiment variant, not shown, the start zone 5 can also be arranged centrally on the device 1, the analysis fields 10 being able to be located on all sides of the start zone 5. A plurality of target zones 7 can also be arranged on the device 1.

FIG. 4 shows a round embodiment of the device 1 as described in FIG. 1. Adjacent to the starting zone 5 there is another zone 6 in which the detection of the at least one analyte 13 takes place.

Fig. 5 shows a round embodiment of the device 1, being adjacent to the Starting zone 5 are a conjugate field 8, a dye id 12, several running fields 9, at least one analysis field 10 and / or a control field 11 and a target zone 7.

6 and 7 show star-shaped designs of the device 1, the device 1 in FIG. 6 being formed only by a start zone 5 and an adjacent zone 6 and in FIG. 7 comprising a start zone 5, a target zone 7 and a zone 6 from a conjugate field 8, a dye id 12, several running fields 9, at least one analysis field 10 and / or a control field 11 are shown. The different zones 6 are arranged in a circle on the device 1.

8 and 9 show octagonal embodiments of the device 1, the device 1 in FIG. 8 being formed only by a start zone 5 and an adjacent zone 6 and in FIG. 9 comprising a start zone 5, a target zone 7 and a zone 6 from a conjugate field 8, a dye id 12, several running fields 9, at least one analysis field 10 and / or a control field 11 are shown.

The different zones 6 or fields can have different forms, e.g. linear, circular, triangular, quadrangular, hexagonal, pentagonal, heptagonal, octagonal, trapezoidal, rhomboid, etc., can be arranged on the device 1.

The capillary-capable material, which constitutes a filter, consists of cellulose and / or its derivatives, of organic polymers and their derivatives, in particular of materials that adsorb little protein on their surface, such as e.g. Glass fiber, ceramic, polytetrafluoroethylene (PTFE), nylon, polyvinylidene fluoride (PVDF), materials based on silicon, materials of biological origin, acetates or from mixtures or modifications thereof

Materials. The filters are of a pore size selected from a range with an upper limit of 50 μm, preferably 40 μm, in particular 35 μm and a lower limit of 0.20 μm, preferably 5 μm and in particular 10 μm. Filters with a pore size have also been selected particularly advantageously from a range with an upper limit of 30 μm, preferably 25 μm, in particular 20 μm and a lower limit of 12 μm, preferably 15 μm, in particular 17 μm. Pre-filters with an undefined pore size can also be used. In particular in the area of the start zone 5 and in the area of the running fields 9 or in the area of the target zone 7, the capillary-capable material can be formed by a filter with an arbitrarily defined pore size. The capillary-capable material 3 of the device 1 has a thickness selected from an area with a lower limit of 0.05 mm, preferably 0.08 mm, in particular 0.15 mm and an upper limit of 2 mm, preferably 1.5 mm. in particular 1 mm. In particular, a thickness has been selected from a range with a lower limit of 0.1 mm, preferably 0.15 mm, in particular 0.2 mm and an upper limit of 0.9 mm, preferably 0.8 mm, in particular 0, 5 mm. The thickness of the capillary material 3 can vary in the different zones 6 or the different fields; ie that the device 1 does not have to have a uniform thickness over its entire extent.

The different zones 6 of the device 1 can consist of the same capillary material 3 or of different capillary materials 3. As shown in FIG. 2, the device 1 in the start and target zone 7 can be made of a different material than in the zone 6 consisting of fields. The thickness and the pore size of the capillary material 3 can also be different in the different zones 6.

To saturate the free non-specific binding sites, the capillary material is treated with solutions containing proteins such as BSA, milk powder, casein, gelatin, fat-free milk powder, calf serum, etc. and / or with commercial blocking reagents, e.g. Blotto or Superblock (from Pierce) in a concentration of 0.1-10 mg / ml and / or detergents, e.g. Tween, Triton, Nonidet P-40, Chaps, etc. in a concentration of 0.005 to

5% by weight blocked.

In the analysis field 10, a predeterminable amount of the at least one analyte 13 is selected in a concentration from a range with a lower limit of 0.5 ng / ml, in particular 1 ng / ml, preferably 2 ng / ml, and an upper limit of 5 mg / ml, in particular

3 mg / ml, preferably 1 mg / ml, immobilized. The at least one analyte 13 can be detected both directly and indirectly, for example by the analyte 13 itself, a specific analyte binding partner or a labeled binding partner. Indirect detection is carried out by binding analyte 13 to a first specific binding partner, eg primary antibody. In the analysis field either this or the analyte (at another location) is then recognized and bound by a second specific binding partner, for example secondary antibody, which is immobilized in the analysis field 10. The presence of the analyte in the analysis field 10 is finally linked to a further specific binding partner, which is conjugated to a substance, which either binds the analyte itself (at another location) or the analyte to the first specific binding partner or recognizes the second specific binding partner.

At least one analyte 13 is immobilized in the analysis field 10. As an alternative to analyte 13, an analyte-specific binding partner can also be immobilized in analysis field 10. In this case, for the detection of the analyte 13, the specific binding partner which is conjugated with a substance must be specific for the analyte and bind it to another location. A large number of different analytes 13 can be detected. The at least one analyte 13 can be selected from a group comprising drugs or drug substitutes including cannabis products, e.g. Marijuana, hashish and / or cannabinol, cocaine, e.g. Benzoylecgonine, crack and / or crystal, opiates, such as e.g. Morphine,

Acetylmorphine, heroin, codeine, propoxyphene and / or fentanyl, nicotine, cotinine, d-lysergic acid diethylamide (LSD), psilocybin and psilocin, mescaline and peyote, methadone or methadone metabolites, or a designer drug such as e.g. Amphetamines (MDA, dimethoxybromamphetamine, etc.), methamphetamines (such as e.g. ecstasy, MDMA), phencyclidine (angel dust), γ-hydroxybutyric acid (liquid ecstasy), anti-epileptics, e.g. phenytoin,

