WO2008141351A1

WO2008141351A1 - Method for quantitatively determining analytes using a test element and test system and use thereof

Info

Publication number: WO2008141351A1
Application number: PCT/AT2008/000172
Authority: WO
Inventors: Alexandra Molinelli; Rudolf Krska; Eva Binder; Johann Binder
Original assignee: Erber Aktiengesellschaft
Priority date: 2007-05-21
Filing date: 2008-05-15
Publication date: 2008-11-27

Abstract

The invention relates to a method and a test system for quantitatively determining at least one analyte using a test element, a quantifiable conversion being performed on an immunochromatographic membrane (5) fixed on a carrier, at least one test or reaction zone (6) being applied to said membrane, and to a use thereof, such that the at least one analyte and an associated anti-analyte labeled with a substance bringing about a color or fluorescence reaction, or the analyte and an analyte labeled with a substance bringing about a color or fluorescence reaction, are brought into contact with the immunochromatographic membrane (5) applied in a defined quantity to the carrier (5); and that the at least one analyte and the associated labeled anti-analyte, or the analyte and the associated labeled analyte, are absorbed by the immunochromatographic membrane (5) and allowed to flow through the at least one test or reaction zone (6), containing either the at least one analyte or the associated anti-analyte; and that the intensity, particularly the color or fluorescence intensity, of the at least one analyte and/or the associated anti-analyte in the reaction zone (6) is determined.

Description

METHOD FOR THE QUANTITATIVE DETERMINATION OF ANALYTS WITH A TEST ELEMENT AND TEST SYSTEM AND USE THEREOF

The present invention relates to a method for the quantitative determination of at least one analyte with a test element in which a quantifiable reaction is carried out on an immunochromatographic membrane fixed on a support on which at least one test or reaction zone is applied. Furthermore, the present invention relates to a test system for the quantitative determination of at least one analyte by means of a quantifiable test element, and to a use of a test system for the quantitative determination of at least one analyte by means of a quantifiable test element.

A large number of quantitative or semi-quantitative methods are known in the art with which analytes, in particular biological analytes, can be detected quantitatively or semi-quantitatively. Most of these methods are either time consuming or allow only qualitative or semi-quantitative detection of substances contained in a sample or pollutants. In particular, a large number of methods are known in which a thin-film technique is carried out in which test strips or test plates are used and an immunochromatographic membrane is fixed on the test plates with which semi-quantitative or qualitative detection of pollutants contained in samples can be carried out ,

Such a semi-quantitative method, which uses a thin-layer or strip test with conventional lateral flow, can be found, for example, in WO 00/42434 or GB 2 300 914. In this device, more than two detection zones are provided on a test strip or element, which are defined along the length of the test strip at distances spaced from each other from the start line, and the amount or semi-quantitative amount of the target analyte can be selected from the number Zones showing a positive result after the sample has been run are determined.

EP 0 462 376 discloses an experiment using a double read-out system having a receiving side on which a receiving reagent is applied, which is coated with the analyte with respect to the binding of the labeled Conjugate competes. Further, a conjugate receiving side is provided to which a conjugate-receiving agent is fixed. Immobilization of the conjugate at this point is then related to the amount of analyte in the test sample, with a decrease in the detectable conjugate on the receiving side and a corresponding increase in the detectable conjugate on the conjugate receiving side indicating that an increasing amount of analyte is present in the test sample the sample is present.

Finally, WO 97/09620 describes quantitative and semi-quantitative experiments in which the signal generated by the target analyte in a detection zone is compared to a signal generated in a range of calibration zones to determine if the signal is Amount of analyte present in the sample is above or below the levels equivalent to those determined by the calibration zones. Standard curve data can be used to calculate the amount of analyte in the sample.

The present invention now aims to provide a simple and, in particular, rapid method for the quantitative determination of at least one analyte in a sample. Furthermore, the present invention aims, in addition to the quantitative detection of an analyte with a test element, to provide the quantitative detection of a plurality of analytes contained side by side in a sample safely, quickly and reliably.

