JP2005069997A - Method for detecting allergen protein, detecting chip and detecting apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for repeatably detecting an allergen protein on a chip, the chip and an apparatus. <P>SOLUTION: The method for detecting the allergen protein is provided with an extraction process of extracting the allergen protein from particles taken from an external environment and providing it to a carrier liquid, and a complex creating step of making the carrier liquid containing the allergen protein flow into a reaction part in which a recognition material specifically reacting to the allergen protein are maintained that it does not flow, reacting the allergen protein and the recognition material and forming a recognition material-allergen protein complex. A method for introducing the extracting carrier liquid in the complex creating step is a method of making the extracting carrier liquid containing the allergen protein circulate and flow into the reaction part twice or more. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for detecting allergen protein, a chip for detecting allergen protein using this method, and such an apparatus.

  In recent years, airborne pollutants, mites, molds, and other suspended micro substances have a great influence on the human body as a cause of allergies. Among such allergies, in particular, so-called hay fever patients who cause allergies to pollen such as cedar and cypress are increasing year by year. Conventionally, as a means for measuring pollen particles that cause this hay fever, optically Various methods using the means have been proposed (see, for example, Patent Documents 1 to 5).

JP 7-43299 A (summary) JP 10-318908 A (summary) JP 2001-183284 A (summary, FIG. 3) JP 2001-264234 A (summary) JP 2002-82038 A (summary)

  However, the method described in the above-mentioned patent document simply measures the number of particles having the same particle size (around 50 microns) as pollen, and detects allergen proteins (antigens) that directly cause allergies. There was a problem of not doing.

  For this reason, an antigen detection method using an antigen-antibody reaction that specifically reacts with an antigen of pollen has been proposed. For example, Patent Document 6 discloses a collection process, a concentration / reaction process, and a detection process of suspended micro substances. And reacting with a fluorescent antibody label that reacts specifically with the floating minute substance in the reaction and detection process, and then irradiating the labeled floating minute substance with ultraviolet light and receiving the fluorescence emission to prevent allergy. Techniques have been proposed for detecting airborne particulate matter that is the cause.

JP-A-11-14511 (Summary)

  Patent Document 7 proposes an immunoassay microchip capable of performing an antigen-antibody reaction in the chip. Based on FIG. 10, the outline | summary of the apparatus of patent document 7 is demonstrated. A plurality of injection holes 61 for allowing an antigen, a labeled antibody (gold colloid-labeled antibody), and a buffer solution to flow into the chip on a single substrate 60, and a microchannel reaction tank 62 for causing an antigen-antibody reaction, respectively. In addition, a waste liquid part 63, a microchannel inflow part 64 that connects the plurality of injection holes and the microchannel reaction part, and a microchannel separation part 65 that connects the microchannel reaction part and the waste liquid part are provided.

  In this antigen detection method using a microchip, antigen and gold colloid-labeled antibody are injected together with a buffer solution from the injection hole, moved to the microchannel reaction section, and the antibody and antigen antibody previously flowed into the microchannel reaction section After the reaction has occurred, a new buffer solution is injected from the injection hole, unreacted substances are poured into the waste liquid part, and then the presence or amount of the antigen is examined by photothermal conversion analysis in the microchannel reaction part. It is.

JP 2001-4628 A (summary)

  Although the technique described in Patent Document 6 can capture and detect a plurality of types of allergens present in the atmosphere, the apparatus is complex as shown in FIG. 9, so that it is difficult to reduce the size. have.

  On the other hand, although the method described in Patent Document 7 can reduce the size of the apparatus, the antigen-antibody reaction is carried out while injecting a pre-extracted antigen and a labeled antibody into a microchip and standing still in the chip. As a result, the contact between the antigen and the antibody becomes insufficient, and the antigen-antibody reaction varies. Therefore, this technique can be used for qualitative determination of the presence / absence of an antigen, but has a problem of poor reproducibility for quantitative measurement. In addition, this technique detects an antigen using a single microchip. However, this method cannot easily and reliably detect an antigen in a suspended fine substance containing a plurality of different types of antigen substances. have.

  The present invention is intended to solve the above-mentioned problem, and its object is to provide an allergen protein detection method capable of reproducibly detecting an antigen in a suspended fine substance containing a plurality of antigen substances, It is another object of the present invention to provide an allergen detection chip capable of simply carrying out such a detection method, and an allergen detection apparatus incorporating the chip.

  1st invention concerning the detection method of allergen protein for solving the said subject contains the extraction step which extracts allergen protein in a carrier liquid from the allergen substance taken in into the extraction part from the external environment, and the said allergen protein An extraction carrier liquid is introduced into a reaction part that is held in a state where a recognition substance that specifically reacts with the allergen protein is not washed away, and the allergen protein and the recognition substance are reacted to form a recognition substance-allergen protein complex. A method of detecting an allergen protein comprising an allergen protein detection pretreatment step comprising: a complex generation step of generating a body, wherein the extraction carrier liquid introduction method in the complex generation step includes the allergen protein 2 carrier liquid into the reaction part Characterized in that it is a method for flowing circulating above.