Doping agents from a group comprising anabolics, such as testosterone and its derivatives, somatotropin, ephedrine derivatives, analeptics, such as strychnine, amphetamine derivatives, analgesics, antitussives, agents for increasing the oxygen transport capacity and the oxygen availability for the skeletal muscles, such as eg erythropoietin and / or diuretics, drugs, analgesics or psychopharmaceuticals from a group comprising, neuroleptics such as phenothiazine, butyrophenones and / or thioxanthenes, antidepressants such as MAO (monoamine oxidase) inhibitors, imipramine, desipramine, amitriptyline, etc., tranquilizers, such as. B. benzodiazepines (diazepam), barbiturates and or psychoanaleptics, stimulants such as phenylethylamine, antiepileptics and / or hypnotics, antibodies, formed as a reaction to viral, viroid, bacterial, mycotic, parasitic or prion-based infections and / or formed as a result of immunizations (vaccination in humans or animals or polyclonal antibody production in animals) and / or formed as a result of autoimmune diseases or allergic reactions, hormones such as, for example, HCG (human chorion gonadotropin), proteins as tumor markers from a group comprising squamous cell carcinoma antigen (SCC), thyroglobin (Tg), steroid hormone receptors, prostate-specific antigen (PSA), neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CYFRA 21-1, CA 125, 19-9, 72-4, 15-3 and / or human calcitonin (hCT), MCA (Mucine-like cancer associated antigen), toxins from a group comprising botulinum toxin, toxins from animals, Pflan zen, such as: fungi, bacteria, algae, plants and their constituents, foods and / or toxins of artificial origin, eg combat poisons, and / or heavy metals, herbicides, fungicides, pesticides, bactericides, antibiotics, preservatives, etc. can be selected. Furthermore, allergens such as. e.g. pollen, dust mite droppings, histamine, etc. can be detected. The detection of prions from the nervous system of various mammals as well as from feed or food is of increasing importance in order to prevent the spread of Creutzfeldt-Jakob disease. Markers or species-specific proteins which are used in genetic engineering to identify genetically modified foods, seeds, etc. can also be detected as analyte 13. Various antibody (sub) types can also be detected.

By immobilizing the at least one analyte 13 in different concentrations in the analysis fields 10, a semi-quantitative determination of the analyte 13 can be carried out. The concentration of the at least one analyte 13 in the dilution series decreases with a factor selected from a range with a lower limit of 1, in particular 2, preferably 3 and an upper limit of 100, in particular 10, preferably with a factor 5.

The analyte 13 can be obtained from a sample of biological origin, such as body fluids from humans and animals, e.g. Blood, plasma, serum, urine, saliva, sweat, sperm, etc. and / or of vegetable origin, e.g. Leaf, fruit, seeds, etc. and / or microbiological

Of origin. The analyte 13 can also be detected from the soil or from water. In order to carry out an analysis of substances from the air, the analyte 13 has to be concentrated, e.g. by sucking in larger air volumes by means of a pump, it being possible for the analyte 13 to be enriched directly on the material of the starting zone 5.

One or more dye components or their precursors are temporarily immobilized in the dye id 12. However, the dye components do not necessarily have to be located in a zone 6, but can also be divided into several zones 6, such as, for example, the start zone 5, the dye id 12, the conjugate field 8 and the running field 9. The dye components are in undissolved form in front. The dye components or their precursors used are: tyramine and its derivatives, X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside), S-gal (3,4 cyclohexenoesculetin-ß-D- Galactopyranoside), ONPG (o-nitrophenyl-ß-D-galactopyranoside), CPRG (chlorophenol red-ß-D-galactopyranoside), bluogal (5-bromo-3-indolyl-ß-D-galactoside), MUGal (4-methylumbelüferyl -beta-D-galacto- pyranoside), NBT (nitroblue tetrazolium chloride), BCIP (5-bromo-4-chloro-3-indolyl phosphate), PNPP (p-nitrophenyl phosphate), OPD (o-phenylenediamine), ABTS (2,2'-azino -di- (3-ethylbenz-thiazoline sulfonic acid), DAB (3,3-diaminobenzidine), TMB (3,3'5,5 '~ tetramethylbenzidine), phenazine methosulfate, potassium ferrocyanide, potassium ferricyanide, distant ammonium citrate, VectorBlackTM , Chemiluminescent dyes, such as Galacton-Star substrate, Lumi-PhosTM plus, LuciGLOTM, DuoLuXTM, Lumigen PS-3, Chemiluminescent HRP (horseradish peroxidase) substrate, 2 component system from BioFXTM, and / or Lu- mi-Gal 530 and / or fluorescent dyes , eg 3-carboxyumbelliferyl-β-D-galactopyranoside (CUG) and / or MUP (4-methylumbelliferyl phosphate) The concentration of the dye components and their precursors is, in each case in% by weight, from a range with a lower limit of 0.01%, in particular 0.1%, preferably 1%, and an upper limit of 20%, in particular 10%, preferably 4%.

In the field with the dye components, other substances, such as BSA, maltrine, milk powder, casein, gelatin, in the concentrations selected from a range with a lower limit of 0.1%, in particular 1%, preferably 5%, and an upper limit of 50%, in particular 30%, preferably 10% , are added in order to reduce the adsorption of the dye components onto the device 1 or to increase the solubility of the dyes.

In conjugate field 8 there are in particular specific binding partners such as e.g. Antibodies against analyte 13, which are combined with a catalyst, e.g. an organic or inorganic catalyst or enzymes or proteins, e.g. β-galactosidase, peroxidase, alkaline phosphatase, streptavidin / avidin, protein A or similar molecules capable of high-affinity specific bonds, etc., are conjugated. Alternatively, several different substances can also be conjugated to a specific binding partner simultaneously or in a predefined sequence. The concentration of the specific binding partner to which the substance is conjugated is selected from a range with a lower limit of 0.5 ng / ml, in particular 1 ng / ml, preferably 2 ng / ml, and an upper limit of 5 mg / ml, in particular 2 mg / ml, preferably 1 mg / ml. Concentrations have also been selected particularly advantageously from a range with a lower limit of 0.1 mg / ml, preferably 0.2 mg / ml, in particular 0.3 mg / ml, and an upper limit of 2 mg / ml, preferably 1 mg / ml, especially 0.5 mg / ml.