To achieve this object, the method according to the invention for the quantitative of at least one analyte with a test element is essentially characterized in that the at least one analyte and one associated with a dye or fluorescence reaction substance causing associated anti-analyte or the analyte and an analyte labeled with a substance causing a color or fluorescence reaction is brought into contact with the immunochromatographic membrane fixed in a defined amount on the support; in that the at least one analyte and the associated labeled anti-analyte or the analyte and the associated labeled analyte are taken up by the immunochromatographic membrane and by the at least one test or reaction zone containing either the at least one analyte or the corresponding anti-analyte, to be flowed; and that the intensity, in particular the color or fluorescence identity of the at least one analyte and / or the associated anti-analyte in the reaction zone is determined. By, as provided for in the invention, the at least an analyte which may be dissolved or suspended, and either the corresponding anti-analyte labeled with a substance causing a staining or fluorescence reaction or the substance labeled with a staining or fluorescent reaction via an immunochromato graphic membrane is run and is provided on the immunochromatographic membrane, a test or reaction zone in which either the at least one analyte or the corresponding anti-analyte is included succeeds in the course of a competing reaction between either the labeled anti-analyte or effecting a color reaction in the reaction zone on the labeled analyte and its associated non-labeled anti-analyte or analyte, the color or fluorescence intensity being inversely proportional to the amount of analyte present in the sample. This can be done by matching the determined color or fluorescence intensity with a calibration curve or color table or statistical evaluations, a quantitative detection of the amount of analyte contained in the sample. It is particularly favorable or advantageous for the method according to the invention that for measuring the intensity for quantification of the analyte a relative signal comparison with a further test or control zone is not required, but rather a direct, absolute measurement of the intensity of the reaction zone for quantification of an analyte an external calibration function or a calibration element is sufficient, whereby a particularly extremely rapid, quantitative measurement of the analyte is possible.

According to a preferred development of the invention, the process is carried out such that the substance causing a color or fluorescence reaction is selected from colored particles, such as gold, latex or silica gel, fluorescent substances, magnetic particles and / or carbon. In particular, by selecting the colored or fluorescent reaction substance from colored particles, magnetic parts and / or carbon, it is possible to achieve a precise grading of the intensities of the at least one or more reaction zones, so that a very accurate detection of the amount in the sample, at least one analyte becomes possible.

According to a development of the method, the method according to the invention is preferably carried out so that the contact time or reaction time of the analyte on the immunochromatographic membrane is determined for the quantitative determination of the amount of analyte absorbed by the immunochromatographic membrane. By the contact time or turnover time or transit time of the analyte on the set immunochromatographic membrane, succeeds in further clarification of the present method, since in addition to defined amounts of reactants used and the reaction time and thus the amount of solution or suspension, which can be recorded on the immunochromatographic membrane is set. By setting the contact time or reaction time to 15 s to 30 min, in particular 1 min to 10 min, it is ensured that an extremely rapid and reproducible, quantitative detection of at least one analyte is possible. To the recorded by the immunochromatographic membrane Flüssigkeitssbzw. To further clarify the amount of suspension, optionally a receiving pad, which is designed for receiving the suspension or solution of the analyte, may be fixed on the carrier after the immunochromatographic membrane, thereby determining the amount of liquid which has passed over the immunochromatographic membrane, to ensure and in particular to avoid reflux of this solution or suspension with certainty, which reflux the reproducibility of the process and in particular the accuracy of detection in the quantitative range would be adversely affected. Optionally, an adsorbent pad can also be used for this purpose.

By applying quantitative amounts of substances interacting or reacting with the respective analyte or the corresponding anti-analyte to defined regions as test or reaction zones, as is the case with a preferred development of the present invention, not only the amount of For a reaction available substances are set, but also, for example, in adjacent or consecutive areas different amounts of reactive analyte or anti-analyte are set, whereby the test accuracy can be further increased by providing appropriate comparisons.

Finally, as this corresponds to a preferred embodiment of the method according to the invention, as a test or reaction zone lines or beams extending over the entire width of the immunochromatographic membrane, punctiform or other geometric shapes having areas which at least over part of Width of the immunochromatographic membrane extend, are used. Such a selection of the geometric shape of the reaction zone makes it possible not only to detect a single analyte at once on a test strip or a test plate, but to obtain a plurality of possible analytes. At the same time qualitatively detect analytes contained in a sample to be examined qualitatively. In addition, as already stated, the intensity of the respective detection reaction can also be influenced by selecting different amounts of analyte or anti-analyte contained in the reflection zones, and thus an even more accurate quantification of the substances to be investigated contained in a sample.

