JP4621583B2 - Protein test body, production method thereof, and protein detection apparatus using protein test body - Google Patents

Description

  The present invention relates to a protein test body for detecting a protein, a method for producing the same, and a protein detection apparatus using the protein test body.

  In recent years, hay fever patients and food allergy patients are rapidly increasing in Japan. In particular, with regard to food allergies, it is said that nearly 10% of the total population in Japan is allergic to some foods, and it tends to increase further. Food allergies can cause anaphylactic shock such as dyspnea, decreased blood pressure, and loss of consciousness in some cases, and there is a risk of death if the response is delayed. For these reasons, food safety is strongly demanded for food allergies.

  Under these circumstances, the Food Sanitation Law Enforcement Regulations were amended to prevent health hazards caused by foods containing allergens (allergens), and in April 2002, allergens were given to food manufacturers. Regardless of the amount of food contained, all food used during processing and production must be labeled. As the allergic substances for which labeling is mandatory, five specific raw materials (eg, egg, milk, wheat, buckwheat, peanut) are designated in particular because of the large number of allergic cases and the severity of the symptoms.

  As an allergen detection method, an immunoassay (immunological assay) has been conventionally used. In this method, a specific protein is specifically identified from a mixture of various substances by specifically binding a protein such as an allergen (antigen) and a substance (antibody) that specifically binds to the protein. Immunoassays are widely used in determining whether foods contain allergen proteins.

  An immunochromatographic method has been proposed as a method for simply performing an analysis using an immunoassay (an immunological measurement method) (see, for example, Patent Document 1).

JP 11-326325 A

  In the technique according to Patent Document 1, one is fixed to a base material that can be chromatographed, and the other is bonded to colored fine particles and is held on the base material in a form that can be dissolved in front of chromatographic development. An immunoassay comprising an antibody pair is characterized in that the detection part is formed of a plurality of detection bands separated along the rear side of the chromatographic development.

  According to this technique, since it has a plurality of detection bands, the antigen-containing liquid is chromatographed to produce a labeled antigen (an antibody having colored fine particles bound to an antigen) and an antibody immobilized on the substrate. It is said that the amount of the antigen dissolved in the test solution can be known from the position of the detection band colored by binding.

  In this technique, an immunological measurement method is used. In this method, a specific protein (antigen) is injected into a living body such as a rat or mouse, and an antibody against the antigen is formed in the living body. It is necessary to separate, extract and purify the antibody. Therefore, there is a problem that it takes a long time to produce an antibody.

  In addition, since antibodies must be generated using a living body, it is complicated and expensive to raise and manage living bodies (rats, mice, etc.). Furthermore, the antibody thus obtained is easily denatured by heat, acid or alkali, and may be deactivated by long-term storage, resulting in loss of recognition ability.

  For this reason, it is desired to develop a protein test body that can specifically recognize a specific protein as much as or more than an antibody, is easy to manufacture, and has good storage stability.

  The present invention has been made to solve the above-described problems existing in the prior art, and an object of the present invention is to use a protein recognition structure (imprint polymer) synthesized by a molecular imprint method using a protein as a template. Thus, by providing a protein test body that does not use an antibody as in the past, a production method that can easily produce such a protein test body, and a protein detection device using such a protein test body is there.

First invention according to the protein inspected for solving the above problems is configured as follows. A protein recognition structure formed by molecular imprinting using a protein as a template is a protein test body in which a plurality of solutions containing proteins are arranged on a substrate that can move by capillary action, and the substrate includes a plurality of protein recognition structures. A protein recognition structure that recognizes a specific type of protein is formed and arranged in each of the partition areas, and the protein recognition structure is formed on the surface and inside of the partition area. It is characterized by.

According to this configuration, unlike the conventional case, since a protein recognition structure that is molecularly imprinted instead of an antibody is used, a protein test body that is simple and inexpensive to manufacture and that has good storage stability can be produced. Furthermore, in this configuration, the substrate is partitioned into a plurality of divided areas, each divided area each, protein recognition structures (imprinted polymer) is arranged capable of recognizing each specific type of protein Therefore, a specific type of protein contained in the sample solution can be separated by a simple operation of dropping the sample solution containing the protein onto the substrate . Therefore, it is possible to easily detect a specific type of protein by incorporating this protein test body into a detection apparatus.
Furthermore, in this configuration, since the protein recognition structure is formed on the surface and inside of the base material, the bond between the base material and the protein recognition structure becomes stronger and the capillary action acts in the base material. Since the sample solution passes through, the recognition reaction between the protein in the sample solution and the protein recognition structure is naturally performed. Therefore, with this configuration, the protein separation sensitivity can be increased.