In control field 11 there is an immobilized specific binding partner, for example a Antibodies for at least one substance, such as, for example, β-galactosidase, alkaline phosphatase, peroxidase, streptavidin / avidin, protein A or similar molecules capable of high-affinity specific bonds, etc., in a concentration selected from a range with a lower limit of 0.5 ng / ml, in particular 1 ng / ml, preferably 2 ng / ml, and an upper limit of 5 mg / ml, in particular 3 mg / ml, preferably 1 mg / ml.

It is not necessary to immobilize a substance or an analyte 13 in the target zone 7 and the running fields 9.

Several analytes or combinations of e.g. B. specific binding partners, substances and / or dye components or their precursors are present.

To carry out the method, a small amount of a sample with an absorbent material, such as e.g. collected a cotton swab. The analyte 13 adhering to the absorbent material is taken up in a reagent, in particular an organic reagent. Three to seven drops of this reagent provided with analyte 13 are applied to the device 1. The reagent with the analyte 13 migrates in the capillary-capable material 3 of the device 1 at a speed of 1 to 12 cm / minute from the starting zone 5, for example through the zone 6 consisting of at least one conjugate field 8, dye id 12,

Run field 9, analysis field 10 and control field 11 and finally into the target zone 7. If there is a sufficient amount of the analyte 13 in the reagent with the sample and all binding sites of the specific binding partners are saturated, they can no longer bind to the immobilized analytes 13 in Bind analysis field 10. A detectable amount of the analyte 13 only results in a marking in the control field 11 on the device 1 because the binding partners are already saturated and do not react with the immobilized analytes 13 on the device 1. In the event that there is no analyte 13 in the sample or an insufficient amount for the detection of analyte 13, there are two markings on the device 1, the first marking resulting from the binding of the analyte-specific and / or substance-conjugated analyte-specific binding partner in the analysis field 10 results and the second label results from the binding of the immobilized binding partner specifically for the substance, such as β-galactosidase in reaction with a dye component or its precursor.

In the absence of the analyte 13 to be determined in the sample, the analyte-specific The binding partner in analysis field 10 and the analyte-specific binding partner to which at least one substance is conjugated, such as, for example, β-galactosidase, reacts with the passing dye component or its precursors in such a way that the dye or dye complex formed is either insoluble or poorly soluble on Analysis field 10 remains and / or can be detected by antibodies against the dye complex formed. The specific binding partner, to which at least one substance is conjugated, can carry several binding sites for dyes or dye complex molecules that produce a clearly visible label.

If a sufficient amount of the analyte 13 is present, it binds to the analyte-specific binding partners which are conjugated with a substance, and these binding partners can therefore no longer bind to the immobilized analyte 13 in the analysis field 10 and run into the target zone 7 without formation of a visible marking of the device 1. Regardless of the presence of the analyte 13 to be detected in the sample, either the analyte-specific binding partners which are conjugated to a substance or the substance itself bind to the control field 11 to corresponding substance-specific binding partners, the substance thus bound also react the passing dye components or their precursors in such a way that the dye formed remains complex insoluble or poorly soluble in place and / or by an antibody against the dye complex formed, which is immobilized in the control field 11, with the

Continuity of the test can be bound in large numbers and form a clearly visible marking there, can be verified.

An immobilized antibody against the substance, e.g. ß-galactosidase. As already mentioned, the reaction takes place in the control field 11, regardless of whether a sufficient amount of the analyte 13 was present in the eluent.

The detection of an analyte 13 with the enzyme ß-galactosidase as an enhancer system is carried out by the cleavage of natural or artificial ß-D-galactosides in galactose and the corresponding residual compounds. Unphysiological substrates for the enzyme, such as ONPG or X-gal, give colored reaction products after hydrolysis and oxidation and allow visual and spectrophotometric detection.

As an alternative amplification system, streptavidin and avidin, proteins with multiple Binding sites for biotin are used. For example, a specific binding partner can be labeled with biotin. Proteins such as streptavidin and avidin bind to one of their four binding sites in a highly specific manner to ^' biotin. Free biotin binding sites are then saturated with biofin-labeled enzyme, eg alkaline phosphatase. Due to the addition of chromogenic substrates, a color mixture precipitates, so that the binding can be demonstrated. Since the biotin-binding proteins contain four biotin binding sites and the enzymes used can also carry several biotin groups, complexes can form with many protein-enzyme-biotin molecules, which significantly increases the sensitivity of detection. Alkaline phosphatase is used as a marker enzyme, since a sensitive, histochemical one is found in connection with suitable substrates

Color reagent and signal amplifier system can be developed. Substrates such as After hydrolytic cleavage by the enzyme, p-nitrophenyl phosphate gives colored, photometrically easily measurable reaction products, or, as in the case of 5-bromo-4-chloro-3-indolyl phosphate (BCIP = X-phosphate), a deep blue colored insoluble, easily recognizable precipitation of indigo. BCEP is usually used together with nitro blue tetrazolium (NBT) as a color enhancer.

The enzyme peroxidase (HRP) with derivatized tyramine can be used as a further alternative amplification system (Super-CARD, Catalytic deposition of derivatized tyramine, Bhattacharya, R., Bhattacharya, D., and Dhar, TK (1999) Journal of Immunological Method 227, 31-39). Here is e.g. the specific binding partner is coupled to the enzyme peroxidase (HRP) and catalyzes the covalent binding of the derivatized tyramine to the analysis field (specifically to specially modified proteins for this purpose) using H2O2 (which is present in the reagent or is formed directly by a chemical reaction). p-OH-PPA-casein, p-OH-PPA-gelatin or p-OH-PPA-BSA; p-OH-PPA = 3- (p-hydroxyphenyl) propionic acid) which are immobilized on the analysis field). As a molecule which is coupled to tyramine, either a dye, gold particles (NanoGold) or a catalyst is considered. In the latter case e.g. Peroxidase (HRP), a histochemical color reagent gives a signal that is amplified many times over.