In order to ensure a particularly precise task of the amount of reactive substances in the test or reaction zone, the process according to the invention is preferably carried out such that the test or reaction zone (s) is (are) applied by spraying, dropping or masking. ,

By, as corresponds to a preferred development of the method according to the invention, the analyte or the anti-analyte interacting or reacting substances from the analyte itself, protein conjugates of the at least one analyte, anti-analyte antibodies, fragments of anti-analyte -Antikörpern, proteins, aptamers and conjugates of biological polymers are selected, it is possible, a variety of to be examined, biological or organic substances quickly and accurately, ie quantitatively to investigate by means of the method according to the invention.

By carrying out the process in such a way that at least one separate, discrete reaction zone is applied to the immunochromatographic membrane for each analyte or its corresponding anti-analyte to be determined, on the one hand it is ensured that a mixture of substances to be investigated and thus a superimposition of reactants in the case where several substances to be examined are contained in one and the same sample, it is certainly avoided in the boundary region of the reaction zones. In addition, it is possible to ensure discrete reaction areas, which show a clear and reproducible intensity of the reaction, so that the analyte to be detected can not only be quantitatively detected, but the process can be performed at any time reproducible.

For a particularly clear, and in particular a high accuracy exhibiting quantitative detection, the inventive method is preferably performed so that the color or fluorescence intensity of the reaction zone by comparison with a background is determined. By determining the intensity, in particular the color intensity and fluorescence intensity, of the reaction zone against a background, a comparison zone automatically exists in the method according to the invention, which facilitates the intensity adjustment of the reaction zone in such a way that in the test itself a standard is measured against can, is provided. With the presence of such a standard, it is possible, for example, to determine the amount of substance contained in an analyte of interest by simply comparing the sample to be examined with an existing color chart or fluorescence intensities.

For a more precise and thus more precise quantitative detection, the method according to the invention is preferably carried out so that the color and fluorescence intensity of the reaction zone in comparison with the background with a photometric or fluorometric measuring device, in particular a photometric or fluorometric reflection device Spectrometer or a CCD camera is measured. By using a photometric or fluorometric measuring device, optical illusions, which can happen when compared with the naked eye, can be avoided with certainty. By indicating the measurement result on a display device of the device, as is the case in accordance with a further preferred method, a completely automated method is provided with which the quantitative amount of an analyte to be examined in a sample can be detected quickly and reliably, and the detection time overall can be lowered further.

By determining the content of the at least one analyte in the liquid or suspension by comparison with standardized values, such as a calibration curve, as is the case with a preferred process procedure, the error rate of the method according to the invention can be further lowered and the accuracy of the detection further increased become.

To ensure that the inventive method has worked correctly and not for example, the immunochromatographic membrane and / or the reaction zone, which is fixed on the immunochromatographic membrane, for example, by improper storage malfunctions or is no longer active, the inventive method is preferably performed in that additionally a control line or control zone is applied and that as a substrate for the control line or control zone, an immobilized anti-species-specific antibody of the anti-analyte antibody is used. By providing a control line or control zone can be ensured that both the immunochromatographic membrane and at least the at least one reaction zone on this membrane are active and not a faulty test system is present and thus the inventive method was carried out correctly and trouble-free.

In particular, to further increase the accuracy of the method and to ensure that cross-reactions are excluded with similar, contained in a sample substances or analytes, the inventive method is preferably performed so that the at least one analyte and each having at least two anti-Ana - lyten is contacted, wherein at least one of the anti-analyte is labeled with a dye or fluorescence-causing substance, and the analyte and the associated anti-analytes with the defined in a defined amount on the support immunochromatographic membrane in contact to be brought.

The present invention further aims at a rapidly and reliably responding test system for the quantitative determination of at least one analyte by means of a quantifiable test element, with which not only fast but also reliable and reproducible analytes can be detected and identified in biological samples.