  The specific type of protein is a concept including both a single protein whose type is specified and a plurality of proteins whose types are specified.

  In order to solve the above-mentioned problem, the second invention according to the protein test object is the first invention, wherein the protein test object recognizes different types of proteins for each of the partition regions of the base material. Different protein recognition structures are arranged.

  According to this configuration, the presence of a plurality of proteins contained in the sample solution can be separated and specified by a single operation.

  Here, the meaning of the different protein recognition structures will be described. For example, a protein recognition structure that recognizes protein A and a protein recognition structure that recognizes protein B are different protein recognition structures. The protein recognition structure that recognizes protein A and protein B and the protein recognition structure that recognizes protein C and protein D are different protein recognition structures. Furthermore, a protein recognition structure that recognizes protein A and protein B and a protein recognition structure that recognizes protein A and protein C are also treated as different protein recognition structures.

In order to solve the above problems, a third invention relating to a protein test object is characterized in that a protein recognition structure formed by molecular imprinting using a protein group consisting of a plurality of proteins contained in a specific kind of food raw material as a template is a protein. Is a protein test body in which a plurality of solutions are arranged on a base material movable by capillary action, and the base material is partitioned into a plurality of partition regions, and for each of the partition regions, A protein recognition structure for recognizing a protein group contained in different types of food raw materials is formed and arranged, and the protein recognition structure is formed on the surface and inside of the partition region .

  According to this structure, the presence or absence of the food raw material contained in the sample solution can be separated and specified by a single operation.

  In the configurations of the first to third inventions, the base material has a disk shape, and the plurality of partition regions are partitioned radially from the center of the base material toward the outer direction of the circle, and the partitioning is performed. The protein recognition structures can be concentrically arranged in the plurality of partitioned regions.

  According to this configuration, when the sample solution is dropped at the center of the circle, the sample solution moves concentrically within the base material and contacts the protein recognition structure disposed in each partition region of the base material at the same time. . Therefore, since a plurality of proteins can be separated more easily by one sample dropping, the examination time can be shortened and the operation can be simplified.

  The said structure WHEREIN: Except for the center vicinity of the said base material, the said some division area can be set as the structure divided by the groove | channel formed radially toward the outward direction of a circle | round | yen.

  According to this configuration, protein recognition structures that recognize different types of proteins can be prevented from intermingling with each other. Therefore, protein separation accuracy is improved.

  The said structure WHEREIN: The said base material can be set as the structure which has the alignment part formed by notching the part.

  According to this configuration, the cut-out portion can be used for alignment for installing the protein test object in a detection apparatus described later, and alignment during installation on the detection apparatus becomes easier.

  In the first invention, the specific type of protein may be an allergen protein.

  In the third aspect, the food raw material may be any one of the food raw materials selected from the group consisting of eggs, milk, wheat, buckwheat, and peanuts.

  The present invention according to the method for producing a protein test object for solving the above-described problem is that a prepolymer solution containing a functional monomer, a cross-linking agent, and a specific type of protein as a template is converted into a solution containing the protein. A polymerizing step that is placed on a substrate that can be moved by capillary action and polymerized, and a polymer that is polymerized by the polymerizing step is brought into contact with a solution that can dissolve the specific type of protein. A template removing step of removing the protein to produce a protein recognition structure.

  According to this configuration, since the protein recognition structure can be arranged on the substrate simultaneously with the formation of the protein recognition structure (imprint polymer), the productivity is excellent.

  The said structure WHEREIN: The said specific type protein can be set as the structure which is a some protein group contained in a specific type food raw material.

  In the above-described configuration, the polymerization step arranges a prepolymer solution containing a functional monomer, a cross-linking agent, and a specific type of protein as a template on a substrate on which the protein-containing solution can move by capillary action. And it can be set as the structure which is the process of superposing | polymerizing a prepolymer, after making the said prepolymer solution osmose | permeate in a base material.

  According to this configuration, the protein recognition structure is formed inside and on the surface of the substrate, and the bond between the substrate and the protein recognition structure can be made stronger. In addition, simply dropping the sample solution onto the substrate causes the sample solution to move by capillary action and reliably contact and react with the protein recognition structure formed inside the substrate, thus improving the separation accuracy.