The reagent used to take up the sample consists of monohydric alcohol with 1 to 5 carbon atoms, ketones with 3 to 8 carbon atoms, polyhydric alcohols, especially ethylene glycol and polyethylene glycol. The concentrations of these chemical compounds are from a range with a lower limit of 0.1%, in particular 5%, preferably 10%, and an upper limit of 40%, in particular 30%, preferably 20% selected.

The reagent can additionally be a detergent, in particular (octylphenoxyl) polyethoxyethanol, alkylphenol polyglycol ether, Tween 20, sodium deoxycholate, Nonidet P-40 (Igepal CA-630), Triton X-100, cholic acid, deoxycholic acid and / or Zwittergent® in a concentration selected from a range with a lower limit of 0.001%, in particular 0.005%, preferably 0.01%, and an upper limit of 1%, in particular 0.5%, preferably 0.2%, as a solubilizer.

A buffer can also be added to the reagent, which keeps the reagent constant in a pH range. Buffer solutions such as e.g. Citrate buffer, acetate buffer, maleate buffer, phosphate buffer, collidine buffer, triethanolamine-HCl-EDTA buffer, tris buffer, ammediol buffer, glycine buffer, diethanolamine buffer or tris-boric acid-EDTA buffer, or buffer according to Good, N.E. et al. (1966) Biochemistry 5, 467. The concentration of the buffer solutions is in one percent by weight

Has a range with a lower limit of 0.1%, preferably 1% and in particular 2% and with an upper limit of 20%, preferably 15%, in particular 10%. The buffer solution has a pH value selected from a range with a lower limit of 5.5, in particular 6.0, preferably 6.5, and an upper limit of 9.5, in particular 9.0, preferably 8.5. Buffer solutions with a pH

Value with a lower limit of 6.8, preferably 7.0, in particular 7.2 and an upper limit of 8.0, preferably 7.8, in particular 7.6, has been proven.

By adding a mixture of the buffers mentioned, it is possible to buffer the reagent over a larger pH range and thus to ensure a correct analysis result even when examining contaminated samples. Contamination from the existing buffer system is also buffered with certainty to such an extent that it does not interfere with the analysis reaction of the at least one analyte 13.

Furthermore, at least one dye component or its precursor can be added to the reagent, the dye components being selected from a group comprising tyramine and its derivatives, X-Gal (5-bromo-4-chloro-3-indolyl-β-D- Galactoside), S-Gal (3,4 cyclohexenoesculetin-ß-D-galactopyranoside), ONPG (o-nitrophenyl-ß-D-galactopyranoside), CPRG (chlorophenol red-ß-D-galactopyranoside), Bluogal (5- Bromine-3-indolyl-β-D-galactoside), MUGal (4-methylumbelliferyl-β-D-galactopyranoside), NBT (Nitroblue Tetrazolium chloride), BCE? (5-bromo-4-chloro-3-indolyl phosphate), PNPP (p-nitrophenyl phosphate), OPD (o-phenylenediamine), ABTS (2,2'-azino-di- (3-ethylbenzothiazoline sulfonic acid) ), DAß (3,3-diaminobenzidine), TMB (3,3'5,5'-tetramethylbenzidine), phenazine methosulfate, potassium ferrocyanide, potassium ferricyanide, ferric ammonium citrate, VectorBlackTM, chemiluminescent dyes such as Galacton-Star substrate, Lumi -PhosTM plus,

LuciGLOTM, DuoLuXTM, Lumigen PS-3, chemiluminescent HRP (horseradishperoxidase) substrate, 2 Component System from BioFXTM, and / or Lumi-Gal 530 and / or fluorescent dyes, e.g. 3-carboxyumbelliferyl-β-D-galactopyranoside (CUG) and / or MUP (4-methylumbelliferyl phosphate). The concentration of the dye components is selected from a range with a lower limit of 1 μM, in particular 10 μM, preferably 50 μM, and an upper limit of 300 mM, in particular 150 mM, preferably 100 mM.

The dye components and / or their precursors are dissolved in the reagent and are dissolved there by adding the components of the reagent, BSA, maltodextrin, milk powder, calf serum, casein and / or gelatin, or are transported via the device 1 by the capillary action ,

To increase the osmolarity of the reagent, sodium salts, potassium salts, phosphate salts, magnesium and / or manganese salts, BSA, maltodextrin (maltrin) or milk powder in a concentration from a range with a lower limit of 0.001%, in particular

0.1%, preferably 1%, and an upper limit of 30%, in particular 20%, preferably 10%, can be added.

Furthermore, a preservative, in particular sodium azide, sodium benzoate, sorbic acid, pentachlorophenol, sorbate and / or preservative based on mercury, can be added to the reagent in a concentration from a range with a lower limit of 0.01%, in particular 0.05%, preferably 0.1%, and an upper limit of 5%, in particular 3%, preferably 1%, in order to extend the shelf life of the reagent. The dye components and / or their precursors are in the reagent or partially in the reagent or are completely or partially in the dye id 12 or completely or partially in the starting zone 5 of the device 1 in undissolved form.

The analysis kit according to the invention comprises a device 1 for detecting at least one analyte 13, the reagent an absorbent substance, such as a cotton swab or the like. To further simplify the implementation of the analysis process, the analysis kit also contain other aids, such as gloves, stopwatch, pipettes, etc.

The two following exemplary embodiments show possible ways of carrying out the method, it being noted at this point that the invention is not limited to the specifically mentioned exemplary embodiments, but rather all conceivable exemplary embodiments which are included in the scope of protection by combinations of individual details of the possibilities described

Example 1:

Device 1 for the determination of benzodiazepines in saliva

The start 5 and finish zone 7 are made of cellulose absorbent paper (Pall Corporation, type 165 for the start zone 5 (BSP165PK) and type 197 for the target zone 7 (BSP197PK)). The format of start 5 and finish zone 7 is 25 x 80 mm. The filter paper is used without further impregnation to produce the device 1.

The dye id 12 is made from glass fiber microfilter type GF / D (Whatman). The format of the dye field 12 is 5 x 80 mm and is evenly charged with 80 μl of the dye solution. This is followed by drying at 20 ° C in the absence of light.