Such a test system according to the present invention essentially characterized in that a support is provided, on which a defined amount of an immunochromatographic membrane is fixed; in that at least one reaction zone is defined on the immunochromatographic membrane and that a photometric or fluorometric measuring instrument is included for determining the amount of analyte passed through the reaction zone of the test element, the labeled analyte and / or the labeled anti-analyte. By defining at least one reaction zone on a defined amount of an immunochromatographic membrane and providing a photometric or fluorometric measuring device for determining the amount of an analyte or anti-analyte passed through the reaction zone of the test element, a clearer, faster and more reproducible, quantitative approach is achieved Detection of the analyte. By, as corresponds to a preferred development of the test system according to the invention, as a photometric or fluorometric measuring device, a photometric or fluorometric reflection device, a spectrometer or a CCD camera is used, fine color differences of photometric reactions or small reflection differences of fluorometrically reacting substances can be clearly and reproducibly be detected.

By using a portable device as a photometric or fluorometric measuring device, as is the case with a further development of the invention, it is not only possible to provide a small-scale test arrangement, but also a test system with which it is possible to work directly on site, i. For example, at the end user to examine substances for the presence of a questionable analyte.

By, as this corresponds to a development of the present invention, the measurement result is displayed directly on a display of the measuring device, in particular a display, an even further simplification of the test system is provided and it is possible in particular to provide a test system available , which does not require any special technical training, but can be applied by anyone at any location.

By, as in a further development of the invention, a test system is used in which a plurality of test strips is used simultaneously and that the plurality of test strips is fixed in a common holder, a quantitative detection of a plurality of simultaneously in a sample to be examined present, various analytes can be provided in any combination. In addition, such a system can of course be used for a negative proof or multiple negative and positive proofs side by side.

By applying a plurality of reaction zones to one and the same test element, as is the case with a preferred development, a further simplification of the test system is achieved and, in particular, test systems can be provided, for example, in which analytes commonly occurring together on one and the same Test element are applied, so that no more separate detection methods must be performed or different test systems must be used side by side for use, but One and the same test system and method provides the quantitative detection, for example, of several analytes to be examined.

By, as corresponds to a preferred embodiment of the invention, the plurality of reaction zones side by side, i. at a level at which the test element is applied, it is ensured that, in addition to the detection of possibly several analytes contained in a sample, each analyte can be reproducibly detected over the immunochromatographic membrane after one and the same running time and moreover, for example, by applying different lent concentrations of one and the same substance to be detected or their anti-analytes in adjacent reaction zones, the accuracy of detection is further increased by making a clear assignment of the obtained intensity of the reaction possible.

By, as already stated, the plurality of reaction zones for the detection of different analytes is formed, the simultaneous quantitative detection of several analytes to be examined succeeds.

The present invention further aims to use a test system for the quantitative determination of at least one analyte by means of a quantifiable test element, which test system is used for the detection of low molecular weight substances, peptides, fragments of antibodies, aptamers, biological polymers, protein conjugates and protein fragments.

In particular, the test system is used for the quantitative detection of undesired contaminants and drugs as well as chemical residues, in particular toxins, in particular mycotoxins, in particular trichothecenes, such as nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, fusarenone X, T-2 Toxin, HT-2 toxin, fumonisins such as fumonisin B1, B2 or B3, ochratoxins such as ochratoxin A, B, C or D, zearalenones, moniliformin or aflatoxins such as aflatoxin B1, B2 G1 or G2, M1, M2, alkaloids, Drugs, as well as allergens, pesticides, hormones, antibiotics, additives and additives, ingredients / active ingredients, acrylamides, genetically modified organisms and environmental pollutants and residues used. With such a use, therefore, the rapid and reliable detection of a variety of pollutants, pharmaceuticals or toxins, for example, in food, animal feed or the like. The invention will be explained in more detail with reference to embodiments schematically illustrated in the accompanying drawings and examples. 1 shows the illustration of a test system according to the present invention with a detection zone and a control zone; Figure 2 is a comparison of standard curves in the detection of deoxynivalenol. Fig. 3 is a regression line for the calibration of a deoxynivalenol strip test; FIG. 4 shows the T2 toxin concentration of extracts measured with a T2 test system; FIG.

5 shows another example of a strip test in which a plurality of punctate reaction areas are shown side by side for the detection of fumonisin B1, aflatoxin B1, chloramphenicol and clenbuterol, wherein FIG. 5a shows a test plate without a control line and FIG. 5b shows the same test plate with a control line; and Fig. 6 shows a test plate in which three allergen-specific monoclonal antibodies, namely hazelnut, walnut and almond, were immobilized on an immunochromatographic membrane.