  In the above configuration, the base material has a disk shape, has a plurality of grooves formed radially outward except for the vicinity of the center of the base material, and is an area partitioned by two of the grooves. The prepolymer solution containing different types of proteins is placed in each of the compartment regions, and after the prepolymer solution has permeated, the polymerization step and the template removal step subsequent thereto are performed. Can do.

  According to this structure, since a groove | channel exists between each division area | region, the prepolymer solution containing a different kind of protein does not cross. Moreover, by making the base material into a disk shape, a plurality of proteins can be separated and specified simultaneously by dropping the sample solution onto the center of the base material.

  In order to solve the above-described problems, a first invention according to a protein detection apparatus includes an installation unit in which a protein test body is installed, and a protein test body according to any one of the first to third inventions installed in the installation unit. And a detector for detecting whether or not a protein is present in each compartment region of the protein test object.

  The said structure WHEREIN: The said protein detection apparatus can be set as the structure provided with the number of detectors corresponding to the number of division areas which the said protein test body has, and detecting simultaneously the presence or absence of the protein in each division area. .

  According to this configuration, since the number of detectors corresponding to the number of partition regions of the protein test object is provided, detection can be performed in parallel on a plurality of protein recognition bodies included in the protein test object. Time can be shortened.

  In order to solve the above problems, a second invention relating to a protein detection apparatus includes: an installation unit provided with a rotation unit that rotationally moves a protein test object; and a protein having a disk-like base material installed in the installation unit A protein detection apparatus comprising: a test body; and a detector that detects whether or not a protein is present in each compartment of the protein test body, wherein the protein test body is rotated by the rotating means, The presence or absence of the protein in each compartment area | region of a protein test body is detected sequentially.

  According to this configuration, it is possible to sequentially align the partition area and the detector by rotation. Therefore, protein can be detected efficiently.

  In order to solve the above problems, a third invention according to the protein detection apparatus is provided with an installation part having a projection part for aligning the protein test body, and a position where the notch part is fitted to the projection part of the installation part. It is characterized by comprising a protein test body having a notch and a detector that detects whether or not a protein is present in each compartment area of the protein test body.

  According to this configuration, since the detector can be easily and accurately arranged on the partition region where the protein recognition structure is arranged, the usability of the apparatus is improved and the detection accuracy is also improved.

  In the first to third inventions, the detector may have an optical detection means.

  There are various types of protein detection methods, but the optical detection means is the simplest, and the apparatus can be simplified by using the optical detection means.

  ADVANTAGE OF THE INVENTION According to this invention, the protein test body with easy operation which can isolate | separate and specify a specific kind protein easily and accurately from the solution containing a some protein or protein group is realizable. In addition, it is possible to provide a remarkable effect that it is possible to provide a protein detection apparatus that is easy to use and excellent in detection accuracy.

  The best mode for carrying out the present invention will be described below with reference to the drawings.

(Embodiment 1)
In the first embodiment, a protein test object and a manufacturing method thereof will be described.
As shown in FIG. 1, the protein test object according to the first embodiment has four protein recognition structure partition regions for each protein recognition structure that recognizes different types of proteins on a substrate 1 on a rectangular flat plate. 4a-4d are formed, and the groove | channel 2 by which a part of base material was notched is formed between the adjacent division area | regions. In each compartment, protein recognition structures 5a to 5d that recognize different types of proteins are arranged.

  The substrate is selected from materials suitable for allowing a sample solution containing protein to permeate and develop by capillary action. Examples of such materials include water-absorbing materials such as filter paper, cellulose membranes, glass filters, nylon fibers, and polyethylene fibers.

  The base material 1 is provided with four partition regions 4a to 4d, and each partition region has a different type of allergen protein (cedar pollen, cypress pollen, ragweed pollen, camo grass pollen) as a template, A protein recognition structure (imprint polymer) obtained by molecular imprinting is disposed (5a to 5d).

(Producing method of protein recognition structure)
Next, a method for producing a protein recognition structure will be described.
In the molecular imprint method, a functional monomer that interacts with and polymerizes a protein as a template and a crosslinking agent that is a crosslinkable monomer are polymerized in the presence of the template protein to remove the protein, A protein recognition structure (imprint polymer) is obtained by leaving a specific binding site (carboxyl group, amino group, etc.) complementary to the shape and protein.
The protein recognition structure does not simply take the form of a protein, but also includes positional information of functional groups such as carboxyl groups and amino groups present on the protein surface. As a result, a recognition action including interactions such as hydrogen bonding, ionic bonding, and hydrophobic bonding is performed between the functional group on the protein surface and the functional group in the protein recognition structure. Recognition is possible.