The dye solution consists of: X-Gal (100 mM), NBT (50 mM), Phenazine Methosulfate (1 mM), BSA (4%), Maltrin (1) in PBS (5 mM KH ₂ PO ₄ , 15 mM Na ₂ HPO ₄ , 120 mM NaCl, 2.3 mM KC1, 2 mM MgCl ₂ , 0.05% NaN ₃ , 5% ethanol, 0.1% Triton X-100). The starting reagents are manufactured by SIGMA-Aldrich.

The conjugate field 8 consists of blocked Accuwick® membrane (Pall. AW 14-20-10). The membrane is blocked with 1% BSA in PBS solution for 1 hour at room temperature, then drying at 20 ° C. The format is again 5 x 80 mm. 80 μl of the antibody solution are pipetted on evenly and then drying is carried out at 20 ° C. The antibody solution consists of: primary monoclonal antibodies (host mouse) against benzodiazepines (25 nM), (Fitzgerald, cat. No .: 10-B12) secondary antibodies anti-mouse (25nM) conjugated with beta-glactosidase (from Fisher Scientifϊc) ß-galactosidase (25 nM), (Röche Applied Science), BSA (4%), maltrin (1%) in PBS (5 mM KH ₂ PO ₄ , 15 mM Na ₂ HPO ₄ , 120 mM NaCl, 2, 3mM KCL, 2mM MgCl ₂ , 0.05% NaN ₃ , 0.1% Triton X-100). Strips of cellulose acetate filter (from Osmonics Inc.) in the format 20 x 80 mm are used for the production of the running fields 9. To block binding sites, the strips are placed in a 1% BSA-containing PBS solution (5 mM KH ₂ PO ₄ , 15 mM Na ₂ HPO ₄ , 120 mM NaCl, 2.3 mM KC1, 2 mM MgCl ₂ , 0.05 % NaN ₃ , 0.1% Titron X-100) incubated for 30 minutes at room temperature with stirring.

A BS A-benzodiazepine conjugate (from Fitzgerald) is immobilized in the test field before binding sites are blocked. For this purpose, the cellulose acetate filter (AcetatePlus) is incubated in 100 mM sodium periodate (SIGMA-Aldrich) for 20 min, then washed with H2O and with a 0.5 μM BSA-benzodiazepine solution in 0.1 M borate buffer (pH 9.0 ) incubated for 10 minutes. Then 4 mg sodium cyanoborohydride (SIGMA-Aldrich) are added per ml and incubated for 2 h at room temperature. Unbound BSA benzodiazepine is washed away using H2O.

In control field 11, a specific polyclonal antibody against the enzyme β-galactosidase (Fitzgerald, host: Rabbit) is immobilized before the binding sites are blocked. 0.25 μM specific polyclonal antibody is immobilized using the same method as in the test field.

The reagent used, in which the sample is taken up, consists of PBS (5 mM

KH ₂ PO ₄ , 15 mM Na ₂ HPO ₄ , 120 mM NaCl, 2.3 mM KC1, 2 mM MgCl ₂ , 0.1% BSA, 0.1% maltrine, 0.05% NaN ₃ , 5% ethanol, 0.05% Triton X-100) with a pH of 7.1.

The starting zone 5, the fields of zone 6 and the target zone 7 are cut according to the specifications described and by means of double-sided adhesive film (from Tesa AG)

Carrier films Xeroperm (Rank Xerox Limited) attached. Of these, strips of 4 mm are cut with a roll cutter. The strips are stored at room temperature in the absence of light and moisture.

Embodiment 2

Device 1 for the determination of testosterone in saliva

The start 5 and finish zone 7 are made of cellulose absorbent paper (Pall Corporation, type 165 for the application cushion (BSP165PK) and type 197 for the absorption cushion (BSP197PK)) manufactured. Strips of 25 x 80 mm are cut to size, which are used to produce the device 1 without further impregnation or pretreatment.

Dye 12 is made from 'Glass Fiber Media' (Pall, A / D Glass). Strips in the format of 5 x 80 mm are cut and evenly charged with 80 μl of the dye solution. The drying is then carried out at 20 ° C in the absence of light. The dye solution consists of: X-Gal (100 mM), NBT (50 mM), phenazine methosulfate (1 mM), BSA (4%), maltrine (1%) in PBS (5 mM KH ₂ PO ₄ , 15 mM Na ₂ HPO ₄ , 120 mM NaCl, 2.3 mM KC1, 2 mM MgCl ₂ , 0.05% NaN ₃ , 5% ethanol, 0.1% Triton X-100) (all solutions are from SIGMA -Aldrich).

The running field 9, analysis field 10 and control field 11 consist of a 5 μm Immunodyne® ABC membrane in the format 20 x 80 mm (Pall., BC500H5R). A 0.5 μM testosterone-3-CMO-BSA solution (from Fitzgerald, 80-IT49) is placed on the device 1 at the location of the analysis field 10 and a 0.25 μM biotin-BSA at the location of the control field 11 solution

(SIGMA-Aldrich) applied. The remaining binding sites on the membrane are then blocked for 2 hours at room temperature with a 1% BSA in PBS solution.

In place of the conjugate field 8 on the device 1, 10 nM of the primary specific monoclonal antibody to testosterone (host: mouse, Fitzgerald: 10-T07), 25 nM

Biotinylated anti-mouse IgG antibodies (host: Pferd, Vector Laboratories: BA-2080) and 100 nM of an avidin-beta-galactosidase conjugate (SIGMA-Aldrich) applied directly to the blocked Immunodyne® ABC membrane between start zone 5 and analysis field 10.

The reagent used, in which the sample is taken up, consists of PBS (5 mM

The cushions and the populated Immunodyne® ABC membrane are cut according to the specifications described and attached to the by means of double-sided adhesive film (Tesa AG)

Carrier material (film Xeroperm type 003R96094 from Rank Xerox Limited) attached. Of these, strips of 4 mm are cut with a roll cutter. The strips are stored at room temperature in the absence of light and moisture.

