EP3183575A1 - Testing device for examining a food for an analyte and test strips intended therefor - Google Patents

Abstract

The invention relates to a testing device (10) for examining a food for an analyte, which testing device has an accommodating space for the sample of the food to be examined and a reservoir, which contains a running buffer. The testing device (10) is designed to comminute the sample accommodated in the accommodating space and to mix said sample with the running buffer. Furthermore, a holder for a test strip is provided, on which test strip an optically detectable signal is produced in the manner of a lateral flow assay, which signal indicates whether the analyte can be detected in the sample. The testing device is also designed to dispense the liquid sample that has been comminuted and mixed with the running buffer onto the test strip.

Description

 Test device for testing a food for an analyte

 and dedicated test strips

The present invention relates to a test device for testing a food for an analyte and a test strip for such a test device.

Many people are allergic or allergic to certain foods or food ingredients. This is especially true for nuts, soy, milk, egg, wheat, fish protein and, for example, histamine. The consumption of foods containing such substances as e.g. Containing allergens, haptens and other small organic molecules can lead to allergic reactions or allergy-like symptoms. The allergic reactions can become life-threatening in individual cases. These substances are generally referred to as analytes in the context of the present application.

Common symptoms are headache and abdominal pain, vomiting, circulatory problems, redness of the skin, itching, etc.

The analytes of interest in the context of the present application, which trigger allergic reactions or allergy-like symptoms, are contained in the same foods in very different concentrations due to the different storage, aging and production of the food in question. Affected individuals can therefore avoid the onset of these symptoms only through a radical avoidance strategy that avoids the consumption of all foods containing potentially harmful substances.

However, this also means that even foods are avoided, which may also be well tolerated. In particular, during restaurant visits, it is also difficult to avoid that analyte-contaminated foods are consumed, especially if the court is not to see what foods it contains in detail. All this leads to a strong impairment of the quality of life of the persons concerned, so that there is a strong demand for a test device that can be applied easily and quickly in the restaurant or the purchase of food and reliable information about whether the for the person important analyte is contained in the food or whether the content of this analyte is below a certain threshold.

US 2010/0317033 A1 describes in this context a lateral-flow test for the detection of allergens in food, wherein a liquid sample of the food saliva of the tester is added to check whether the allergen in the food binds an antibody present in the saliva.

With this test, multiple allergens can be detected simultaneously using antibodies binding to the allergen conjugated to a detectable label.

WO 2009/048895 A2 describes a portable device for detecting possible allergens in a sample. The device comprises a housing in which an allergen detection chip can be placed, on which an antibody against the potential allergen is provided, which is provided with a detectable marker. Via an input port, the sample is introduced into the housing, wherein the detection is carried out in the manner of an immunoassay. For this purpose, for example, be used with a particulate tracer conjugated antibodies against the allergens.

EP 1 767 938 B1 describes a detection method for histamine in foods in which histamine is first extracted from a sample of the food and then detected via a color change.

The company Immundiagnostik, 64652 Bensheim, offers a histamine ELISA kit for the determination of histamine in EDTA plasma and urine. The test is based on the method of the competitive enzyme immunoassay. In preparation for that becomes assaying sample with a derivatization reagent for coupling the histamine contained added. Subsequently, in a histamine-derivative-coated ELISA plate, the derivatized sample is incubated together with a peroxidase-labeled polyclonal histamine antibody. During the incubation, the target antigen in the sample competes with the histamine derivative bound to the plate for binding of the polyclonal antibodies. The concentration of the antibody bound to the histamine derivative is therefore inversely proportional to the concentration of the target antigen in the sample.

This method requires extensive laboratory equipment and experienced users to carry out.

[0013] The company Labordiagnostika Nord in 48531 Nordhorn sells a histamine test called His- taure ™, which must also be carried out in the laboratory and only by experts.

The test is for detection of histamine in samples of foodstuffs, wherein during the preparation of the samples the histamine is derivatized by an acylating reagent in N-acylhistamine. Subsequently, a competitive ELISA test is performed using a microtiter plate.

The company Labordiagnostika North also offers a rapid test, with the use of a lateral-flow cassette histamine can be detected in fish.

For this purpose, 10 g of the fish sample are removed, mixed with 240 ml of distilled water and homogenized. Thereafter, the homogenate is filtered, then 100 .mu.l of the filtered homogenate are placed in a tube with acylation buffer, after which the tube is shaken and incubated for five minutes at room temperature. Thereafter, 100 μΙ of the thus prepared sample are mixed with a running buffer and shaken again. Subsequently, 100 .mu.Ι of the sample mixed with the running buffer are added to the lateral-flow cassette and incubated for five minutes at room temperature, whereupon the test result can then be evaluated by means of a formation of colored lines in a detection zone and a control zone.

After this complicated sample preparation, the test indicates whether histamine is present in a concentration above or below a threshold of 50 ppm in the sample.

For the detection on the lateral-flow-carrier labeled with gold particles antibodies are used, which bind to solid phase histamine. The amount of bound labeled antibodies is inversely proportional to the histamine concentration in the sample.

Two distinct test lines indicate that the histamine concentration is below the threshold, one line indicates that the test was successful and the histamine concentration is above the threshold.

This test is a competitive assay in which histamine bound to the support competes with the derivatized histamine from the sample for binding to the antibody labeled with gold particles.

Test strips and cassettes, which in the manner of a lateral flow assay in an immunological reaction indicate an analyte by an optically visible color reaction, are also widely known in the art.

For example, EP 0 291 194 B1 describes competitive immunoassays and sandwich assays for detecting the pregnancy hormone in urine. The described test strips have a deposition zone to which the liquid sample is applied. Downstream of the application zone, a first zone is provided on which, in the dry state, an antibody to the pregnancy hormone labeled with gold particles is stored. By adding the liquid sample is the Gold-antibody complex resolubilizes and migrates with the liquid sample through a detection zone into a control zone. Downstream of the control zone may still be provided a suction zone to receive excess liquid.