(Polymerization process)
For polymerization, a functional monomer and an allergen protein as a template are mixed in a polymerization solvent (solvent used in carrying out the polymerization reaction) to form a protein-functional monomer complex, and then a prepolymer is added with a crosslinking agent. A solution is formed, and this prepolymer solution is dropped at different positions 5a to 5d of the partition regions 4a to 4d formed on the substrate 1, and polymerization is performed.

  For the polymerization, thermal polymerization, photopolymerization by ultraviolet irradiation, or the like can be used. These may be used alone or in combination. The polymerization temperature at this time is preferably a temperature at which the protein is not thermally denatured, and specifically, it is preferably 4 ° C to 40 ° C.

  There is no limitation on the polymerization time. If the solution is gelled visually, it can be determined that the polymerization is complete. In general, the polymerization is completed in 0.5 to 48 hours.

(Mold removal process)
Thereafter, the protein as a template is removed from the polymer body obtained by polymerizing the prepolymer solution. As the method, the polymer body may be brought into contact with a solution that is soluble in protein. Examples of such a solution include a solution in which an acid or alkali is added to a pH different from that of the solvent during polymerization, and a solution in which a salt such as sodium chloride or ammonium sulfate is added to increase the salt concentration.

(Protein separation)
A sample solution containing allergen protein is dropped onto the sample solution dropping unit 7. Since the base material 1 is made of a material capable of moving the sample solution by capillary action, the sample solution moves through the base material 1 and recognizes and reacts with the protein recognition structures 5a to 5d provided in the partition regions 4a to 4d. . As a result, a specific type of protein reacts with the protein recognition structure and is captured in each compartment region, and the protein is separated.

Here, when a protein recognition structure (imprint polymer) is produced by polymerization, the polymerization solvent may be an inert solvent in the system. For example, an organic solvent such as chloroform, toluene, acetone, acetonitrile, tetrahydrofuran, various alcohols, or an aqueous solvent can be used. However, since the allergen protein may be denatured when it comes into contact with an organic solvent, the polymerization solvent is preferably an aqueous solvent.
In addition, since the surface charge of protein varies depending on the pH of the solvent, thereby changing the positional information of the functional group on the protein surface, it is preferable to keep the pH of the solvent constant. For this reason, the polymerization solvent is more preferably an aqueous buffer solution. Examples of such buffers include acetate buffer, citrate buffer, phosphate buffer, borate buffer, Tris-HCl buffer, HEPES (2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid ) Etc. can be used.

  As the functional monomer, a compound having a linking group capable of interacting with a protein and having a polymerizable vinyl group is preferably used. For example, acrylic acid, methacrylic acid, acrylamide, 2- (dimethylamino) ethyl methacrylate, hydroxyethyl methacrylate and the like can be mentioned.

  As the crosslinking agent, it is preferable to use a compound having at least two polymerizable vinyl groups in the molecule. For example, ethylene glycol dimethyl acrylate, N, N′-methylenebisacrylamide, divinylbenzene and the like can be mentioned.

A polymerization initiator may be added to the polymerization of the protein recognition structure. As the polymerization initiator, for example, a peroxide such as ammonium persulfate or potassium persulfate, or an azo polymerization initiator such as azobisisobutyronitrile can be used.
Further, a polymerization accelerator such as N, N, N ′, N′-tetramethylethylenediamine may be added.

(Embodiment 2)
As shown in FIG. 2, the protein test object according to the second embodiment includes five protein recognition structure partition regions 4 a for each protein recognition structure that recognizes different types of proteins on a disc-shaped substrate 1. -4e are formed, and each partition region is partitioned by the groove 2 in which a part of the base material is cut out except for the region 6 near the center of the circle. Moreover, the notch part 3 used for the alignment for installing in the protein detection apparatus mentioned later is formed in parts other than protein recognition structure division area | region 4a-4e and the center vicinity area | region 6. FIG.