In the following embodiments, which refer to the above figures, the invention will be further explained.

example 1

In Example 1, as shown in Fig. 1, a test plate or a test strip for the quantitative detection of deoxynivalenol (DON) in wheat or wheat flour in a concentration range of 250 to 1,600 ppb is shown. The test strip, which is generally indicated by 1, has at its start end 2 a release pad 3, which dips into a well, not shown, or a vessel containing the analyte to be examined and either a labeled anti-analyte or a labeled analyte. Starting from this release pad, the sample or analyte to be examined runs in the direction of the arrow 4 over an analytical nitrocellulose membrane 5 which, like the release pad, is applied to a carrier, for example a plastic plate. In the middle region of the nitrocellulose membrane 5, a reaction line 6 consisting of a protein conjugate of the target analyte deoxynivalenol is applied in the present case. The proteins used for the conjugation were here, for example, bovine serum albumin, ovalbumin min or konalbumin. The reaction zone in the present case was applied to the nitrocellulose membrane 5 using a spray device. At a distance from the reaction line 6, in the example of Fig. 1, a control line 7 consisting of a rabbit anti-mouse antibody was applied to confirm the correct test development since mouse antibodies were used in the experiment.

At the end of the immunochromatographic membrane 5 is provided for the complete absorption of excess, finally an adsorbent pad 8, in which not only the excess solution is taken up, but also the reflux of the solution is prevented with certainty. As Absorbenskissen 8 a cotton pad is applied in the present case.

The experimental procedure was conducted as follows. Monoclonal anti-DON antibodies were conjugated to colloidal gold and used as a selective uptake reagent. As a sample to be examined containing the analyte, a soil sample was extracted in a ratio of 1: 4 (weight / volume) with distilled water using a mixer for 3 minutes. The sample was allowed to sit for 5 to 10 minutes. The extract was further diluted 1.2 with distilled water and 50 μl was used for the test. In the case of too high contaminations with the analyte, obvious dilution series are readily possible.

For the labeled anti-analyte, a sufficient amount of monoclonal antibody, in the present mouse monoclonal antibody, is used for the coupling reaction with colloidal gold, which has particle sizes of about 40 nm. For a first dilution, the calculated amount of antibody was added to 1 ml of distilled water at pH 8.5. The antibody solution was then mixed with a colloidal gold solution in a centrifuge tube and mixed for 90 minutes at room temperature with gentle stirring on a shaking platform. The unreacted residual binding sites on the mouse monoclonal antibody were bound by the addition of 2% BSA or bovine serum albumin and incubation of the mixture for a further 90 min. To separate unbound antibody was centrifuged for 30 min at 8000 G and washed with 30 mL of distilled water at pH 8.5.

The test extract was thoroughly mixed with the receiving reagent buffer mixture in a microtiter well by repeated pipetting. The strip test was then vertically immersed in the well containing a well-defined amount of solution and uptake reagent for a test period of 3 minutes. The colloidal gold allowed the development of colored red lines after selective retention by the immobilized reagents. The reaction line 6 was applied at 8.0 ± 5 mm from the bottom of the immunochromatographic membrane 5 and had a width of about 1.0 ± 1 mm. Control line 7 had a constant intensity independent of the presence or absence of the target analyte for rapid visual confirmation of the correct test performance and to confirm that the test plate used is active and effective. Control line 7 was applied at about 13 to 14 mm from the bottom of the immunochromatographic membrane 5 and had a distance from the absorbent pad of about 3 mm.

A positive sample resulted in a slightly colored line in the reaction zone 6. Alternatively, a negative sample would result in the formation of a clearly visible line in the reaction zone 6, since in the present case a competing reaction between the analyte to be detected and the labeled anti-analyte and the Analyte in the reaction zone 6 took place. The test strip 1 was then developed with a portable reader to scan the reaction zone 6. The test results can be read immediately after the three-minute test development time.

FIG. 2 shows the comparison of two standard curves in the range from 0 to 2,000 ppb DON, wherein in particular in the curve 9 a straight region 10 clearly occurs, in which the experiment can be carried out in a quantitatively reproducible manner.