In the detection zone, an antibody is immobilized, which binds to the pregnancy hormone in the manner of a sandwich reaction, to which in turn the gold-labeled antibody complex is bound. In this way, gold particles in the detection zone are concentrated when pregnancy hormone is present in the discontinued sample. The concentrated gold particles in the detection zone lead to an optically detectable colored line.

The gold-labeled antibodies which are not bound in the detection zone migrate further into the control zone, where antibodies directed against the gold-labeled antibodies are immobilized. In this way, the gold-labeled antibodies (also) are concentrated in the control zone, which also leads there to an optically detectable colored line.

A colored line in the control zone indicates that the assay has worked in principle, with a colored line in the detection zone indicating that pregnancy hormone was present in the sample.

EP 291 194 B1 and the WO 2012/069610 A1 based thereon deal in detail with the structure of the test strips, the suitable materials, the immobilization of the antibodies in the detection zone and the control zone, the dry storage of the labeled antibodies in the first Zone, the way in which the resolubilization of the dry, gold-labeled antibodies can be done etc. For further details on structure, function and interpretation of lateral-flow tests and the corresponding test strips is based on this state of the art and on there referenced prior art. The structure and function of lateral flow assays is now part of the prior art, so that a detailed description in the present application is not required. The same applies to the generation of polyclonal or monoclonal antibodies to certain analytes. Furthermore, antibodies against analytes of interest in the context of the present application are widely available on the market, so that the specific generation of antibodies is also assumed to be known in the context of the present application as known in the art.

Furthermore, it is also known in the prior art that derivatized or modified antigens achieve greater binding to antibodies than is achieved with antibodies which have been generated against the unmodified analytes.

In fact, the unmodified analytes often lack sufficient immunogenic properties, so that it is difficult to generate specific antibodies against the unmodified analytes, which is why the analytes are modified before the generation of the antibodies. An example of this is described in Wolthers et al .: "Evaluation of urinary metanephrines and normetanephrine enzyme immunoassay (ELISA) kits by comparison with isotope dilution mass spectrometry", in Clinical Chemistry 1997, 43: 1, pages 14-155.

Against this background, the present invention seeks to provide a test device of the type mentioned above, which is simple and with a quick and easy to be performed test procedure is possible, even the non-professional users in the restaurant or at Can use everyday shopping.

According to the invention, this object is achieved by a test device for examining a foodstuff for an analyte, having a receiving space for a sample of the foodstuffs to be examined, a reservoir containing a running buffer, the tester for comminuting and mixing the foodstuffs received in the receiving space Sample is set up with the running buffer, and with a receptacle for a test strip, on the manner of a lateral-flow-assays, an optically detectable signal is generated, which indicates whether the analyte is detectable in the sample, wherein the test device is set up to dispense the comminuted and mixed with the running buffer, liquid sample on the test strip.

A test strip provided for the test device comprises a porous support on which in a first zone a labeled binding reagent that is specific for the analyte and an immobilized binding agent are arranged in a detection zone such that the liquid sample applied to the porous support contains the picks up labeled binding reagent and moves downstream therewith to the detection zone where an optically detectable signal is generated indicating whether the analyte is detectable in the sample.

The object underlying the invention is completely solved in this way.

The test device can be a cheap disposable product or a reusable and once-usable components containing product that can be carried in the pocket or handbag due to its small dimensions similar to a pregnancy test or a pen.

It is only necessary to enter a sample of the food to be examined in the receiving space of the test device, whereupon then by appropriate manipulation of the test device, the recorded in the receiving space sample is crushed and mixed with the running buffer.

The grinding and mixing of the sample can be carried out by twisting two parts of the test apparatus relative to one another, as is the case with a pepper or salt mill or also with a ballpoint pen, in which the mine is turned by rotating the upper section. and is extended.

It is also possible to arrange two parts of the test device longitudinally displaceable against each other, so that by compressing the test device in one of its axes, the corresponding crushing and mixing of the sample takes place. After the sample has been prepared accordingly, the sample can be applied to the test strip by further manipulation of the test device. This manipulation can be that an opening connecting the receptacle with the test strip is released, for example, by twisting or displacing two parts of the test device, pulling a film from the opening, or pulling a closure plug out of this opening.

By building up an overpressure or by corresponding spatial orientation of the test device, the liquid sample can then reach the test strip, where it leads in the manner of a lateral-flow-assays to a colored line indicating the result of the test carried out.

On the one hand, a successful test can be that a colored line is displayed when the analyte is contained in the sample. The immunological reaction then proceeds e.g. in the manner of a sandwich assay.

On the other hand, the colored line can arise even if the sample is sufficiently free of the analyte, the immunological test then runs e.g. in the manner of a competitive test.

The test device and the test strip may be adapted to detect any analytes in food, but the analyte is preferably histamine, because in particular the histamine intolerance in large parts of the population causes great problems. Histamine is found, for example, in cheese, fish, vegetables, meat, and also in red wine, where widely varying histamine levels can be found in the same foods, and consumers also respond individually to different levels of histamine-loaded foods.

Against this background, there is a great need for an easy-to-use test device with which the histamine content (or lack of histamine content) in foods can be detected quickly and easily. This requirement is met by the new test device with the appropriate test strip. The new test equipment is simple and therefore inexpensive to manufacture, furthermore, it has small dimensions, because it only needs to absorb a small amount of food and additionally only need to provide the reservoir for the running buffer and the recording for the test strip.

It is preferred if the test device has a base body and a cap attachable to the body, wherein the receiving space provided in the cap and on the base body a plunger is arranged, which protrudes when plugged into the receiving space or is hineinbewegbar and is actuated such that a sample located in the receiving space is crushed and mixed with the running buffer.

These measures are structurally advantageous, the test device consists of a possibly reusable body with a plunger, which is provided for crushing and mixing the sample with the running buffer. In the cap, however, the receiving space is provided in which the sample is crushed and mixed with the running buffer.

While the cap can be discarded after a successful test, it is possible to reuse the body, only the tappet must be cleaned under running water before reuse.

Further, it is preferred if the reservoir is provided with the running buffer in the receiving space and closed by a film which is pierced when placing the cap and / or crushing the sample.