  2nd invention concerning the detection method of allergen protein for solving the above-mentioned subject includes the extraction step which extracts allergen protein in the carrier liquid from the allergen substance taken into the extraction part from the external environment, and the allergen protein An extraction carrier liquid is introduced into a reaction part that is held in a state where a recognition substance that specifically reacts with the allergen protein is not washed away, and the allergen protein and the recognition substance are reacted to form a recognition substance-allergen protein complex. An allergen protein detection method comprising an allergen protein detection pretreatment step comprising: a complex generation step for generating a body, wherein a carrier liquid is circulated between the extraction unit and the reaction unit, thereby providing an allergen protein Extraction, allergen protein and recognition And performing repeated more than once in the same carrier liquid reaction and with.

  In the first or second invention relating to the method for detecting an allergen protein, after the complex generation step, a carrier liquid containing a recognition substance with a labeled substance is used instead of the carrier liquid containing the allergen protein. 2 or more times, a labeling substance reaction step for reacting the recognition substance with the labeling substance and the recognition substance-allergen protein complex in the reaction part can be provided.

  Moreover, it can be set as the structure further equipped with the washing | cleaning process which flows a carrier liquid between the said complex production | generation step and the said label | marker substance reaction step, and wash | cleans the said reaction part.

  3rd invention concerning the detection method of allergen protein for solving the above-mentioned subject includes the extraction step which extracts allergen protein in the carrier liquid from the allergen substance taken into the extraction part from the external environment, and the allergen protein A mixing step of mixing an extraction carrier liquid and a carrier liquid containing an allergen protein with a labeling substance whose content is known in advance into a mixed carrier liquid, and a recognition substance that reacts specifically with the allergen protein, The mixed carrier solution is introduced into a reaction part that is fixed in a state where the recognition substance whose amount is known does not flow out, and the allergen protein and the allergen protein with the labeling substance are competitive with the recognition substance. To the recognition substance-allergen protein complex and the recognition substance A complex production step for producing an allergen protein complex with a labeling substance, and an allergen protein detection pretreatment step comprising: a method for introducing the mixed carrier liquid in the complex production step. The method is characterized in that the mixed carrier liquid is circulated into the reaction section two or more times.

  In the third invention according to the method for detecting an allergen protein, the mixing in the mixing step is performed in the reaction unit, and the mixed carrier liquid mixed in the reaction unit is circulated into the reaction unit at least twice and flows into the reaction unit It can be set as the structure made to do.

  The fourth invention according to the allergen protein detection chip for solving the above-mentioned problem is that an introduction part having an introduction port for introducing a carrier liquid from the outside of the chip into the chip, and the carrier liquid can flow from the introduction part In this way, there is an opening that communicates with the introduction part and has an opening for taking in allergen-containing particles from the outside of the chip, and extracts the allergen protein from the allergen-containing particles into the carrier liquid, and the carrier liquid from the extraction part A reaction part that is communicated with the extraction part so as to be able to flow in and is held in a state in which a recognition substance that specifically reacts with allergen protein is not flowed out, and is located downstream of the reaction part, and a carrier liquid is disposed on the chip. An allergen detection chip formed on a single substrate includes a lead-out portion having a lead-out port for lead-out to the outside.

  Here, “one substrate” includes not only one substrate originally but also one in which two or more substrates are joined to form a substantially single substrate. “Communication” refers to a relationship in which liquids are circulated and includes both a directly connected state and a state in which another element such as a flow path is interposed.

  In the fourth aspect of the invention for the allergen protein detection chip, a circulation channel for circulating the carrier liquid may be provided between the introduction part and the lead-out part. .

  The labeling substance temporary storage part further includes a second introduction part and a labeling substance temporary storage part for temporarily storing a recognition substance with a labeling substance, and the labeling substance temporary storage part introduces a carrier liquid into the chip from outside the chip. The carrier part communicated with the second introduction part and also communicated with the reaction part so that the carrier liquid introduced from the second introduction part can flow into the reaction part.

  The apparatus further comprises a second introduction part and a labeling substance allergen protein temporary storage part for temporarily storing the labeling substance-attached allergen protein, and the labeling substance-added allergen protein temporary storage part is provided inside the chip from outside the chip. Communicating with the second introduction part for introducing the liquid and also communicating with the reaction part so that the carrier liquid introduced from the second introduction part can flow into the reaction part, and exists in the reaction part The amount of the recognition substance to be recognized can be configured in advance.

  In addition, a mixing unit that mixes the carrier liquid containing the allergen protein that has flowed out of the extraction unit and the labeled allergen protein that has flowed out of the temporary allergen protein storage unit with the labeled substance is between the extraction unit and the reaction unit. And it can be set as the structure which has between the said allergen protein temporary storage part with the said labeled | labeling substance, and the said reaction part.

  Moreover, it can be set as the structure which has the carrier part which flowed out from the said extraction part, and the dilution part diluted with the carrier liquid introduce | transduced from other than the said extraction part between the said extraction part and the said reaction part.

  Moreover, it can be set as the structure which has an opening / closing means which interrupts | blocks or allows the flow of a carrier liquid between the said mixing part and the said reaction part.

  Moreover, it can be set as the structure which has the opening / closing means which interrupts | blocks or allows the flow of a carrier liquid between the said dilution part and the said reaction part.

  Moreover, it can be set as the structure by which the separation means which removes the insoluble substance in a carrier liquid is provided between the said extraction part and the said reaction part.

  In addition, the separation means is a channel configured such that the minimum diameter of the channel existing between the extraction unit and the reaction unit is smaller than the maximum diameter of the insoluble substance existing in the carrier liquid. Can do.