Finally, it should also be mentioned that in particular the antibodies which are used in the anti body solution are available, are each adapted to the analyte to be detected.

For the sake of order, it should finally be pointed out that, for a better understanding of the structure of FIGS. 1 to 9, these or their components have been partially shown to scale and / or enlarged and / or reduced.

The object on which the independent inventive solutions are based can be found in the description.

Above all, the individual versions shown in FIGS. 1 to 9 can form the subject of independent solutions according to the invention. The relevant tasks and solutions according to the invention can be found in the detailed descriptions of these figures.

Regarding eilun

Device Carrier material Capillary material Connection layer Start zone Zone Target zone Conjugate field Run field Analysis field Control field Dye analyte Binding partner

Claims

Patent claims

1. A method for the detection of at least one analyte from a sample by an immunochemical reaction with a device consisting of several zones, characterized in that the at least one analyte in a reagent, in particular an organic reagent, is applied to a starting zone and by capillary forces in at least a further zone with one or more fields runs, at least one specific binding partner, to which at least one substance is conjugated, temporarily immobilized in one field.

2. The method according to claim 1, characterized in that the at least one analyte is catalyzed by a chemical or physical reaction of the at least one substance or by a chemical reaction by the substance which to the specific binding partner, such as. B. an antibody conjugated is detectable.

3. The method according to any one of claims 1 or 2, characterized in that the at least one substance from a group comprising alkaline phosphatase, peroxidase, e.g. horseradish, ß-galactosidase, streptavidin avidin, protein A or molecules capable of high affinity specific bonds and / or a catalytically active substance.

4. The method according to any one of claims 1 to 3, characterized in that the at least one analyte runs through a zone consisting of at least one field selected from a group comprising analysis fields, control fields, running fields, dye fields and / or conjugate fields.

5. The method according to any one of the preceding claims, characterized in that the at least one analyte from a group comprising active ingredients, hormones, proteins, peptides, allergens, antigens, antibodies, neurotransmitters, nucleic acids, carbohydrates and / or lipids is detected.

6. The method according to claim 5, characterized in that active substances from a group comprising drugs, drug substitutes, performance-enhancing substances such as doping agents, drugs, toxins or their metabolites are detected.

7. The method according to claim 5, characterized in that drugs or drug substitutes from a group comprising cannabis products, such as. Marijuana, hashish and / or cannabinol, cocaine, e.g. Benzoylecgonine, crack and / or crystal, opiates, such as e.g. Morphine, acetylmorphine, heroin, codeine, propoxyphene and / or fentanyl, nicotine, cotinine, d-lysergic acid diethylamide (LSD), psilocybin and psilocin, mescaline and peyote, methadone or methadone metabolites, or a designer drug such as e.g. Amphetamines (MDA, dimethoxybromamphetamine), methamphetamines (ecstasy, MDMA), phencyclidine (angel dust), γ-hydroxybutyric acid (liquid ecstasy), anti-epileptics, e.g. Phenytoin.

8. The method according to claim 5, characterized in that doping agents from a group comprising anabolic agents, such as. Testosterone and its derivatives, somatotropin, epedrin derivatives, analeptics, such as e.g. Strychnine, amphetamine derivatives, analgesics, antitussives, agents for increasing the oxygen transport capacity and the oxygen availability for the skeletal muscles, e.g. Erythropoietin and / or diuretics.

9. The method according to claim 5, characterized in that the medicament comprises analgesics or psychotropic drugs from a group comprising neuroleptics, such as e.g. Phenothiazine, butyrophenones and / or thioxanthenes, antidepressants such as e.g. MAO (monoamine oxidase) - inhibitor, imipramine, desipramine, amitriptyline, etc., tranquilizers, such as. B. Benzodiazepines

(Diazepam), barbiturates and / or psychoanaleptics, stimulants, e.g. Phenylethylamine, anti-epileptics and / or hypnotics.

10. The method according to claim 5, characterized in that antibodies formed in response to viral, viroid, bacterial, mycotic, parasitic or based on prions

Infections and / or formed as a result of immunizations (vaccination in humans or animals or polyclonal antibody production in animals) and / or formed as a result of autoimmune diseases or allergic reactions.

11. The method according to claim 5, characterized in that hormones such as e.g. HCG

(human chorion gonadotropin).

12. The method according to claim 5, characterized in that as proteins tumor markers from a group comprising squamous cell carcinoma antigen (SCC), thyroglobin (Tg), steroid hormone receptors, prostate-specific antigen (PSA), neuron-specific enola- se (NSE), Carcinoembryonic Antigen (CEA), Alpha-Fetoprotein (AFP), CYFRA 21-1, CA 125, 19-9, 72-4, 15-3, MCA (Mucine-like cancer associated antigen) and / or human cal-citonin (hCT).

13. The method according to claim 5, characterized in that intoxications caused by toxins from a group comprising botulinum toxin, toxins from fungi, bacteria, algae, plants and their constituents, foods and / or toxins of artificial origin, e.g. Combat poisons and / or heavy metals are detected.

14. The method according to any one of the preceding claims, characterized in that the at least one analyte from a biological sample of human, animal or vegetable origin, such as e.g. Blood, plasma, serum, urine, saliva, sweat, sperm and / or leaves.

15. The method according to any one of the preceding claims, characterized in that the at least one analyte is detected from a surface of an object or a human, animal or plant body.

16. The method according to any one of the preceding claims, characterized in that the at least one analyte from the soil, water or air is detected.

17. The method according to any one of the preceding claims, characterized in that a concentration of the at least one analyte in the sample selected from a range with a lower limit of 0.01 ng / ml, preferably 0.1 ng / ml, in particular 0.2 ng / ml and an upper limit of 1000 ng / ml, preferably 200 ng / ml, in particular 100 ng / ml.

18. Device for detecting at least one analyte, in particular for carrying out the method according to one of claims 1 to 17, with one or more capillary-capable materials on which at least one starting zone and an adjacent zone consisting of one or more fields in that or those At least one immobilized reagent is arranged, characterized in that at least one analyte-specific binding partner (14) to which at least one substance is conjugated is temporarily immobilized on the capillary-capable material (3).