(Producing method of protein recognition structure)
In the present embodiment, as a method of placing a protein recognition structure (imprint polymer) on a substrate to which a protein-containing solution can move by capillary action, the prepolymer solution is sufficiently immersed in the substrate. A polymerization method is employed. When this method is employed, since the polymer is formed by cross-linking polymerization with the prepolymer entering the base material, the protein recognition structure (imprint polymer) is firmly fixed on the base material. Further, when the sample solution passes through the substrate, the protein in the sample solution and the protein recognition structure are surely recognized and reacted, so that the protein separation / specific performance can be improved.

  In addition, since the protein recognition structure can be synthesized and the protein recognition structure can be placed and immobilized on the substrate at the same time, antibody production and antibody immobilization can be achieved as in the case of a protein test body using conventional antibodies. Need not be done separately. Therefore, the production time of the protein test object can be greatly shortened.

  In order to perform polymer polymerization in the substrate, a prepolymer solution in which functional monomers necessary for polymer polymerization, a crosslinking agent, a protein serving as a template, and the like are mixed is added to the substrate 1 and then polymerized. . At this time, in order to be able to detect a plurality of proteins on a single substrate, prepolymer solutions containing different types of proteins are respectively dropped onto positions 5a to 5e in different compartments of the single substrate. Then, after sufficiently soaking, polymerize.

  In addition, since the base material is solution-absorbing, the added prepolymer solution may permeate and spread in the base material, and may be mixed with the prepolymer solution having a different template added to the adjacent partition region. . In order to prevent this phenomenon, it is preferable to form a groove 2 in the base material between the adjacent partition regions so that the prepolymer solution does not spread into the adjacent partition region. The groove 2 may have a structure formed on the surface of the base material, but in order to prevent the prepolymer solution from spreading into the adjacent compartment area, the base material 1 is notched to completely separate each compartment. It is more preferable to employ a configuration that separates the regions. At this time, a groove 2 is formed between adjacent partition regions, but a groove is formed between each partition region 4a to 4e and the central vicinity region 6 so that the sample solution reaches each partition region by development. Do not.

In the second embodiment, the circular flat plate-shaped (disk-shaped) base material 1 is used. However, the present invention is not limited to this shape, and may be any shape that does not hinder the development of the sample liquid. For example, the rectangle used in the first embodiment may be used, and other polygons, ellipses, and indeterminate shapes may be used.
However, when protein recognition structures are arranged concentrically using a circular base material and the sample solution is dropped at the center of the circle, the sample solution that has developed concentrically from the dropping point differs in protein recognition structure. Since it reacts simultaneously, efficiency increases. Therefore, it is preferable that the shape of the base material 1 is a disk shape.

(Embodiment 3)
In Embodiment 3, a protein test body using a protein group contained in a specific food raw material as a template and a method for producing the same will be described. Since the shape and the like of the protein test object are all the same as those in the second embodiment, the description thereof is omitted.

(Producing method of protein recognition structure)
In the present embodiment, for example, a plurality of proteins contained in one kind of food raw material are used as one protein group as one template. First, a food raw material is ground, mixed with a buffer solution, stirred, allowed to stand for a while, and then filtered to extract a plurality of types of proteins contained in the food raw material into the filtrate. This filtrate is mixed with a functional monomer and a crosslinking agent to form a prepolymer solution. Subsequent polymerization and template removal are the same as in the second embodiment.

(Embodiment 4)
Next, the detection device will be described.
As shown in FIG. 3, the detection apparatus according to the present embodiment includes a protein test body setting unit 13, a protein test body 12 produced in the second embodiment, and the number of partition regions of the protein test body ( And five detectors 11 corresponding to five).

  The detector 11 is arranged at a position where the protein recognition structure arrangement unit 10 in a plurality of partitioned regions in the protein test body 12 can be detected. In the present embodiment, the detector has an optical detection means, and detects an optical change in the protein recognition structure arrangement unit 10. Examples of such optical detection means include a photodiode array and a CCD camera.

  In such a detection apparatus, it is necessary to arrange the protein recognition structure arranging unit 10 at a position that can be detected by the detector 11. For this purpose, as shown in FIG. 2, a part of the base material 1 is cut out and a cutout portion 3 is formed in the protein test body. In the detection device, as shown in FIG. 4, a protrusion 24 is formed on the protein test body setting portion 23 at a position matching the notch. And if it installs so that the notch part of the protein test body 22 and the projection part 24 may be match | combined, position alignment with the detector 11 and the protein recognition structure arrangement | positioning part 10 can be performed easily.