  A fifth invention according to an allergen detection device for solving the above-mentioned problems includes any of the above-described allergen detection chips, and further a carrier liquid storage section for storing a carrier liquid, and a distribution for distributing the carrier liquid. A pipe group, a liquid feeding means for circulating the carrier liquid in the flow pipe group, and a detection means for detecting the amount and / or type of the allergen protein present in the reaction part of the allergen detection chip, The carrier liquid storage part, the liquid feeding means, the introduction part of the allergen detection chip, and the lead-out part of the allergen detection chip are connected by the flow pipe group, and the carrier liquid can circulate and flow. This is an allergen detection device in which a path is formed.

  In the fifth invention related to the allergen detection device, the allergen detection device further comprises a recognition substance temporary storage unit with a labeling substance for storing a recognition substance with a labeling substance that specifically reacts with the allergen protein. Can do.

  In addition, the allergen detection device may further include a labeled substance-recognized substance temporary storage unit that stores a known amount of labeled allergen protein in advance.

  Moreover, the said distribution piping group shall be provided with the connection means which can be connected with the introduction part and derivation | leading-out part of the said allergen detection apparatus so that attachment or detachment is possible respectively.

  In the first aspect of the present invention, the extraction carrier liquid containing the allergen protein is circulated into the reaction part at least twice, and this method holds the allergen protein in the extraction carrier liquid and the reaction part. Increases contact opportunities with recognized substances. Therefore, the allergen protein that could not be reacted by the first inflow also reacts by the subsequent repeated inflow, so that the reaction between the allergen protein and the recognition substance is sufficiently performed. As a result, since there is no detection variation due to the reactivity between the allergen protein and the recognition substance, qualitative and quantitative reproducibility is improved.

  In the second aspect of the present invention, the carrier liquid is circulated between the extraction part for extracting the allergen protein in the carrier liquid and the reaction part for reacting the allergen protein and the recognition substance. The extraction and the reaction of the allergen protein and the recognition substance are repeated twice or more in the same carrier solution. In this method, not only the reaction step in the reaction part but also the extraction step in the extraction part is repeated. Done. Therefore, the allergen protein is sufficiently extracted from the allergen substance, and the reaction between the extracted allergen protein and the recognition substance is also sufficiently performed, so the qualitative and quantitative reproducibility and detection accuracy of the allergen protein are remarkable. improves.

  In the third aspect of the present invention, a so-called competitive reaction is used. In this method as well, the mixed carrier liquid containing both the measurement antigen and the labeled antigen is circulated, so the chance of antigen-antibody reaction increases. As a result, the reproducibility and detection accuracy of detection are improved.

  In addition, since the allergen protein detection chip according to the fourth aspect of the present invention has a simple structure, for example, a microallergen protein detection chip of about 50 mm in length, about 30 mm in width, and about 2 mm in thickness can be realized. Since such a microchip is easy to be hermetically sealed, it has a remarkable effect such that a disposable chip capable of long-term storage can be realized at low cost. By using such an allergen protein detection chip, the above-described method for detecting an allergen protein according to the present invention can be easily carried out.

  In addition, the allergen detection device according to the fifth aspect of the present invention has an effect that the method for detecting an allergen protein according to the present invention can be easily carried out. This allergen detection apparatus has the detection chip according to the present invention as an essential element, but various types of allergen proteins can be obtained by a simple operation such as selecting the type of detection chip according to the measurement target at the time of measurement. Can be qualitatively and quantified with high reproducibility.

  The best mode for carrying out the present invention will be described with reference to the drawings, taking as an example allergen protein contained in cedar pollen.

[Embodiment 1]
FIG. 1 is a conceptual diagram showing an allergen detection chip and an allergen detection device using the same according to the first embodiment. As shown in FIG. 1, the allergen detection apparatus according to the first embodiment includes a detection chip 101 and an apparatus main body (portion excluding the chip) 201.

  The allergen detection chip 101 has an introduction part 41, a lead-out part 42, an extraction part 43, a reaction part 44 and flow paths 45, 46, 47 connecting them on the substrate 40. A separation portion 49 is provided between the two and 46. Here, for example, the introduction part 41 and the lead-out part 42 are holes having a diameter of 100 μm or more and a depth of 50 μm to 1 mm, and the extraction part 43 is a hole having a diameter of 100 μm or more and a depth of 50 μm to 1 mm. The shape of these holes (the shape viewed from the vertical direction of the substrate) may be any of a circle, a square, a rectangle, and the like.

  The separation unit 49 has a structure in which a plurality of cylinders are spaced with a gap of about 5 to 50 microns. The separation unit 49 has pollen particles, broken pollen shells, insoluble particles such as sand in the reaction unit 44. This is to prevent inflow. That is, when the carrier liquid flows from the extraction unit 43 to the flow path 46, pollen shells and the like are stopped by the pillars of the separation unit 49. Therefore, insoluble components such as pollen shells and soluble components such as antigens (allergen proteins) can be separated. Here, the gap of the separation portion 49 is preferably configured to be smaller than the maximum diameter of insoluble substances such as pollen shells and sand, and more preferably configured to be smaller than the minimum diameter of insoluble substances. .