19. The apparatus according to claim 18, characterized in that free non-specific binding sites on the one or more capillary materials (3) selected by reagents from a group comprising proteins, such as. B. BSA, milk powder, casein, gelatin, fat-free milk powder, calf serum, commercial blocking agents such as Blotto or Superblock (from Pierce), and / or detergents, such as e.g. Tween-20, Triton X-100, No- nidet P-40 and / or Chaps are blocked.

20. Device according to one of claims 18 or 19, characterized in that the substance from a group comprising alkaline phosphatase, peroxidase e.g. from horseradish, ß-galactosidase, streptavidin / avidin, protein A or molecules capable of high affinity specific bonds and / or a catalytically active substance.

21. Device according to one of claims 18 to 20, characterized in that the zone (6) from at least one field selected from a group comprising analysis fields (10), control fields (11), running fields (9), dye fields (12) and / or conjugate fields (8).

22. Device according to one of claims 18 to 21, characterized in that there is at least one field with dye components or their precursors between the starting zone and the at least one analysis and / or control field (10, 11).

23. The device according to claim 22, characterized in that the dye components or their precursors from a group comprising tyramine and its derivatives, X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), S-Gal (3,4 cyclohexenoesculetin-ß-D-galactopyranoside), ONPG (o-nitrophenyl-ß-D-galactopyranoside), CPRG (chlorophenol red-ß-D-galactopyranoside), Bluogal (5-bromo-3-indolyl -ß-D-galactoside), MUGal (4-methylum-belliferyl-ß-D-galactopyranoside), NBT (Nitroblue Tetrazolium Chloride), BGB? (5-bromo-4-chloro-3-indolyl phosphate), PNPP (p-nitrophenyl phosphate), OPD (o-phenylenediamine), ABTS (2,2'-azino-di- (3-ethylbenzothiazoline sulfonic acid), DAB (3,3-diaminobenzidine), TMB (3,3 5'-tetramethylbenzidine), phenazine methosulfate, potassium ferrocyanide, potassium ferricyanide, distant ammonium citrate, VectorBlack ™, chemiluminescent dyes, such as Galacton-Star substrate, Lumi-Phos ™ plus, LuciGLO ™, DuoLuX ™, Lumigen PS-3, chemiluminescent HRP (horseradishperoxidase) substrate, 2 component system from BioFX ™, and / or Lumi-Gal 530 and / or fluorescent dyes, eg 3-carboxyumbelliferyl-ß-D-galactopyranoside (CUG ) and / or MUP (4-methylumbelliferyl phosphate) are selected.

24. Device according to one of claims 22 or 23, characterized in that the concentration of the dye components and their precursors, in each case in% by weight, from a range with a lower limit of 0.1%, in particular 1%, preferably 2%, and an upper limit of 20%, in particular 10%, preferably 4%, is selected.

25. The device according to any one of claims 22 to 24, characterized in that on the at least one dye (12) substances selected from a group comprising, bovine serum albumin (BSA), casein, gelatin, maltodextrin (maltrin), milk powder and / or the reagent and / or constituents of the reagent according to one of claims 35 to 56 are present.

26. The apparatus according to claim 25, characterized in that the concentration of the substances, each in% by weight, from a range with a lower limit of 0.1%, in particular 1%, preferably 5%, and an upper limit of 50% , in particular 30%, preferably 10%, is selected.

27. The device according to one of claims 18 to 26, characterized in that in the control field (11) at least one specific binding partner (14) for the at least one substance is immobilized.

28. The device according spoke 27, characterized in that the concentration of the substance-specific binding partner from a range with a lower limit of 0.5 ng / ml, in particular 1 ng / ml, preferably 2 ng / ml, and an upper limit of 5 mg / ml, in particular 3 mg / ml, preferably 1 mg / ml, is selected.

29. Device according to one of claims 18 to 28, characterized in that the control field is arranged distally from the analysis field with respect to the starting zone.

30. Device according to one of claims 18 to 29, characterized in that a predeterminable amount of the at least one analyte (13) is immobilized in the analysis field (10).

31. The device according to one of claims 18 to 30, characterized in that the at least one analyte (13) is selected from a group according to one of claims 5 to 13.

32. Device according to one of claims 30 or 31, characterized in that the amount of at least one analyte (13) from a range with a lower limit of 0.5 ng / ml, in particular 1 ng / ml, preferably 2 ng / ml , and an upper limit of 5 mg / ml, in particular 3 mg / ml, preferably 1 mg / ml, is selected.

33. Device according to one of claims 30 to 32, characterized in that the at least one analyte (13) is present in a dilution series with different concentrations, each concentration of the dilution series being present in a predeterminable analysis field (10).

34. Device according to claim 33, characterized in that the concentration of the at least one analyte (13) in the dilution series with a factor selected from a range with a lower limit of 1, in particular 2, and an upper limit of 100, in particular 10 , preferably decreases by a factor of 5.

35. Reagent for performing the method according to one of claims 1 to 17, characterized in that the reagent consists of at least one alcohol and / or ketone and / or at least one buffer solution and / or at least one detergent and / or at least one preservative.

36. Reagent according to claim 35, characterized in that the reagent is prepared with at least one analyte-specific binding partner (14).

37. Reagent according to one of claims 35 or 36, characterized in that the reagent is prepared with one or more dye components or their precursors.