In this measure, it is advantageous that the cap can be provided with an individual running buffer for the respective analyte to be detected. In this case, the running buffer may be provided not only for diluting the sample so that it can participate in the capillary flow on the test strip, but it may also contain extraction reagents that facilitate the dissolution of the respective analyte from the sample of the food or only allow in individual cases. While the base body of the test device can thus be used for various analytes, a separate cap with a specific running buffer can be used for each analyte.

Also, the test strip can not be reused, the test strip is also designed for the particular analyte.

The test strip can be delivered either together with the cap or together with the entire test device when the test device is designed as a total disposable part.

The test strip can also be provided as a separate asset, if the main body and possibly also the cap are designed to be reusable.

Against this background, the present invention relates not only to a test device with body, cap and test strips, but also a test strip provided for this test device as well as a main body and a cap in which a running buffer buffer contained is arranged.

The reservoir can be closed by a film which is installed after the running buffer has been placed in the reservoir in or next to the receiving space. This film can be pierced when attaching the cap to the body or at the latest at the actuation of the plunger, so that the running buffer then comes into contact with the sample of the food.

Further, it is preferred if a modifying reagent for the analyte is provided, wherein the modifying reagent is presented either on the test strip or in a reservoir in the cap.

If the reservoir is provided with modifying reagent in the cap, so this reservoir can be completed by a film. On the other hand, if the modifying reagent is provided on the test strip, it may be placed between the application zone and the first zone in which the labeled specific binding reagent is stored. When the prepared sample is applied to the test strip, it then first migrates to the area of the modifying reagent where the modification of the analyte contained in the sample takes place before it comes into contact with the labeled binding reagent.

The modifying reagent and optionally also the labeled binding reagent can be arranged on a glaze provided on the porous support in order to provide sufficient time for the modification of the analyte and to provide for the resolubilization of the reagents. The latter is described in detail in the above-mentioned EP 291 194 B1, so that reference is also made in this regard to this document.

The reservoir of running buffer and / or modifying reagent may preferably be formed by spheres stored in the receiving space, in which the running buffer and / or the modifying reagent are enclosed, and which are crushed when the sample is comminuted.

Such beads, which are also referred to as microcapsules, serve for the encapsulation of liquid substances, they are available for example from the company Inducap GmbH, 38302 Wolfenbüttel and have a diameter of, for example, 1 mm or 5 mm.

Another example of microcapsules, which can accommodate liquid components and are readily degradable, can be found in US 2010/00033300 A1.

The provision of the running buffer and optionally of the modifying reagent in beads or microcapsules has the advantage that, on the one hand, modifying reagent and running buffer can be stored separately from one another, and, on the other hand, the cap can also be reusable, if after a previous use Not only the main body, but also the cap is rinsed out and before the next use provided for the analyte reservoirs to be introduced into the cap and the corresponding test strip is attached to the test device.

It is preferred, alternatively, if the base unit, cap and test strips are designed as a disposable device, so that all reagents and mechanical components are provided in this disposable article, which are provided for the detection of a particular analyte, in particular of histamine.

The beads, which form the reservoir of running buffer or possibly the reservoir of modifying reagent, can be fixed by a film in the receiving space, this film as in a liquid-stored running buffer and / or modifying reagent by attaching the cap on the Body and / or crushing of the then abandoned on the foil food sample is destroyed, so that a mixing of the sample with the running buffer and possibly the modifying reagent can take place.

When the test device is to be assayed for histamine, the modifying reagent is preferably 2-iminothiolane (Traut's reagent). For histamine modified with 2-amino-thiolane, the inventors were able to generate several monoclonal antibodies which, in the immunoassay to be described below, allow detection of histamine in food samples in the order of a few ppb, the time for modification of the histamine contained in the food only takes a few minutes. The detection of histamine in foods using a modification with Traut's reagent can therefore be carried out very quickly and very sensitively, which is why this method is suitable for use in the restaurant or in everyday life shopping.

Further, it is preferred if an actuating knob is provided on the base body, via which the plunger is displaceable in its longitudinal direction relative to the base body, preferably between plunger and actuating button a rotary mechanism is provided which converts a longitudinal movement of the actuating knob into a rotary movement of the plunger, wherein more preferably the rotary mechanism then converts the longitudinal movement of the actuating knob into a rotary movement of the plunger when the actuating knob moves in the longitudinal direction relative to the plunger.

These measures are structurally advantageous, they allow actuation in the manner of a ballpoint pen, wherein the plunger is first moved into the receiving space by pressing the operating knob until it then encounters the food there or the bottom of the receiving space. Upon further pressing of the actuating button then takes place a relative movement between the actuating knob and the plunger in the longitudinal direction, which is converted by the rotation mechanism in a rotation of the plunger.

In this way, the plunger not only serves to crush or crush the food, it also serves to swirl the food in the receiving space, whereby the swirling speed is determined by the gap that forms between the head of the plunger and the receiving space becomes.

In this way, a very fast and efficient comminution and mixing of the sample of the food can be achieved, so that after a short time a prepared sample is available, which can then be applied to the test strip.

The receptacle for the test strip can be arranged on the base body or on the cap, wherein the receptacle for the test strip is preferably connected via at least one opening with the receiving space, which is closed by a removable seal against the receiving space, wherein Preferably, the opening is connected via a filter with the receiving space.

These measures are constructive and advantageous in terms of ease of operation. In particular, when the receptacle for the test strip is arranged on the cap, the opening connecting the receptacle with the receptacle for the test strip can also be provided in the cap, so that no longer channels are required to the liquid sample to guide the test strip.

The arrangement of the test strip on the cap is also advantageous if the cap - possibly with the test strip - to be provided as a separate supplier part, so if the main body is reused, but cap and test strips are disposable items.

About the removable seal of the test strip is completed over the receiving space as long as the sample is comminuted in the receiving space and mixed with the running buffer. Furthermore, after this manipulation, it may be necessary to wait a certain period of time for the modification buffer, if it has also been initially introduced in the receiving space, to have sufficient time to react with the analyte, provided it is present in the sample.

Thereafter, the seal is removed so that the receiving space is in fluid communication with the test strip. Via a filter in this opening, when the sample is applied to the test strip, it is then not possible to retain sufficiently homogenized sample material so that only liquid sample reaches the test strip.