  The reaction unit 44 is a site for reacting an allergen protein (antigen) and a recognition substance (antibody), and a plurality of beads 50 having a diameter of about 50 microns are arranged in the reaction unit 44. A recognition substance that reacts specifically is immobilized and retained. Further, the flow path 47 on the downstream side of the reaction section 44 is formed so that the width thereof is smaller than the diameter of the beads 50. Therefore, the beads 50 arranged in the reaction unit 44 do not flow out to the flow path 47 side. In this example, an antibody that specifically reacts with an antigen of cedar pollen (for example, an anti-Cry j-1 monoclonal antibody) is fixed and held on the surface of the beads 50, extracted into a carrier solution, and moved to the reaction unit 44. The cedar pollen antigen binds to the antibody on the bead surface and stays there.

  Such a chip structure can be easily formed by processing a glass substrate or the like using wet etching or the like. As the substrate 40 for chip formation, for example, a photocurable resin, a thermosetting resin, Glass and plastic materials can be used. Further, as the cross-sectional shape of the flow paths 45, 46, 47 formed on the chip substrate 40, formation of a square, rectangle, trapezoid or the like can be exemplified as a general shape, and the bottom of the flow path is a part of a circle. It may be rounded like

  Although not shown in FIG. 1, this chip is covered on a substrate 40 with a cover plate (the material is the same as that of the substrate 40). The cover plate is provided with holes corresponding to positions corresponding to the introduction part 41, the lead-out part 42, and the extraction part 43. Among these, the hole on the extraction part 43 is for taking in allergen containing particle | grains from outside air. The allergen-containing particles may be taken into the extraction unit 43 by a method of forcibly taking in allergen-containing particles from the outside air via an external suction device. It is good also as a structure to provide.

  Here, as the structure of the separation part 49, a structure in which a plurality of cylinders are arranged is shown, but the structure is not limited to this. For example, a gel-like polymer having an average pore size equal to or smaller than the size of the insoluble matter may be provided in the boundary between the extraction part and the reaction part or in the flow path connecting both. Moreover, you may arrange | position the filter whose opening is below the size of an insoluble matter in the same location. Further, as shown in FIG. 6, a channel having a depth equal to or less than the size of the insoluble material may be provided in the flow path 46 connecting the extraction unit 43 and the reaction unit 44.

  Further, the derivation unit or the like is not limited to the size described above. Further, in order to prevent the beads in the reaction unit 50 from flowing away, the flow path 47 is formed smaller than the diameter of the beads 50, but the structure is not limited to this. For example, a bottleneck like the separation unit 49 may be provided on the downstream side of the reaction unit 44. Alternatively, the beads 50 may be magnetic beads, and may be fixed to the reaction portion by applying a magnetic force from the outside.

  In addition, the reaction unit 44 does not place the beads 50 on which the recognition substance is fixed, but directly fixes and holds the recognition substance on the wall of the reaction unit 44 (including the inner wall of the lid positioned above the reaction part 44). May be. Examples of the fixing method include adsorption and covalent bonding, but the covalent bonding is preferable because it is more firmly fixed.

  If there is no need to provide a means for opening and closing the liquid flow between the extraction unit 43 and the reaction unit 44, the flow path 46 may not be provided between the extraction unit 43 and the reaction unit 44.

  The allergen detection chip 101 is configured to be detachable from the apparatus main body 201, and it is necessary to prevent mixing of other substances and to inactivate antibody activity until it is used for allergen detection. . Therefore, it is preferable to remove it from the apparatus main body 201, hermetically package the chip 101 alone, and store it in a refrigerator or frozen. In this case, it may be in a solid state (a state not containing a carrier liquid) or in a state dissolved in a carrier liquid.

  Next, the apparatus main body 201 connected to the detection chip 101 will be described. The apparatus main body 201 is connected to the introduction port of the introduction part 41 of the chip 101 so as to flow the carrier liquid into the chip 101, and the distribution that guides the carrier liquid flowing out from the outlet port of the lead-out part 42 to the outside of the apparatus main body. Pipe 51b, 51c in which the carrier liquid flows in the apparatus main body 201, cocks 48a to 48d for controlling the flow of the carrier liquid, a pump 52 (liquid feeding means) for feeding the carrier liquid, and a carrier liquid tank 53 for storing the carrier liquid ( A carrier liquid storage unit), a recognition substance solution tank with a labeling substance containing a recognition substance with a labeling substance (a recognition substance temporary storage part with a labeling substance) 54, a detection means 55 for detecting a label, an excess of a recognition substance with a labeling substance, etc. A waste liquid tank 56 is provided. In this example, the recognition substance solution tank with labeling substance contains the recognition substance with labeling substance (labeled antibody) dissolved in the carrier liquid, but it may exist in a solid state.

  The pump 52 is used to circulate the carrier liquid in the flow path, and a liquid pump such as a micro pump or a syringe type that can flow a liquid of microliter / second to milliliter / second can be used.

  As for the cock that is the flow path changing means, there is no particular restriction, so that a normal one can be used. The same applies to the distribution piping group.

  In order to connect the apparatus main body 201 and the chip 101 without causing liquid leakage, various connectors that can connect the flow paths can be used. For example, a sucker-shaped rubber connector can be used, and when such a rubber connector is provided at the end of the piping group, the connector is pressed against the inlet or outlet of the chip 101. Both can be easily connected and the liquid can be circulated between them. Such a connecting tool may be provided on the chip 101 side, and in this case, the same convenience can be obtained.

  The specific configuration of the detection means 55 varies depending on the measurement method to be applied. For example, when detecting by a fluorescent labeling method, a light source that generates ultraviolet light, an optical system for irradiating the reaction part of the chip with ultraviolet light, And a detector for detecting fluorescence from the labeled antibody.