By comparison with such a calibration curve, the portable reader can perform an evaluation of the experiment immediately and without delay. It is irrelevant to note that when a fluorescent reagent is used instead of colloidal gold, an evaluation can be made immediately using, for example, a naked-eye UV lamp and comparison with test cards.

Fig. 3 shows a calibration of a selected working range of the test system using deoxynivalenol. The analyte quantification can be carried out using naturally contaminated samples using a formula Y = -AX + P for a competition trial format, as shown in FIG. The method used may be matrix-dependent and must be chosen according to the matrix to be investigated. The reaction zone signal is immediately measured at the end of the test period with a reader and the data are processed with available software, which immediately yields a quantitative result.

Example 2

In Example 2, which is conducted essentially as Example 1, instead of the quantitative detection of deoxynivalenol, that of a concentration of T2 toxin extract is detected, which is measured with a specific T2 strip test. The test strip for the assay plate is hereby identical to that of Example 1 and as shown in Fig. 1, except that anti-T2 monoclonal antibodies were conjugated to colloidal gold and the labeled anti-analytes in the represent sample to be examined. The test procedure was otherwise carried out as in Example 1, except that the test duration was 5 minutes. The range of activity of the quantitative test for T2 toxin is in the range of 1 to 100 ppb.

Example 3

The plate-shaped test systems used in the present example are shown in FIG. FIG. 5 a shows a test system in which the immunochromatographic membrane 5 is brought into direct contact with the analyte to be detected or the labeled anti-analyte or the labeled analyte. A release zone is not provided. In Fig. 5a, a plurality of punctiform reaction zones 6 are applied to the immunochromatographic membrane 5, each of the reaction zones containing an analyte-protein conjugate. Finally, the test system according to FIG. 5a also has an absorbent pad 8 for receiving excess reagent or excess solution containing the substance to be detected.

FIG. 5b shows the analogous immunochromatographic membrane 5, in which in turn reaction zones or test points 6 containing four different analyte-protein conjugates are applied. In addition to the test points 6, Fig. 5b has a control line 7 for proving whether the test is positive, which is formed as in Example 1. Finally, an absorbent pad 8 for receiving excess analyte solution or suspension is also provided in FIG. 5b in order to avoid a backflow of the analyte into the reaction system. More specifically, Example 3 is executed as follows.

Protein conjugates coupled to BSA and / or ovalbumin and conalbumin A, of fumonisin B1 and aflatoxin B1, chloramphenicol and clenbuterol were applied as circular surfaces 6. Optionally, a rabbit anti-mouse antibody was used to check the correctness of the test procedure. The concentrations of the conjugates were used for test optimization in the range of 0.05 mg / ml to 5 mg / ml. A mixture of monoclonal or polyclonal antibodies conjugated to colloidal gold and each with specific affinity for each analyte was used to selectively recognize and bind the target analytes. The intensity of the circular zones was measured with a suitable reader and quantified by an externally created calibration function.

The target analytes were in diluted form, on the one hand as a standard dissolved in methanolic solution (solution 1, as shown in Table 1), and on the other hand on a real sample extract from animal feed, as shown in Table 2, tested with the target substances. The results are shown in the form of signal intensities as follows, converted into concentration values by means of the external calibration function.

Table 1

Table 2

Example 4

In the present example, a test was carried out for the simultaneous detection of allergenic hazelnut, walnut and almond proteins. In this example, as shown in FIG. 6, a nitrocellulose analytical membrane 5 was used as the immunochromatographic membrane in combination with a release pad 3. The samples to be examined ran in the direction of arrow 4. On the nitrocellulose membrane 5 were three reaction or test zones 6 of one reagent at the same height from the bottom of the test plate 1 for simultaneous detection of the three allergenic nut proteins of aqueous Extracts from foods believed to contain these nuts are applied. Reaction zones 6 were monoclonal antibodies to the following target proteins, selected from Cor a11 for hazelnut (48 kDa allergen), Jug r2 for walnut (45 kDa allergen) and Pru du4 for almond (14 kDa allergen). The reaction zones 6 had a maximum area of 5 mm × 5 mm. The applied concentrations ranged from 0.5 to 5 mg / L to bind the available target allergenic protein. For control line 7, a rabbit anti-mouse antibody was used to confirm the correct test development. Control line 7 at 0.75 mg / ml was sprayed 1 μl / cm onto a contact point delivery platform.