As the detection method, a so-called sandwich method can be used.
In the sandwich method, first, a target substance (allergen protein) and a recognition substance (an antibody, a protein recognition structure, etc.) attached with a labeling substance are reacted to form a target substance-recognition substance complex. A sample solution containing this complex is dropped onto the center of the circle of the protein test object shown in FIG.
The sample solution moves in the base material 1 by capillary action, and binds to the protein recognition structure fixed to the protein recognition structure arrangement portions 5a to 5e by a specific recognition reaction. Thereby, a target substance is fixed to the protein recognition structure area | region of a base material. Thereafter, the presence and amount of the target substance are measured by detecting the amount of the labeling substance using the detector 11.

  Examples of the labeling substance include dyes, fluorescent substances, and radioisotopes. As a technique for attaching a labeling substance to the recognition substance, physical adsorption or a technique of chemically bonding to a functional group on the antibody surface can be used.

  When the recognition substance is a protein recognition structure, polymerization may be performed in a state where a dye or a fluorescent substance is mixed as a labeling substance at the time of preparing the protein recognition structure. Thereby, the process of attaching the labeling substance can be omitted.

  The present invention will be further described below using examples.

Example 1
In this example, a protein test body that recognizes allergen proteins contained in eggs, milk, wheat, buckwheat, and peanuts was prepared and used for protein detection.
As shown in FIG. 2, circular filter paper (manufactured by Advantech, diameter 110 mm) was used as the base material 1, and grooves 2 were formed radially from the center of the filter paper. Moreover, the notch part 3 of 1 cm x 1 cm was provided.

A protein recognition structure (imprint polymer) that recognizes an egg allergen was prepared as follows.
100 g of eggs were taken, ground and suspended in 100 ml of 50 mM Tris-HCl buffer (pH 8.0). After filtration of the suspension, 6 ml of the filtrate was taken, 7 μl of acrylic acid was added as a functional monomer, mixed well, and allowed to stand for 1 hour to form an egg allergen-functional monomer complex.

  To this solution, 22 mg of acrylamide and 154 mg of methylenebisacrylamide as a cross-linking agent were added and mixed well, and nitrogen substitution was performed for 5 minutes. Thereafter, 100 μl of a 5% potassium persulfate aqueous solution as a polymerization initiator and 10 μl of N, N, N ′, N′-tetramethylethylenediamine as a polymerization accelerator were added and mixed well, and 100 μl was dropped on a filter paper as a base material. This dropping position was dropped at a position 2 cm from the end (outer periphery) of the filter paper.

Immediately after the addition, it was placed in a constant temperature bath at 36 ° C. and thermal polymerization was carried out for 2 hours.
Thereafter, the polymer body after polymerization was washed three times with a 1 mM sodium chloride solution to remove the protein as a template to obtain a protein recognition structure.
The protein recognition structure thus prepared can recognize a protein group (egg albumin, lysozyme, ovomucoid) contained in an egg.

In the same manner as the above eggs, for milk, wheat, buckwheat, and peanuts, an imprint polymer (protein recognition structure) was synthesized in another compartment on the same filter paper to obtain a protein test body shown in FIG. In this protein test body, eggs are shown in the partition area indicated by reference numeral 4a in FIG. 1, milk is displayed in the partition area indicated by reference numeral 4b, wheat is displayed in the partition area indicated by reference numeral 4c, soba is adjacent to the partition area indicated by reference numeral 4d, A peanut protein structure was placed in the region (5a-5e).
The protein recognition structure produced in this way contains a protein group (casein, lactalbumin, lactoferrin), etc. contained in milk, and a protein group (gliadin, glutenin, wheat albumin), etc., contained in wheat. Protein groups (buckwheat globulin, buckwheat albumin) and the like, and protein groups (Arah1, Arah2, peanut lectin) and the like contained in peanuts, respectively.
In addition, the protein recognition structure can recognize and separate proteins contained in approximately 10% or more of the total amount of proteins contained in each food raw material.