  The allergen detection apparatus according to Embodiment 1 includes the chip 101 and the apparatus main body 201. Next, a method for detecting an allergen protein using this allergen detection apparatus will be described.

Process 1
First, as shown in FIG. 1, the distribution pipe 51 a led out from the cock 48 c of the apparatus main body 201 is connected to the introduction port of the introduction part 41 of the chip 101, and the circulation pipe 51 b is connected to the introduction port of the extraction part 42.

Process 2
The cocks 48a, 48b, and 48c are adjusted, and the introduction part 41, the flow path 45, the extraction part 43, the flow path 46, the reaction part 44, the flow path 47, the lead-out part 42, the flow pipe 51b, the flow pipe 51c, the pump 52, and the flow A circulation channel composed of the pipe 51a is formed. The pump 52 is driven, the cock 48d is opened, the carrier liquid in the carrier liquid tank 53 is supplied to the circulation channel, and the circulation channel is filled with the carrier liquid. This step is a step of expelling air in the flow path, thereby preventing air from entering the chip.

Process 3
Next, after taking in cedar pollen from the upper opening of the extraction part 43, the pump 52 is driven, a carrier is introduce | transduced into the extraction part 43, and the pollen surface and internal allergen protein are extracted in carrier liquid. The extraction carrier liquid is circulated in the flow path at least two times or more. After circulating the carrier liquid as many times as desired, the cock 48a is operated so that the carrier liquid in the chip flows into the waste liquid tank, and the cock 48d is opened and fresh carrier liquid is supplied from the carrier liquid tank 53. So that In this state, the inside of the flow path is replaced with a new carrier liquid while the carrier liquid in the chip is discharged into the waste liquid tank over several minutes.

  A fresh carrier liquid is stored in the carrier liquid tank 53, and the tank internal pressure is higher than that of the circulation channel. Therefore, the carrier liquid in the tank 53 is not contaminated with the circulated carrier liquid.

  Here, as a method for taking in cedar pollen, it may wait for cedar pollen or the like to naturally fall into the upper opening of the extraction unit 43, or may be collected by suction or the like. Moreover, the method of putting the cedar pollen etc. which were collected separately may be used. The carrier solution may be any solvent that does not denature the allergen protein and the recognition substance. However, when the object to be detected is cedar pollen, a weak alkaline ammonium bicarbonate solution is used in terms of extractability and stability. Is good. Furthermore, as a method for efficiently extracting allergen protein from pollen particles, a ball mill method may be employed in which a magnetic ball is arranged in the extraction unit 43 and a magnetic force is applied from the outside to swing the ball.

  Further, as a preferable method for further improving the reaction efficiency in the reaction unit 44, for example, a method in which the beads 50 are made of a magnetic material and the beads themselves are randomly swung by applying a magnetic force from the outside can be mentioned. In this method, the opportunity for the reaction between the antigen and the antibody is remarkably increased, and the reaction efficiency is increased.

Process 4
Next, by operating the cocks 48b and 48c, the labeling substance-attached recognition substance stored in the labeling substance-added recognition substance tank 54 is introduced into the flow path, and the same operation as described above is performed. A carrier liquid containing (labeled antibody) is circulated in the flow path. Thereby, the allergen protein couple | bonded with the reaction part in process 1-3 and the recognition substance with a labeled substance react. Here, as a recognition substance with a labeling substance, for example, a Cry j-1 monochrome antibody with a fluorescent dye labeling substance is used. Cry j-1 is an allergen protein contained in cedar pollen.

Process 5
After circulating the carrier liquid containing the labeled antibody a desired number of times, the cock 48a is operated so that the carrier liquid in the chip flows into the waste liquid tank and the fresh carrier liquid is supplied from the carrier liquid tank 53. Then, the excess labeled antibody remaining in the reaction part is washed away.

Step 6
The detection unit 55 provided on the reaction unit 44 irradiates the reaction unit 44 with ultraviolet rays, and measures the fluorescence intensity. Thereby, the amount of allergen protein can be quantified.

Step 7
Thus, one measurement is completed. When the next measurement is performed with a new chip, the chip 101 is replaced.

Step 7-2
On the other hand, when the same chip is used for subsequent measurement, pollen shells remaining in the extraction unit 43 are removed. In addition, when adopting a method of performing measurement a plurality of times using the same chip, it is preferable to provide means for removing pollen shells remaining on the chip 101. As this means, for example, a pollen shell removal port having a large opening is attached to the extraction unit 43, and an open / close valve is provided between the extraction unit 43 and the pollen shell removal port. A method of rinsing the pollen shell from the pollen shell removal port can be exemplified.

Step 7-3
After removing the pollen shell, the allergen protein (antigen) and the allergen with labeling substance from the recognition substance-allergen protein complex and the recognition substance-labeling substance allergen protein complex bound to the reaction part from the introduction port of the introduction part 41 A solution having an ionic concentration for separating the protein (labeled antibody) is flowed and discharged to the waste liquid tank 56 through the outlet 42. After this operation, the carrier liquid is flowed to wash the extraction unit, the reaction unit, and the associated flow path. As a result, all labeled antibodies used in the reaction are removed. Therefore, in order to perform continuous measurement, the labeled substance with the labeled substance is added again to the recognized substance tank 54 with the labeled substance, or the recognized substance tank with the labeled substance is removed. It is necessary to provide a plurality. The order of the steps 7-2 and 7-3 may be reversed.