Generally, it is advantageous if a punching sleeve for removing a sample of the food and for transferring the sample is arranged in the receiving space on the base body, wherein preferably the plunger is arranged in the punching sleeve.

Here it is advantageous that the removal of the sample from the food can be done in a simple, fast and hygienic way. It is only necessary to remove the cap from the body and then pierce the punching sleeve in the food so as to take a sample of the food. By the diameter of the punching sleeve and the immersion depth in the food, which can be specified for example by outside provided on the punch sleeve color marking, the volume of the recorded sample can be precisely defined, so that the volume of the sample on the amount of running buffer and the test still to be performed can be coordinated.

For the sensitivity and reproducibility of the result of a yes / no-result test, the amount of the sample of the food must be known within certain limits, and further the volume of the sample must be matched to the volume of running buffer and possibly Modifizierungsreagenz. By means of the marking rings on the outside of the jacket of the punching sleeve, the volume of the various foods can be set so far that, despite the simple mechanical construction and the simple test procedure, a sufficiently accurate test can be carried out for the purposes of the present application.

In this case, it is possible to provide different markings for different foods on the outside of the punching sleeve, because, for example, a smaller amount of cheese is required than for fish in order to successfully carry out the test.

Because the plunger extends inside the punching sleeve, the sample is automatically pushed from the punching sleeve into the receiving space after placing the cap on the base body and the advancement of the plunger, where the film is then pierced during further advancement of the plunger, the There, the reservoir of running buffer and / if necessary. Modifying reagent covers or holds.

In this case, it is preferred if the receptacle for the test strip is arranged on the cap and at least one opening is provided in the cap, which connects the receiving space with the receptacle for the test strip, wherein the punch sleeve has a front cylindrical jacket, which at plugged cap which closes at least one opening. In this measure, it is advantageous that the sealing of the opening takes place through the punching sleeve when the cap is pushed onto the base body.

By simply turning the cap, this opening can then be released, so that the prepared sample can then pass over to the test strip, which can be provided that the cap can be further pushed onto the base body in this twisted position to in the Recording space to build up an overpressure, which supports the abandonment of the now liquid sample and / or accelerated.

It is further preferred if the punching sleeve is mounted longitudinally displaceably on the base body, in its extended basic position with its front cylindrical shell covering at least one opening, and in its retracted position which releases at least one opening, wherein preferably the punching sleeve on a pushable latch is held in the home position.

In these measures, it is advantageous that the opening is not released by a possibly inaccurate rotation of the cap relative to the base body, but by pushing back the punching sleeve.

Further, it is preferred if the plunger is connected via a shaft with the actuating button, which is sealed by a sealing ring inside against the punching sleeve and lockable with the shaft, so that the liquid sample during advancement of the plunger and released opening by in the overpressure generated in the receiving space is placed on the test strip.

This measure also facilitates the handling of the new test device, because after the release of the opening between the receiving space and the recording for the test strip only the plunger must be advanced again so as to transfer by overpressure the liquid sample on the test strip. In particular, if a filter is still in the opening, this ensures the successful course of the test. It is preferred if the cap has lateral recesses through which the punching sleeve can be grasped with an attached cap by an operator and moved in the direction of the base body.

In this measure, it is advantageous that no further mechanisms for the pushing back of the punching sleeve must be provided, but the lateral openings in the cap are sufficient, so that an operator take the punching sleeve, for example, with thumb and forefinger of one hand and against the Can push body so that the opening between the receiving space and recording for the test strip is released in a quick and easy way.

Further, it is preferable that the labeled binding reagent comprises analyte-specific antibodies bound to particulate tracers and when the binder immobilized in the detection zone has the analyte.

The labeled binding reagent in this embodiment is a conjugate of an antibody and a particle, for example a gold bead, wherein an analyte-protein conjugate is immobilized in the detection zone. If there is no analyte in the applied sample, the antibody-particle conjugates travel through the detection zone to the control zone. On the other hand, if analyte is present in the liquid sample, at least some of the antibody-particle conjugates are captured and concentrated by the analyte immobilized in the detection zone, resulting in a visible line.

Alternatively, it is preferred that the labeled binding reagent comprises the analyte bound to particulate labels and when the analyte immobilized in the detection zone has specific antibodies to the analyte. This test is also a competitive test in which the analyte-particle conjugates compete with the analyte optionally contained in the liquid sample for the antibodies immobilized in the detection zone.

It is preferred in this case if a modifying reagent for the analyte is provided on the carrier, wherein the modifying reagent is preferably 2-iminothiolane.

As already described, the modifying reagent modifies the analyte contained in the liquid sample so that it binds particularly strongly to the antibodies used in the test downstream, which shifts the detection limit to the range of a few ppb.

Further, it is preferred that the labeled binding reagent comprises analyte-specific antibodies bound to particulate labels, biotin-bound analyte on the support, and the binder immobilized in the detection zone binds to biotin.

In this assay, the analyte present in the liquid sample and the biotin-bound analyte presented on the test strip compete for binding with the antibody conjugated to the particulate label. If analyte is not present in the sample, the label antibody-antibody conjugates are captured on the binder in the detection zone, which binder may be streptavidin or avidin.

On the other hand, if analyte is present in the sample, the analyte saturates the antibodies in the label-antibody-conjugate such that no label-antibody conjugates are concentrated in the detection zone.

For the competitive assays to function reliably, a certain ratio must be present between the analyte conjugate presented and the free analyte in the liquid sample. At a ratio of, for example, 1: 1 is still 50% of the label conjugate bound in the detection zone. This ratio is determined once for a desired detection limit of a particular analyte and taken into account in the specification for the volumes of sample, running buffer and possibly modifying reagent.

In general, it is still preferred if downstream of the detection zone on the support a control zone is arranged, is immobilized in the binder for the labeled binding reagent.

In this way, not concentrated in the detection concentrated labeled binding reagent is concentrated in the control zone, wherein a colored line in the control zone indicates that the test has in principle been successful, ie that the labeled binding reagent from the liquid sample and recorded by the detection zone could migrate to the control zone.