A recognition substance with a labeling substance was prepared as follows.
100 g of eggs were taken, ground and suspended in 100 ml of 50 mM Tris-HCl buffer (pH 8.0). After filtration of the suspension, 6 ml of the filtrate was taken, 7 μl of acrylic acid as a functional monomer was added thereto, mixed well, and allowed to stand for 1 hour to form an allergen / functional monomer complex. To the solution, 22 mg of acrylamide, 154 mg of methylenebisacrylamide as a cross-linking agent, and 100 mg of red dye (alizarin) were added and mixed well. This solution was transferred to a separable flask and purged with nitrogen for 5 minutes. While stirring at a speed of 120 revolutions per minute with a mechanical stirrer, 34 mg of 2,2′-azobis (2-methylpropionamidine) dihydrochloride as a polymerization initiator dissolved in 1 ml of distilled water was added. Subsequently, the mixture was polymerized by irradiation with ultraviolet rays at 4 ° C. for 16 hours while stirring. After polymerization, the obtained red protein recognition structure fine particles were washed three times with a 1 mM sodium chloride solution to remove the protein as a template.
Similarly, protein recognition structure fine particles containing red pigment as a labeling substance were synthesized for milk, wheat, buckwheat and peanuts.

The measurement was performed as follows.
A 20 μM ovalbumin solution was prepared as a sample solution, and the above recognition substance was added thereto and mixed for 1 hour. 500 μl of this solution was taken and dropped at the center of the protein test specimen. When left for 30 minutes, the sample solution moved in the substrate by capillary action. After that, the protein test body shown in FIG. 1 is installed in the detection device shown in FIG. 4 by fitting the notch 3 to the protrusion 24 and aligning them, and the protein recognition structure arrangement parts 5a to 5e are arranged. The red color state was measured. The results are shown in Table 1 below.

  The coloration was evaluated as +; colored,-; not colored. Only the egg protein recognition structure arrangement part was colored red, and the presence or absence of the allergen contained in the sample could be detected.

(Example 2)
As a sample solution, a mixed solution of 20 μM casein (milk protein) and 20 μM gliadin (wheat protein) was prepared, and the above recognition substance was added thereto and mixed for 1 hour. 500 μl of this solution was taken and dropped at the center of the protein test specimen. When left for 30 minutes, the sample solution moved in the substrate by capillary action. Then, the protein test body was installed in the detection apparatus shown in FIG. 4, and the red coloration state of each protein recognition structure arrangement | positioning part was measured. The results are shown in Table 2 below.

  From the said Table 2, the milk protein recognition structure arrangement | positioning part and the wheat protein recognition structure arrangement | positioning part are colored red, The presence or absence of the multiple types of allergen contained in a sample was able to be detected.

(Example 3)
As a sample solution, a mixed solution of 30 μM ovalbumin, 10 μM casein, and 10 μM gliadin was prepared, and the above recognition substance was added thereto and mixed for 1 hour. 500 μl of this solution was taken and dropped at the center of the protein test specimen. When left for 30 minutes, the sample solution moved in the substrate by capillary action. Then, the protein test body was installed in the detection apparatus shown in FIG. 4, and the red coloration state of each protein recognition structure arrangement | positioning part was measured. The results are shown in Table 3 below.

The coloration was evaluated as ++; strong coloration, +; weak coloration,-; no coloration.
From Table 3 above, the egg protein recognition structure arrangement part is strongly colored in red, the milk protein recognition structure arrangement part and the wheat protein recognition structure arrangement part are weakly colored in red, It was possible to detect not only the presence or absence of multiple types of allergens but also their amounts.

  In the above examples, an allergen test body was prepared using a food allergen protein. However, the present invention, in addition to food allergens, tests for allergen proteins contained in the air such as pollen and ticks, in vivo proteins, etc. It can also be applied to. Further, in order to detect only a specific protein, a protein recognition structure may be prepared using an isolated protein crystal and used as a protein test object.

As described above, according to the present invention, it is possible to provide a protein test body that can detect a plurality of types of proteins at the same time and has no fear of inactivation at low cost. Therefore, the industrial significance is great.

It is a schematic diagram which shows the protein test body concerning Embodiment 1. FIG. It is a schematic diagram which shows the protein test body concerning Embodiment 2. FIG. FIG. 6 is a schematic diagram illustrating a detector portion of a detection apparatus according to a fourth embodiment. It is a schematic diagram which shows the protein test body installation part of the detection apparatus concerning Embodiment 4.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Base material 2 Groove 3 Notch part 4 Protein recognition structure division area 5 Protein recognition structure arrangement | positioning part 6 Center vicinity area | region 7 Sample solution dripping part 10 Protein recognition structure arrangement | positioning part 11 Detector 12, 22 Protein test body 13, 23 Protein Specimen Installation Section 24 Projection

Claims (17)