  In the above description, cedar pollen has been described as an example. However, it is needless to say that the present invention can be applied to other allergens. Moreover, although the case where one kind of antibody was used was described above, allergen-containing particles (floating microparticles) in the atmosphere are various, and one allergen-containing particle contains two or more kinds of allergens. In some cases, particles in the atmosphere often contain many types of allergens. When multiple types of allergen proteins (antibodies) are detected at the same time, for example, multiple types of antibodies are arranged in the reaction part, and fluorescence having different fluorescent colors depending on the type of antibody as a labeled antibody in the recognition substance temporary storage part 12 with a labeled substance. By preparing a dye-attached one, many types of antigens can be detected simultaneously by the difference in fluorescence color. Alternatively, a method may be employed in which a plurality of reaction units are provided, different antibodies and labeled antibodies are arranged, and corresponding labeled recognition substances are prepared in the labeled recognition substance solution tank.

  In the above description, the case where a so-called sandwich method in which an antigen is sandwiched between an antibody and a labeled antibody is used as an example, but another known method may be used. For example, there is a method using a competitive reaction (competitive reaction method). In this method, an immobilized antibody whose amount is known in advance (immobilized antibody) is the same kind as the antigen to be measured (measurement antigen) and the antigen whose amount is known and labeled. This is a method in which an antigen (labeled antigen) is allowed to compete and react, the amount of immobilized antibody-labeled antigen formed by reacting with the labeled antigen is measured, and the amount of the measured antigen is calculated from the amount of the label. Also in this competitive reaction, by simultaneously circulating both the measurement antigen and the labeled antigen (mixed carrier solution), the chance of antigen-antibody reaction can be increased, so that the reproducibility of detection and the detection accuracy are improved.

  In this case, instead of the labeling substance-attached recognition substance solution tank 54, a labeling substance-containing allergen protein solution tank (a labeling substance-containing allergen protein temporary storage unit) 57 containing a known amount of the labeling substance-containing allergen protein is used, step 3 In the circulation channel, the labeled antigen stored in the allergen protein solution tank 57 with the labeled substance is introduced into the extraction unit, mixed with the antigen extracted in the extraction unit to form a mixed carrier solution, and circulates in the reaction unit It may be configured to flow. When the competition method is used, it is necessary that the amount of the recognition substance contained in the reaction part of the chip 101 is also known in advance. In this case, since it is not necessary to introduce a labeled antibody, step 4 becomes unnecessary.

  It is necessary to know the amount of the recognition substance and the labeled antigen in advance. The amount of the recognition substance contained in the reaction part of the chip 101 is excessive, and the amount of the extracted allergen protein and allergen protein with the labeling substance Is too small, all the allergen proteins react with the recognition substance contained in the reaction part to form a complex, resulting in inconveniences such as failure to detect the exact allergen protein amount.

  In the above description, the antigen-antibody reaction has been described as an example. However, the reaction is not limited to the antigen-antibody reaction as long as it is a reaction that recognizes allergen protein, and a recognition substance that recognizes allergen protein other than an antibody, such as a peptide or DNA. Alternatively, a high molecular compound or a low molecular compound having a reactive group that specifically reacts with RNA or allergen protein may be used.

In addition, the detection method using a fluorescent dye label has been described above, but in addition to this, a conventionally proposed enzyme labeling method, colloidal gold method, radioisotope labeling method, crystal resonator method, chemiluminescent substance A detection method such as a method or a surface plasmon resonance method can be used.
For example, in the case of enzyme labeling, for example, when glucose-degrading enzyme is used as a label, the antibody-antigen-labeled antibody complex formed in the reaction part 44 is obtained by flowing a glucose solution into the reaction part 44 after the above step 5. React with glucose. In order to carry out this method, for example, a tank containing a glucose solution is provided in the apparatus main body 201 in advance, and this is connected to the liquid feeding means to circulate the glucose solution in the same manner as in the case of the carrier liquid, React with antibody-antigen-labeled antibody complex. Then, the hydrogen peroxide generated by this reaction may be detected by a hydrogen peroxide detection electrode provided in the reaction part, and the detected voltage or current may be amplified by a detection means provided in the main body 201 as necessary. In this step, the completion of the reaction can be determined by monitoring the current or voltage during the circulation.
For example, in the case of using the crystal resonator method, the recognition substance with the labeling substance is not necessary, and therefore the recognition substance tank with the labeling substance may not be provided.

[Embodiment 2]
A second embodiment of the present invention will be described with reference to FIG. As shown in FIG. 2, the allergen detection device according to the second embodiment includes at least an allergen detection chip 102 and a device main body 202. Like the first embodiment, the allergen detection chip 102 and the device main body 202 are used. Is configured to be detachable.

  First, the apparatus main body 202 will be described. The apparatus main body 202 has a storage means 2 for storing the liquid to be injected into the chip, a liquid feeding means 3 for flowing the liquid to the chip, a flow rate adjusting means having valves 23a, 23b and 23c for adjusting the flow rate, and changing the flow path. Flow path changing means having cocks 22a, 22b, 22c, 22d, micro valve driving means (not shown) for operating opening / closing means (micro valves 15a, 15b) provided on the chip 102, operation of each of the above means Control means 4 for controlling the flow, flow pipe groups 5, 5 a, 5 b, 5 c, 5 d for flowing the liquid, connection means 6 for connecting the chip and the flow pipe group, the presence or absence of the antigen after the antigen-antibody reaction A detection means 7 for detection and a waste liquid reservoir 24 for discarding the used carrier liquid and cleaning liquid are provided.