38. Reagent according to spoke 37, characterized in that the one or more dye components or their precursors from a group comprising tyramine and its derivatives, X-Gal (5-bromo-4-chloro-3-indolyl-ß-D- Galactoside), S-gal (3,4 cyclohexenoesculetin-ß-D-galactopyranoside), ONPG (o-nitrophenyl-ß-D-galactopyranoside), CPRG (chlorophenol red-ß-D-galactopyranoside), bluogal (5- Bromo-3-indolyl-ß-D-galactoside), MUGal (4-methylumbelliferyl-ß-D-galactopyranoside), NBT (nitroblue tetrazolium chloride), BCIP (5-bromo-4-chloro-3-indolylphosphate), PNPP ( p-nitrophenyl phosphate), OPD (o-Phenylenediamine), ABTS (2,2 ^'-Azino-di- (3-ethylbenz-thiazoüne Sulfonic Acid), DAB (3,3 Diaminobenzidme), TMB (3,3' 5 , 5'- tetramethylbenzidine), phenazine methosulfate, potassium ferrite rocyanid, potassium ferricyanide, distant ammonium citrate, VectorBlack ™, chemiluminescent dyes, such as Galacton-Star substrate, Lumi-Phos ™ plus, LuciGLO ™, DuoLuX ™, Lumigen PS-3, chemiluminescent HRP (horseradish peroxidase) substrate, 2 component system from BioFX ™, and / or Lumi-Gal 530 and / or fluorescent dyes, for example 3-carboxyumbelliferyl-β-D-galactopyranoside (CUG) and / or MUP (4-methylumbelliferyl phosphate), are selected.

39. Reagent according to one of claims 37 or 38, characterized in that the concentration of the dye components, in each case in% by weight, from a range with a lower limit of 0.01%, in particular 0.1%, preferably 1% , and an upper limit of 20%, in particular 10%, preferably 4%, is selected.

40. Reagent according to one of claims 35 to 39, characterized in that at least one substance selected from a group comprising, bovine serum albumin (BSA), casein, gelatin, calf serum, maltodextrin (maltrin), milk powder and / or commercial

Blocking reagents, e.g. Blotto or superblock.

41. Reagent according to claim 40, characterized in that the concentration of the substances, each in% by weight, from a range with a lower limit of 0.1%, in particular 1%, preferably 5%, and an upper limit of 30%, in particular 20%, preferably 10%, is selected.

42. Reagent according to one of claims 35 to 41, characterized in that the reagent can be prepared from monohydric alcohols selected from a group comprising methanol, ethanol, propanol, butanol and / or pentanol.

43. Reagent according to claim 42, characterized in that the concentration of the monohydric alcohols, each in% by weight, from a range with a lower limit of 0.5%, in particular 5%, preferably 10%, and an upper limit of 40 %, in particular 30%, preferably 20%, is selected.

44. Reagent according to one of claims 35 to 43, characterized in that polyhydric alcohols, such as ethylene glycol, polyethylene glycol and / or glycerol are added.

45. Reagent according to claim 44, characterized in that the concentration of the polyhydric alcohols, each in% by weight, from a range with a lower limit of 0.1%, in particular 2%, preferably 5%, and an upper limit of 20 %, in particular 15%, preferably 10%, is selected.

46. Reagent according to one of claims 35 to 45, characterized in that the reagent consists of ketones having 3 to 8 carbon atoms, such as e.g. Propanone, butanone, pentanone, hexanone, heptanone and / or octanone can be produced.

47. Reagent according to claim 46, characterized in that the concentration of the ketones, in each case in% by weight, consists of a range with a lower limit of 0.1%, in particular 2%, preferably 5%, and an upper limit of 20%, in particular 15%, preferably 10%, is selected.

48. Reagent according to one of claims 35 to 47, characterized in that as a further component at least one buffer solution selected from a group comprising citrate buffer, acetate buffer, maleate buffer, phosphate buffer, collidine buffer, triethanolamine-HCl-EDTA buffer, tris buffer, ammediol buffer , Glycine buffer, diethanolamine buffer or tris-boric acid EDTA buffer or buffer according to Good, NE et al. (1966) Biochemistry 5, 467.

49. Reagent according to spoke 48, characterized in that the pH of the buffer solution is selected from a range with a lower limit of 5.5, preferably 6.0 and in particular 6.5 and with an upper limit of 9.5. is preferably 9.0, in particular 8.5.

50. Reagent according spoke 48 to 49, characterized in that the concentration of the at least one buffer solution, each in% by weight, from a range with a lower limit of 0.1%, in particular 1%, preferably 2%, and an upper Limit of 20%, in particular 15%, preferably 10%, is selected.

51. Reagent according to one of claims 35 to 50, characterized in that at least one representative selected from a group comprising (octylphenoxyl) - polyethoxyethanol, alkylphenol polyglycol ether, Tween 20, sodium deoxycholate, nonidet P-40 (Igepal CA-630), Triton as detergent X-100, Cholic acid, Deoxycholic acid and / or Zwittergent® is added.

52. Reagent according to claim 51, characterized in that the concentration of the at least one detergent, each in% by weight, from a range with a lower limit of 0.001%, in particular 0.005%, preferably 0.01%, and an upper limit of 1%, in particular 0.5%, preferably 0.2%, is selected.

53. Reagent according to one of claims 35 to 52, characterized in that at least one substance selected from a group comprising sodium salts, potassium salts, phosphate salts, magnesium and / or manganese salts, bovine serum albumin (BSA), casein, gelatin, maltodextrin , Calf serum and / or milk powder, are added.

54. Reagent according spoke 53, characterized in that the concentration of the substances, each in% by weight, from a range with a lower limit of 0.001%, in particular 0.1%, preferably 1%, and an upper limit of 30%, in particular 20%, preferably 10%, is selected.

55. Reagent according to one of claims 35 to 54, characterized in that at least one preservative selected from a group comprising sodium azide, sodium benzoate, sorbic acid, pentachlorophenol, sorbate and / or preservative based on mercury is added.

56. Reagent according to claim 55, characterized in that the concentration of the preservative, each in% by weight, from a range with a lower limit of 0.01%, in particular 0.05%, preferably 0.1%, and an upper Limit of 5%, in particular 3%, preferably 1%, is selected.

57. Analysis kit comprising at least one absorbent material, a reagent for receiving at least one analyte (13) and a device (1) made of capillary material, characterized in that the reagent according to one of claims 35 to 56 and the device (1) according to one of claims 18 to 34 is formed.

58. Use of the device according to one of claims 18 to 34 for the detection of at least one analyte.

59. Use of the reagent according to any one of claims 35 to 56 for the detection of at least one analyte.

60. Use of the analysis kit according to spoke 57 for the detection of at least one analyte.