Polyclonal antibodies were used as the second antibody in the sandwich experiments and conjugated with colloidal gold for use as a selective uptake reagent. A mixture of uptake reagents for each target allergen was mixed. The sample extract was thoroughly mixed in a well with the uptake-agent buffer mixture in a 1: 1 ratio by repeated pipetting and release. The prepared mixture containing the extract was applied in a metered amount to the release pad 3 of the strip test. The liquid sample was allowed to flow on the membrane for a test period of 10 minutes and the excess was collected on an absorbent pad 8.

The colloidal gold allowed the development of colored red zones after selective retention of the allergen proteins by the immobilized reagents. The control line had a constant intensity independent of the presence or absence of the target allergens for rapid, visual confirmation that the test is functioning correctly. A positive sample resulted in the formation of a visible reaction zone. Alternatively, a negative sample resulted in no signal formation on the Reaction zone. The signal intensity on each reaction zone was proportional to the protein concentration.

Walnut, almond and hazelnut samples were used to determine the specificity of the antibodies. Roasted hazelnuts, white almonds and untreated walnuts were used. For each chosen nut, 200 grams of ground nuts were mixed with 400 ml of ice cold distilled water, dispersed in a mixer at 6N for about 90 seconds, and passed through a 125 μm sieve for 30 minutes. The supernatant was again dispersed with 200 ml of ice-cold distilled water and again passed through a sieve. The sieve fractions were lyophilized overnight to total dryness. The resulting powder was stored at -20 ⁰ C until further use.

The nut strip test was developed for use with a meter which is used to sense the intensity of the reaction zones 6. Depending on the type of meter, a mean, relative intensity or average pixel intensity in the reaction zone 6 is measured. The selection of the area size of the reaction zone makes it possible to adjust or adapt a possible concentration range of the selected target proteins which are scanned. The intensity of reaction zone 6 may decrease from the bottom to the top of the test zone in the direction of flow, depending on the amount of allergenic proteins in the sample. The results may be expressed in μg / ml protein extract or mg nut / kg of tested food.

Control line 7 was visible within one minute, confirming the correct test development, while 10 to 15 minutes were required for complete signal development at reaction zones 6, depending on the matrix chosen.

The test results could be read directly after the test development time.

Claims

Patent claims

1. A method for the quantitative determination of at least one analyte with a test element, wherein carried out on a supported on a support, immunochromatographic membrane (5) on which at least one test or reaction zone (6) is applied, carried out a quantifiable reaction is characterized in that the at least one analyte and in each case one with a color or fluorescence reaction causing substance labeled associated anti-analyte or the analyte and a substance causing a color or fluorescence reaction substance marked with the analyte in a defined amount on the Carrier determined immunochromatographic membrane (5) is brought into contact; in that the at least one analyte and the associated labeled anti-analyte or the analyte and the associated labeled analyte are taken up by the immunochromatographic membrane (5) and by the at least one test or reaction zone (6) containing either the at least one analyte or the associated anti-analyte, to be flowed; and that the intensity, in particular the color intensity or fluorescence intensity of the at least one analyte and / or the associated anti-analyte in the reaction zone (6) is determined.

2. The method according to claim 1, characterized in that the substance causing a color or fluorescence reaction of colored particles, such as gold, latex or silica gel, fluorescent substances, magnetic particles and / or coal is selected.

3. The method according to claim 1 or 2, characterized in that for the quantitative determination of the immunochromatographic membrane (5) recorded amount of the analyte, the contact time or reaction time of the analyte on the immunochromatographic membrane (5) is set.

4. The method according to claim 3, characterized in that the contact time or reaction time is set to 15 s to 30 min, in particular 1 min to 10 min.

5. The method according to any one of claims 1 to 4, characterized in that as test or reaction zones quantitative amounts of interacting with the respective analyte or the corresponding anti-analyte substances or substances are applied to defined areas.

6. The method according to any one of claims 1 to 5, characterized in that as a test or reaction zone (6) lines or bars which extend over the entire width of the immunochromatographic membrane, punctiform or other geomet- rische shapes having areas, which extend at least over part of the width of the immunochromatographic membrane can be used.