  As the storage means 2, a general microtank can be used as it is, and a carrier liquid or the like for extracting an antigen is stored in the tank. As will be described later, since the carrier liquid circulating in the flow path contains antigens, labeled antibodies, and other soluble substances, it is necessary to separate such carrier liquid from fresh carrier liquid in the storage tank. is there. For this reason, the storage means 2 (tank 2) is divided into a portion 2a through which the circulating carrier liquid flows and a portion 2b through which fresh carrier liquid is supplied into the flow path.

  The control means 4 for controlling the operation of the flow rate adjusting means or the like is preferably provided with a computer and can be automatically controlled according to a preset program, but may be controlled manually.

  Moreover, the same thing as the said Embodiment 1 can be used for a liquid feeding means (pump), a cock, and a distribution piping group. A known micro valve may be used as a valve for opening and closing the flow path.

  Further, the connecting means 6a to 6d use various connecting devices capable of connecting the flow paths shown in the first embodiment in order to connect the apparatus main body 202 and the chip 102 without causing liquid leakage. be able to.

  The waste liquid reservoir 24 is a tank for replacing the carrier liquid in the flow path or for discarding the cleaning liquid after washing the flow path, and there are no particular restrictions on the shape and material of the tank. Note that the waste liquid needs to be discarded from the apparatus main body 202 to the outside, but the waste liquid reservoir 24 is not necessarily required.

  The detection means 7 detects the kind and / or amount of the antigen (allergen protein) chemically immobilized on the reaction part 11 (described later) of the chip 102, and the same one as in the first embodiment is used. That's fine.

  In the apparatus main body 202, it is important to supply the carrier liquid to the chip 102 and circulate it. If this is satisfied, the storage tank 2 (storage means 2) and the liquid feed pump 3 (liquid feed means) are satisfied. 3) The number and arrangement of the detecting means 7, the valve group, the cock group, the piping group, the connecting means 6, the waste liquid reservoir 24, etc. are not limited to those shown in FIG.

Next, the detection chip 102 in the second embodiment will be described.
The detection chip 102 according to the second embodiment has an opening that can collect pollen from the atmosphere, an extraction unit 10 that extracts an antigen (allergen protein) from the incorporated pollen, and an antigen that is a detection target. Specifically reacts with the antigen-antibody complex and the reaction part 11 held in a state where the antibody (recognition substance) that specifically reacts with the antibody does not flow away, and forms an antigen-antibody-labeled antibody complex by the reaction. A recognition substance-attached recognition substance temporary storage unit 12 in which a labeled antibody to be stored is stored, an introduction part 13 a having an introduction port for allowing a carrier liquid to flow into the chip 102 from the apparatus main body 202, and the chip 102 from the apparatus main body 202. A second introduction part 13c having an introduction port for allowing the carrier liquid to flow into the recognizing substance storage part 12 with the labeling substance, and the carrier liquid from the chip 102 toward the apparatus main body 202 side. Lead-out portions 13b and 13d having lead-out ports for letting out and channels 14a, 14b, 14c, 14d, 14e, and 14f for flowing the carrier liquid into the chip 102 are formed on the substrate 40, and the liquid in the chip Micro-valves 15a and 15b are provided on the substrate 40 as opening / closing means for blocking or passing the flow of water.

  The substrate 40 is covered with a cover plate (see reference numeral 31 in FIG. 6), and the cover plate has openings at the introduction port and the discharge port portion of the introduction portion and the discharge portion, and at the opening portion of the extraction portion 11. The substrate 40 and the cover plate are integrated. The opening on the extraction unit 11 is a take-in hole 9 for taking in pollen, and an open / close lid that can be opened and closed as necessary may be provided on the take-in hole 9.

  The same material as that of the first embodiment can be used for the substrate 40 and the cover plate. The introduction parts 13a and 13c and the lead-out parts 13b and 13d formed on the substrate 40 are vertical holes with a diameter of 100 μm or more and a depth of 50 μm to 1 mm, and the extraction part 10 has a diameter of 100 μm or more and a depth of 50 μm. It is a 1 mm vertical hole. The extraction unit 10 communicates with the introduction unit 13a through the flow channel 14a, and a separation unit similar to that shown by reference numeral 49 in FIG. 6 is provided between the extraction unit 10 and the flow channel 14b located on the downstream side. ing. The separation part 49 is a bottleneck in which the gap with the cover plate is less than the size of insoluble substances such as pollen, pollen shells, sand, etc. (for example, 20 μm or less). Since the pollen shell and the like are dammed up when flowing from the extraction section 10 to the flow path 14b, the insoluble matter such as the pollen shell and the dissolved matter such as the antigen can be separated.

  The reaction unit 11 is a space where the pollen antigen and the antibody are reacted, and an antibody that specifically reacts with a specific antigen as an object to be detected is fixedly held therein. The method for fixing and holding the antibody may be the same as in the first embodiment. The shape of the reaction part 11 may be the same as that of the extraction part, for example, a cylindrical vertical hole, a hemispherical shape, and a rectangular parallelepiped hole. The size is preferably 100 μm or more, and the depth is preferably 50 μm to 1 mm.