Further advantages will be apparent from the description and the accompanying drawings.

It is understood that the features mentioned above and those yet to be explained below can be used not only in the particular combination given, but also in other combinations or in isolation, without departing from the scope of the present invention.

An embodiment of the invention is illustrated in the accompanying drawings and will be explained in more detail in the following description. Show it:

Fig. 1 is a side view of the new, assembled test device, in a schematic, not to scale true representation;

FIG. 2 shows a longitudinal section through the test device from FIG. 1; FIG. Figure 3 is a longitudinal section through the main body of the test apparatus of Figure 1, wherein the shaft of the actuating knob is not cut ..;

4 shows a longitudinal section through the cap of the test apparatus from FIG. 1;

Fig. 5 is a schematic plan view of a test strip, as in the

 Test device of Figure 1 can be used;

Fig. 6 is a plan view of the test strip of Fig. 5 after a successful

 Test in which no histamine was found in the sample;

Fig. 7 is a plan view of the test strip on Fig. 5 after a successful

 Test in which histamine was present in the liquid sample;

FIG. 8 shows the binding behavior of a monoclonal antibody in a free histamine competition assay; FIG.

FIG. 9 shows the binding behavior of the monoclonal antibody of FIG. 8 in a modified histamine competition assay; FIG. and

FIG. 10 shows the binding behavior of the monoclonal antibody from FIG. 8 in a modified histamine competition assay for two different incubation times during the modification. FIG.

In Fig. 1 is shown in a schematic, not to scale side view of a test device 10 for the investigation of a food on an analyte. The test device 10 comprises a cylindrical base body 1 1, on which a cap 12 is attached with a tip 13. The cap 12 is locked on two legs 14 on an undercut 15 on the base body 1 1 and can be removed from the base body 1 1 and put back on this. At its remote from the undercut 15 rear end 16, the base body on an actuating knob 17 which is guided via a shaft 18 longitudinally displaceable in the base body 1 1, which is indicated by a double arrow 19.

At its rear end 16 opposite the front end 21 protrudes from the base body 1 1 a punching sleeve 22 out, which extends into the circumferentially closed tip 13 of the cap 12.

In the sectional view of the test apparatus 10 in Fig. 2 it can be seen that in the main body 1 1, a cylindrical blind hole 23 is provided, which extends from the rear end 16 to a bottom 24 in which a through hole 25th is arranged.

In the blind hole 23, a shaft 26 which extends through the through hole 25 into the punch sleeve 22, where it carries a plunger 27 extends.

Opposite the plunger 27, a cylinder part 28 is arranged on the shaft 26, on which a radially projecting pin 29 is provided.

The cylinder part 28 is arranged in a blind hole 31 of the shaft 18, wherein the bolt 29 extends radially outward through a helical groove 32 which extends in a cylindrical wall 33 which surrounds the blind hole 31.

The blind hole 31 which is open to the right in FIG. 2 has a bottom 34. Between the bottom 34 and the cylinder part 28, a compression spring 35 is arranged, which presses the shaft 18 and the shaft 26 apart. By running in the helical groove 32 bolts 29, the arrangement of shaft 18, compression spring 35 and shaft 26 is held together.

Projecting into the blind hole 23, the shaft 18 has an end face 36. Between the end face 36 and the bottom 28, another compression spring 37 is attached. orders, which presses the arrangement of shaft 18 and shaft 26 relative to the main body 1 1 in Fig. 2 to the left.

When the operating knob 18 is pressed in Fig. 2 to the right, it is first against the force of the compression spring 37 of the shaft 18 and thus the shaft 26 and pressed over this the plunger 27 to the right, so that the plunger 27 up to the tip 13 of the cap 12 is moved in, where a receiving space 38 is provided for the sample of the food to be examined.

In a manner to be described, a sample of the food is introduced into the receiving space 38 in the case of the cap 12 withdrawn from the base body 11.

When the plunger 27 strikes food in the receiving space 38 or on the bottom 39 of the receiving space 38, the shaft 18 shifts with further actuation relative to the shaft 26, wherein the compression spring 35 is compressed.

By this further longitudinal displacement of the shaft 18 relative to the shaft 26, the shaft 26 is set in rotary motion, because the bolt 29 and the helical groove 32 together form a kind of rotation mechanism, implemented by the longitudinal movement of the shaft 18 in a rotational movement of the shaft 26 becomes.

When the pressure on the operating knob 17 is reduced, first the shaft 18 moves relative to the shaft 26 to the left, whereby the plunger 27 is now rotated in the other direction until the compression spring 35 is again relaxed so far that the bolt 29 in the helical groove 32 abuts, so that a further movement of the shaft 18 in Fig. 2 to the left causes the spring 18 and the shaft 26 are moved to the left via the compression spring 37, whereby the plunger 27 from the receiving space 38 is moved out. So that the functional sequence just described is secured, the pressure force of the compression spring 35 must be greater than that of the compression spring 37th

In Fig. 3, the base body 1 1 is shown cut, in which case the shaft 18 is not cut, so that the helical groove 32, in which the bolt 29 extends, can be seen better. The cap 12 is subtracted in the illustration of Fig. 3 of the main body 1 1, so that the punch sleeve 22 is exposed.

Immediately behind the plunger 27 sits on the shaft 26, a sealing ring 41 which bears sealingly against a cylindrical inner wall 42 of the plunger 22. In Fig. 3, the left side of the sealing ring 41 is followed by a cylindrical holding part 43, in which 44 balls 45 are seated in radially distributed recesses, against which a collar 46 is applied, which is connected to the punch sleeve 22.

The holding part 43 and the collar 46 are arranged in a cylinder chamber 47 open to the right in the front end 21 of the base body 1 1, wherein the cylinder chamber 47 is closed by a bottom 48 which extends parallel to the bottom 24.

The collar 46 is supported on a in Fig. 2 to be recognized compression spring 50, which is arranged in the cylinder chamber 47 between the collar 46 and bottom 48, so that the punch sleeve 22 in its extended basic position, shown in Fig. 3 is locked.