The protein recognition structure formed by molecular imprinting using a protein as a template is a protein test body in which a plurality of solutions containing proteins are arranged on a substrate that can move by capillary action,
The base material is partitioned into a plurality of partition regions, and a protein recognition structure that recognizes a specific type of protein is formed and arranged for each of the partition regions. Formed on and inside the surface,
A protein test body characterized by the above. The protein test body according to claim 1,
In the protein test object, different protein recognition structures for recognizing different types of proteins are formed and arranged for each partition region of the base material,
A protein test body characterized by the above. A plurality of protein recognition structures, which are molecularly imprinted using a protein group consisting of a plurality of proteins contained in a specific kind of food raw material as a template, are arranged on a substrate on which a protein-containing solution can move by capillary action. A protein tester,
The base material is partitioned into a plurality of partition regions, and a protein recognition structure that recognizes a protein group contained in a different kind of food raw material is formed and arranged for each partition region, and the protein recognition structure The body is formed on and inside the compartment area,
A protein test body characterized by the above. In the protein test body according to claim 1, 2, or 3,
The substrate is disk-shaped,
The plurality of partitioned regions are partitioned radially from the center of the substrate toward the outer direction of the circle, and the protein recognition structures are concentrically formed and arranged in the partitioned plurality of partitioned regions. Yes,
A protein test body characterized by the above. The protein test body according to claim 4 ,
The plurality of partition regions are partitioned by grooves formed radially toward the outer direction of the circle, except for the region near the center of the base material.
A protein test body characterized by the above. The protein test body according to claim 4 ,
The base material has an alignment portion formed by cutting out a part of the base material,
A protein test body characterized by the above. The protein test body according to claim 1,
The specific type of protein is an allergen protein,
A protein test body characterized by the above. In the protein test body according to claim 3,
The food ingredient is any one kind of food ingredient selected from the group consisting of eggs, milk, wheat, buckwheat, peanuts,
A protein test body characterized by the above. A polymerization step in which a prepolymer solution containing a functional monomer, a cross-linking agent, and a specific type of protein as a template is placed on a substrate on which the protein-containing solution can move by capillary action and polymerized;
A template removing step of bringing the polymer polymerized by the polymerization step into contact with a solution capable of dissolving the protein, removing the specific type of protein from the polymer body, and producing a protein recognition structure;
A method for producing a protein test body, comprising: In the manufacturing method of the protein test body of Claim 9 ,
The specific type of protein is a plurality of protein groups contained in a specific type of food raw material.
A method for producing a protein test substance. In the manufacturing method of the protein test body of Claim 9 or 10 ,
In the polymerization step, a prepolymer solution containing a functional monomer, a cross-linking agent, and a specific type of protein as a template is disposed on a substrate on which the protein-containing solution can move by capillary action, A step of polymerizing the prepolymer after infiltrating the prepolymer solution inside,
A method for producing a protein test substance. In the manufacturing method of the protein test body of Claim 9 or 10 ,
The substrate is disk-shaped, except for the vicinity of the center of the substrate, and having a plurality of grooves formed radially outward.
A region partitioned by two of the grooves is defined as a partition region, a prepolymer solution containing different types of proteins is placed in each partition region, and after the prepolymer solution has permeated, the polymerization step and Performing the subsequent template removal step,
A method for producing a protein test substance. An installation section for installing a protein test body;
The protein test body according to any one of claims 1 to 3 installed in the installation section,
A detector for detecting whether a protein is present in each compartment region of the protein test object;
A protein detection apparatus comprising: In protein detection apparatus according to claim 1 3,
The protein detection device includes a number of detectors corresponding to the number of compartments included in the protein specimen, and detects the presence or absence of proteins in each compartment simultaneously.
The protein detection apparatus characterized by the above-mentioned. An installation section equipped with a rotating means for rotating the protein test body;
The protein test object according to claim 4 or 5 installed in the installation part,
A detector for detecting whether a protein is present in each compartment region of the protein test object;
A protein detection apparatus comprising:
A protein detection apparatus that sequentially detects the presence or absence of a protein in each compartment area of a protein test object by rotating the protein test object by the rotating means. An installation part having a protrusion for aligning the protein test object;
The protein test body according to claim 6 , wherein the notched portion is fitted and aligned with the protrusion of the installation portion, and installed.
A detector for detecting whether a protein is present in each compartment region of the protein test object;
A protein detection apparatus comprising: The protein detection device according to any one of claims 13 to 16 ,
The detector has an optical detection means;
The protein detection apparatus characterized by the above-mentioned.

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