7. The method according to any one of claims 1 to 6, characterized in that the test or reaction zone (s) (6) is applied by spraying, drops or a mask (is) applied.

8. The method according to any one of claims 1 to 7, characterized in that the interacting with the analyte or the anti-analyte substances or reacting substances from the analyte itself, protein conjugates of at least one analyte, anti-analyte antibodies, fragments of anti- Analyte antibodies, proteins, aptamers and conjugates of biological polymers are selected.

9. The method according to any one of claims 1 to 8, characterized in that for each analyte to be determined or its corresponding anti-analyte least one separate, discrete reaction zone (6) on the immunochromatographic membrane (5) is applied.

10. The method according to any one of claims 1 to 9, characterized in that the color or fluorescence intensity of the reaction zone (6) is determined by comparison with a background.

11. The method according to any one of claims 1 to 10, characterized in that the color and fluorescence intensity of the reaction zone (6) compared with the background with a photometric or fluorometric measuring device, in particular a photometric or fluorometric reflecting device, a spectrometer or a CCD Camera is measured.

12. The method according to claim 11, characterized in that the measurement result is displayed on a display device of the device.

13. The method according to any one of claims 1 to 12, characterized in that the determination of the content of the at least one analyte in the liquid or suspension is determined by comparison with standardized values, such as a calibration curve.

14. The method according to any one of claims 1 to 13, characterized in that in addition a control line or control zone (7) is applied and that as the substrate for the control line or control zone, an immobilized anti-species-specific antibody of the anti-analyte antibody is used.

15. The method according to any one of claims 1 to 14, characterized in that the at least one analyte and is brought into contact with at least two anti-analyte, wherein at least one of the anti-analyte is labeled with a dye or fluorescence-causing substance, and the analyte and the associated anti-analytes are contacted with the immunochromatographic membrane (5) defined in a defined amount on the support.

16. Test system for the quantitative determination of at least one analyte by means of a quantifiable test element, characterized in that a support is provided, on which a defined amount of an immunochromatographic membrane (5) is fixed; in that at least one reaction zone (6) is defined on the immunochromatographic membrane (5) and that a photometric or fluorometric measuring device is used to determine the amount of an analyte, the labeled analyte and / or the labeled analyte passed through the reaction zone (6) of the test element Anti-analyte is included.

17. Test system according to claim 16, characterized in that a photometric or fluorometric measuring device, a photometric or fluorometric reflection device, a spectrometer or a CCD camera is used.

18. Test system according to claim 17, characterized in that a portable device is used as a photometric or fluorometric measuring device.

19. Test system according to one of claims 16, 17 or 18, characterized in that the measurement result is displayed directly on a display of the measuring device, in particular a display.

20. Test system according to claim 16 to 19, characterized in that a plurality of test strips (1) is used simultaneously and that the plurality of test strips (1) is fixed in a common holder.

21. Test system according to one of claims 16 to 20, characterized in that a plurality of reaction zones (6) is applied to one and the same test element.

22. Test system according to claim 21, characterized in that the plurality of reaction zones (6) is applied side by side on the test element.

23. Test system according to one of claims 16 to 22, characterized in that the plurality of reaction zones (6) is designed for the detection of different analytes.

24. Use of a test system for the quantitative determination of at least one analyte by means of a quantifiable test element, characterized in that the test system for the detection of low molecular weight substances, peptides, fragments of antibodies, aptamers, biological polymers, protein conjugates and protein fragments is used.

25. Use according to claim 24, characterized in that the test system for the quantitative detection of unwanted contaminants and drugs and chemical residues, in particular toxins, in particular mycotoxins, in particular trichothecenes, such as nivalenol, deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl Deoxynivalenol, fusarone X, T-2 toxin, HT-2 toxin, fumonisins such as fumosin B1, B2 or B3, ochratoxins such as ochratoxin A, B, C or D, zearalenones, moniliformin or aflatoxins such as aflatoxin B1, B2 G1 or G2, M1, M2, alkaloids, drugs, as well as allergens, pesticides, hormones, antibiotics, additives and additives, ingredients / active ingredients, acrylamides, genetically modified organisms, as well as environmental pollutants and residues.