By pressing on the punching sleeve 22 in Fig. 3 to the left, the compression spring 50 is compressed so far that the balls 45 are pressed radially inwardly into the recesses 44, and the holding part 43 with the shaft 26 lock.

The purpose of this measure will be explained below. The punching sleeve 22 has at its front end a thin-walled portion 49, which serves to sample from a food indicated at 51

52 aus punch, which is indicated in Fig. 3 inside the punch sleeve 22.

On the outside of the thin-walled portion 49 are optical marks

53 and 54, which indicate how far the thin-walled portion 49 may be inserted into the food 51 in order to accommodate a certain volume of sample 52.

Only for better illustration, the markings 53 and 54 are shown in Fig. 3 raised, in reality, they are only optical marks that do not extend beyond the cylindrical shell 55 of the thin-walled portion 49 radially outward.

In Fig. 4, the withdrawn from the main body 1 1 cap 12 is enlarged and shown in side section.

It can be seen that in the top 13 to the left of the receiving space 38, two annular grooves 56 and 57 are arranged, in which seals 58 and 59 are arranged, which are shown in Fig. 2.

The seals 58 and 59 are applied to the base body 1 1 plugged cap 12 on the cylindrical shell 55 and thus seal off a provided with a filter 61 a opening 61, which connects the receiving space 38 with a receptacle 62 for a test strip , which can be inserted into the receptacle 62. The receptacle 62 is formed on one of the two legs 14 of the cap 12.

The receiving space 38 is further closed by a film 63, which seals a reservoir of beads 64, which are arranged in the receiving space 38 and contain a running buffer and optionally a modifying reagent. The running buffer and modifying reagent are needed to perform the test on the test strip, as will be described below. When attaching the cap 12 of FIG. 4 on the base body 1 1 of Fig. 3, the punch sleeve 22 enters into the receiving space 38, wherein the further insertion of the thin-walled portion 49 of the punch sleeve 22 pierces the film 63. The film 63 can also be attached so that it is pierced only when ancestor of the plunger 27 of this.

When the cap 12 is completely pushed onto the base body 1 1, so that the legs 14 engage behind the undercut 15, the cylindrical shell 55 is in contact with the seals 58 and 59 in the annular grooves 56 and 57, so that the Opening 61 is completely sealed from the receiving space 38.

By repeatedly pressing the operating knob 17, the sample 52 is now ground by the plunger 27, wherein at the same time the beads 64 are broken, so that the running buffer and possibly the modifying reagent are released and mixed with the sample 52. As a running buffer, for example, phosphate buffer or PBS 0.1% Tween can be used.

This process, in which the plunger 27 advanced into the receiving space 38 and there first in the one and then pulled back in the other direction, is carried out until it is recognized on the basis of the pressure resistance that the sample 52 homogenized and mixed with the running buffer and optionally modifying reagent.

If there is a reservoir of modifying reagent in the cap 12, then a few minutes must be allowed for the modifying reagent to be able to modify the analyte optionally contained in the sample 52.

Thereafter, the punching sleeve 22 between the two legs 14, which form two opposing recesses 66, taken with thumb and forefinger and pushed in Fig. 3 to the left, so that the balls 45 run into the recesses 44. By this movement of the punching sleeve 22 in Fig. 3 to the left, the thin-walled portion 49 moves so far to the left that the opening 61 is released, so that the receiving space 38 is now in fluid communication with the receptacle 62 for the test strip ,

Returning to FIG. 2, it can be seen that between the collar 46 and the bottom 48, the further compression spring 50 is provided, which biases the punching sleeve 22 in its extended basic position, which can be seen in Figs. 2 and 3.

As long as the balls 45 are radially extended, as shown in Figs. 2 and 3, sealing ring 41 and holding member 43 are held immovably against the collar 46, so that when moving the actuating knob 17 in Fig. 2 to the right Shaft 26 passes through the sealing ring 41 and the holding part 43.

If now by pushing the punch sleeve 22 to the left, the balls 45 are pressed into the recesses 44, sealing ring 41 and holding member 43 are immovably connected to the shaft 26 so that now when moving the actuating knob 17 to the right of the sealing ring 41 and Holding member 43 are moved with the shaft 26 with, which leads to the compression in the receiving space 38.

Now, the operating knob 17 is thus pressed again, so that the plunger 27 and with it the sealing ring 41 are moved to the receiving space 38, which leads to an overpressure in the receiving space 38. As a result of this overpressure, the liquid sample located in the receiving space 38 is conducted through the opening 61 into the receiving space 62 and at the same time filtered through the filter 61 a, so that residual particles of the sample 52 can not reach the test strip. In the receiving space 62, the liquid sample then reaches the test strip.

It will now be explained with reference to FIG. 5 how a lateral flow assay is started by this application of the liquid sample to the test strip 67 shown schematically there in plan view. FIG. 5 schematically shows in plan view a test strip 67 which comprises a porous support 68 which is made, for example, from nitrocellulose and provides for a capillary flow of a liquid sample which is applied to the porous support 68 at an application end 69 is then moved downstream along an arrow 71 in the porous support 68.

Downstream of the applicator end 68, on the porous support 68 are successively a modifying reagent zone 72, a labeled analyte-specific binding reagent zone 73, a capillary flow zone 74, a detection zone 75 in which Binder is immobilized, another capillary flow zone 76, a control zone 77 in which binders for the labeled binding reagent from zone 73 are immobilized, and a suction zone 78 are arranged, which receives excess liquid sample.

FIG. 6 shows the test strip 67 from FIG. 5 after a successful test, in which a colored line 79 or 81 can be recognized both in the detection zone 75 and in the control zone 77.

The colored line 81 indicates that the test was successful, that is, that the liquid sample has passed through the porous support 68, with the sample having migrated the labeled binding reagent from the zone 73 through the support 68.

The colored line 79 also indicates that in the liquid sample no or so little analyte was present that the labeled binding reagent could bind in the detection zone 75.

FIG. 7 shows the test strip 67 from FIG. 5 after a test in which sufficient analyte was present in the liquid sample, so that no colored line can be seen in the detection zone 75, while the colored line 81 in FIG the control zone 77 again indicates that the test was completed successfully. In the following, embodiments are described in which the analyte is histamine, and in the zones 72, 73, 75 and 77, different reagents are presented or immobilized in order to carry out a competition test, as has already been explained in principle at the outset.

option A

In zone 73 histamine specific antibodies are provided in this embodiment, which are bound to a particulate tracer such as gold beads. These are, for example, rat anti-histamine antibodies bound to gold beads.

In the detection zone 75, a histamine-protein conjugate is immobilized, the protein serving to permanently immobilize the conjugate in the detection zone.

In control zone 77, anti-rat IgG is immobilized.

If no histamine is present in the liquid sample applied to the applicator end 69, the anti-rat histamine conjugate from the capillary flow zone 73 passes through the detection zone 75 where it is sandwiched to the immobilized histamine and bound in control zone 77, where the anti-rat IgG concentrate the anti-rat histamine conjugate.

In this test course, the colored lines 79 and 81 according to

Fig. 7.

In contrast, histamine contained in the liquid sample would saturate the rat anti-histamine antibodies to such an extent that no binding to the immobilized histamine conjugate can take place in the detection zone so that the colored line 79 does not arise. If, on the other hand, no histamine is present in the sample applied, the rat anti-histamine conjugate can also bind in the detection zone and form the colored line 79.

Variant B

This embodiment corresponds to the variant A, but in addition a modifying reagent is provided in the zone 72, in this case 2-lminothiolan.

If no histamine is provided in the liquid sample applied to the application end 69, the two colored lines 79 and 81 result again, the modifying reagent is in this case inoperative.

In contrast, when histamine is present in the applied liquid sample, it is first modified by the modifying reagent, resulting in a significantly more effective binding of the modified histamine to the rat anti-histamine conjugate.

In this case, considerably less histamine is required in the liquid sample to prevent formation of the colored line 79.

In other words, the detection reagent makes the detection considerably more sensitive. While the detection limit in the variant A is in the range of ppm, it could be increased with the variant B in the range ppb.

Fig. 8 shows the binding behavior of one of the monoclonal antibodies generated by the inventors in a competition assay with free histamine. Plotted were the maximum normalized levels of inhibition for different concentrations of histamine.

The experiments were carried out for a total of five different monoclonal antibodies, with the half-maximal inhibition between 1 and 10 ppm. Similar to Fig. 8, the binding behavior of the monoclonal antibody in a free modified histamine competition assay is shown in FIG. 9.

The half-maximal inhibition for the five antibodies tested here is between 10 and 30 ppb.

The modification of the histamine by the modifying reagent 2-iminothiolane takes place in a few minutes, as can be seen from the curves shown in FIG. 10, which for the antibody from FIGS. 8 and 9 show the binding behavior in a competition assay with free, modified Histamine, the circles corresponding to an incubation time of 5 minutes and the triangles corresponding to an incubation time of 10 minutes. The longer incubation of the histamine with the modifying reagent did not increase the sensitivity. These experiments show that an incubation period of a few minutes is sufficient to almost completely modify the histamine present in a sample with 2-iminothiolane.

This period of time can be ensured that either the length of the zone 74 is selected accordingly, so that during the capillary flow the discussed reactions can take place, or that the zones 69, 72 and possibly 73 are provided with a sugar glaze , as it is known from the aforementioned EP 291 194 B1. This glaze prevents immediate entry of the liquid into the porous support 68, thus delaying the test period, leaving enough time for modification of the free histamine.

Variant C

This test setup differs from variant B in that streptavidin is immobilized in the detection zone 75, while in the zone 72 biotin-modified histamine is introduced.

Zone 73 rat anti-histamine conjugate binds in zone 75 only when there is no or as little free histamine in the applied liquid sample it is present that sufficient biotin-modified histamine can bind to the rat anti-histamine conjugates in order to concentrate them so far in the detection zone 75, that the colored line 79 is formed.

On the other hand, if sufficient free histamine is present in the liquid sample, the rat anti-histamine conjugates are saturated by the free histamine so that no binding of the rat anti-histamine conjugates in sufficient to form a colored line 79 in the detection zone 75 can form.

In either case, excess or all of the rat anti-histamine conjugate migrates to control zone 77 where the rat anti-histamine conjugates are bound by the anti-rat IgG resulting in colored line 81 leads.

Variant D

In this embodiment, the labeled binding reagent presented in zone 73 comprises a histamine-protein conjugate bound to particulate tracers, particularly colloidal gold.

In the detection zone 75 rat anti-histamine antibodies are immobilized, in the control zone 77 again anti-rat IgG.

If no histamine is present in the applied liquid sample, the labeled histamine-protein conjugates bind in both detection zone 75 and control zone 77. This results in the formation of colored lines 79 and 81.

If, on the other hand, free histamine is present in the applied liquid sample, the rat anti-histamine antibodies in the detection zone 75 are saturated by the free histamine so that no or only a few labeled histamine-protein conjugates can bind there. In this case, only the colored line 81 is formed in the control zone 77. Variant E

Variation E differs from variant D in that 2-iminothiolane is introduced in zone 72, which leads to a higher sensitivity of the detection test, as has already been described above in connection with variant B.

Claims

claims 1 . A test device for testing a food (51) for an analyte, having a receiving space (38) for a sample (52) of the food (51) to be examined, a reservoir (64) containing a running buffer, the testing device (10) for Comminuting and mixing the sample (52) received in the receiving space (38) with the running buffer, and with a receptacle (62) for a test strip (67) on which a visually recognizable signal (79 , 81) indicating whether the analyte is detectable in the sample (52), wherein the testing device (10) is adapted to apply the comminuted and mixed with the running buffer, liquid sample on the test strip (67). 2. Test device according to claim 1, characterized in that the test strip (67) has a porous support (68), to which in a first zone (73) a labeled, specific for the analyte binding reagent and in a detection zone (75) an immobilized Binder are arranged such that the liquid sample applied to the porous support (78) receives the labeled binding reagent and migrates therewith downstream to the detection zone, where an optically detectable signal (79) is generated indicating whether the analyte in the Sample (52) is detectable. 3. Test device according to claim 1 or 2, characterized in that it has a  Base body (1 1) and one on the base body (1 1) attachable cap (12), wherein the receiving space (38) provided in the cap (12) and on the base body (1 1) a plunger (27) is arranged, which in the attached cap (12) protrudes into the receiving space (38) or is hineinbewegbar and is operable such that in the receiving space (38) located sample (52) is crushed and mixed with the running buffer. 4. Test device according to claim 3, characterized in that the reservoir (64) provided with the running buffer in the receiving space (38) and by a film (63) is completed, which is pierced when placing the cap (12) and / or when crushing the sample (52). 5. Test device according to one of claims 1 to 4, characterized in that a modifying reagent is provided for the analyte. 6. Test device according to claim 5, characterized in that the modifying reagent is provided in the receiving space. 7. Test device according to one of claims 4 to 6, characterized in that the reservoir is formed by in the receiving space (38) mounted beads (65), in which the running buffer and / or the modifying reagent are included, and in the comminution of the sample (52) are crushed. 8. Test device according to one of claims 3 to 7, characterized in that on the base body (1 1) an actuating knob (17) is provided, via which the plunger (27) in its longitudinal direction (19) relative to the base body (1 1) is displaceable. 9. Test device according to claim 8, characterized in that between the plunger (27) and the actuating knob (17) a rotary mechanism (29; 32) is provided, which converts a longitudinal movement of the actuating knob (17) in a rotary movement of the plunger (27). 10. Test device according to claim 9, characterized in that the rotary mechanism (29;  32), the longitudinal movement of the actuating knob (17) then in the rotational movement of the plunger (27) converts when the actuating knob (17) in the longitudinal direction (19) moves to the plunger (27). 1 1. Test device according to one of claims 1 to 10, characterized in that the receptacle (62) for the test strip (67) on the base body (1 1) is arranged. 12. Test device according to one of claims 1 to 10, characterized in that the receptacle (62) for the test strip (67) on the cap (12) is arranged. 13. Testing device according to one of claims 1 to 12, characterized in that the receptacle (62) for the test strip (67) via at least one opening (61) with the receiving space (38) is connected, which via a removable seal against the receiving space (38) is closed. 14. Test device according to claim 13, characterized in that the opening (61) via a filter (61 a) with the receiving space (38) is connected. 15. Test device according to one of claims 3 to 14, characterized in that on the base body (1 1) a punching sleeve (22) for taking a sample (52) of the food (51) and for transferring the sample (52) into the receiving space (38) is arranged. 16. Test device according to claim 15 and one of claims 8 to 14, characterized in that the plunger (27) in the punching sleeve (22) is arranged. 17. A test device according to claim 15 or 16, characterized in that the receptacle (62) for the test strip (67) on the cap (12) and at least one opening (61) in the cap (12) is provided which the receiving space (38) connects to the receptacle (62) for the test strip (67), and the punching sleeve (22) has a cylindrical jacket (55) which closes the at least one opening (61) when the cap (12) is fitted. 18. Test device according to claim 17, characterized in that the punching sleeve (22) is longitudinally displaceably mounted on the base body (1 1), in its extended basic position with its front cylindrical shell (55) covering at least one opening (61), and in their retracted position, the at least one opening (61) releases. 19. Test device according to claim 17 or 18, characterized in that the punching sleeve (22) is held in the basic position by means of a push-on lock (44; 20. Test device according to one of claims 17 to 19, characterized in that the plunger (22) via a shaft (26) with the actuating knob (17) is connected, via a sealing ring (41) inside against the punching sleeve (22) sealed and with the shaft (26) is lockable, so that the liquid sample when advancing the plunger (22) and released opening (61) by in the punching sleeve (22) generated overpressure on the test strip (67) is abandoned. 21. Test device according to one of claims 17 to 20, characterized in that the cap has lateral recesses (66) through which the punching sleeve (22) with an attached cap (12) gripped by an operator and in the direction of the base body (1 1) are moved can. 22. Test device according to one of claims 1 to 21, characterized in that the analyte is histamine. 23. The test strip for the test device (10) according to any one of claims 1 to 22, with a porous support (68) on which in a first zone (73) a labeled binding reagent specific for the analyte and in a detection zone (75) immobilized binder such that the liquid sample applied to the porous support (68) receives the labeled binding reagent and travels therewith downstream to the detection zone (75) where an optically detectable signal (79) is generated indicating whether the analyte in the sample (52) is detectable. 24. Test strip according to claim 23, characterized in that the marked Binding reagent for the analyte comprises antibodies bound to particulate tracers and that the immobilized in the detection zone (75) binder has the analytes. 25. Test strip according to claim 23, characterized in that the labeled binding reagent comprises the analyte bound to particulate tracers and that the immobilized in the detection zone binder for the analyte has specific antibodies. 26. Test strip according to claim 24 or 25, characterized in that a modifying reagent for the analyte is provided on the support (68). 27. Test strip according to claim 26, characterized in that the modifying reagent is 2-iminothiolane. 28. Test strip according to claim 23, characterized in that the marked  Binding reagent for the analyte specific antibodies which are bound to particulate tracers, provided on the porous support biotin-bound analyte, and binds in the detection zone (75) immobilized binder to biotin. 29. Test strip according to one of claims 23 to 28, characterized in that downstream of the detection zone (75) on the porous support (68) a control zone (77) is arranged, in which a binder for the labeled binding reagent is immobilized. 30. cap for the test device (10) according to one of claims 1 to 22, characterized in that in the cap a running buffer containing reservoir (64) is arranged. 31. Cap according to claim 30, characterized in that a reservoir (64) containing the modifying reagent is arranged in it. A cap according to claim 30 or 31, characterized in that the or each reservoir (64) is closed by a foil (63). Cap according to one of claims 30 to 32, characterized in that the or each reservoir comprises beads (65) in which the running buffer and / or the modifying reagent are enclosed. Basic body for a test device according to one of claims 1 to 22. Test device according to one of claims 1 to 22 with a test strip according to one of claims 23 to 29 and a cap according to one of claims 30 to 33.