  The labeling substance-attached recognition substance temporary storage unit 12 is a part for storing a labeled antibody (a recognition substance with a labeling substance) to which a labeling substance is bound, and the labeling antibody may be stored as it is, or a carrier such as a bead. It is also possible to hold the However, even when it is held on a carrier such as a bead, it is necessary to prevent the labeled antibody from flowing out of the flow path 14f due to the flow of the carrier liquid, and a structure that prevents the bead from flowing out is preferable. This structure may be the same as the separation part in the first embodiment. Further, in order to prevent the labeled antibody from staying in the reaction part 11, it is also necessary to have a structure that leads to the apparatus main body 202 through the flow path 14c.

  As the opening / closing means (microvalves 15a, 15b) for opening and closing the flow in the chip 102, various known means can be used as long as they can open and close a minute flow. For example, a resin molded product (resin film 21) having a shape as shown in FIG. Since the resin film 21 is provided in a narrow flow path, the resin film 21 expands in volume when irradiated with light by a micro valve driving means (not shown) provided in the apparatus main body 202 or heated by a heater, and passes through the flow path. On the other hand, when light irradiation or heater heating is stopped and the temperature of the resin film decreases, the volume returns to the original and the flow path is opened. Therefore, the opening and closing of the flow path can be controlled.

  The flow paths 14a, 14b, 14c, 14d, 14e, and 14f are grooves having a width of 0.1 μm to 1 mm and a depth of 0.1 μm to 1 mm. The cross-sectional shape is, for example, a rectangle or a trapezoid, and the bottom of the flow path may be round like a part of a circle. However, it is not limited to these sizes and shapes.

In the allergen detection chip 102 according to the second embodiment described above, the recognition substance temporary storage unit 12 with labeling substance (recognition substance tank with labeling substance in the first embodiment) is placed inside the chip 102 instead of the apparatus main body 202. It differs from the chip 101 of the first embodiment in that it is provided. An antigen detection method using such an allergen detection apparatus according to Embodiment 2 will be described below with reference to FIG. 2 taking the sandwich method as an example.
Here, it is assumed that the recognition substance with labeling substance (labeled antibody) contained in the recognition substance temporary storage unit 12 with labeling substance is fixed to an insoluble carrier.

Process 1
First, the chip 102 is attached to the apparatus main body 202. At the time of mounting, the introducing means / leading parts 13a, 13b, 13c, 13d of the chip 102 and the corresponding pipe groups 5a, 5b, 5c, 5d on the apparatus main body side are connected to the connecting means 6a to 6d. Connect through d.

Process 2
Next, the microvalve 15a is opened, 15b is closed, the carrier liquid in the storage means 2 is supplied to the pipe 5b from the path 2b, and the extraction unit 10, the reaction unit 11, and the labeling substance are provided via the introduction units 13a and 13c. It is made to flow into the recognition substance temporary storage part 12. In this case, the flow of the carrier liquid passes from the storage means 20 in the apparatus main body 202 through the pipe 5b and enters the chip 102 through the introduction port of the introduction part 13a. Further, the flow path 14a, the extraction part 10, the flow path 14b, A route that flows into the pipe 5 in the apparatus main body 202 through the reaction unit 11, the flow path 14c, and the derivation unit 13b, returns to the bypass 2a of the storage unit 2 through the liquid feeding unit 3, and passes through the pipe 5b from the storage unit 2. The flow is diverted by the cock 22c, passes through the cock 22b, passes through 5c, enters the chip 102 from the second introduction part 13c, and passes the flow path 14d, the recognition substance temporary storage part 12 with the labeled substance, the flow path 14f, and the lead-out part 13d. The control device 4 causes the cocks 22a, 22 in the apparatus main body 202 to pass through the piping 5d in the main body 202 and return to the bypass 2a of the storage means 2 through the liquid feeding means 3. Controls 22c, 22 d and the valve 23a, 23b, a 23c. The operation of flowing along two routes may be performed simultaneously, or after flowing along one route, the operation may be performed along another route. Since the volumes of the extraction unit 10, the reaction unit 11, the labeled substance-attached recognition substance temporary storage unit 12 and the like are known in advance, the control unit 4 flows in a necessary amount and stops the inflow.

  Here, the width or thickness is larger than the size of the carrier to which the labeled antibody is fixed, between the recognition substance temporary storage unit 12 with the labeled substance and the flow path 14f of the flow path system of the carrier liquid flowing in from the second introduction part 13c. A flow path with a small diameter is provided. With such a configuration, the labeled antibody can be prevented from flowing into the main body 202.

  By the above operation, each flow path in the chip 102, the extraction unit 10, the reaction unit 11, and the recognition substance temporary storage unit 12 with the labeling substance can be filled with the carrier liquid. Thereby, gas, such as air, can be prevented from being mixed in the carrier liquid, and the reaction in the subsequent steps can be performed reliably.

Process 3
Next, in a state where the microvalve 15b is closed, the carrier liquid is stored in the storage means 2, the pipe 5a, the introduction part 13a, the flow path 14a, the extraction part 10, the flow path 14b, the reaction part 11, the flow by the liquid sending means 3. The cocks 22a, 22b, 22c and 22d and the valves 23a, 23b and 23c in the apparatus main body 202 are operated so as to circulate back to the pipe 5 in the apparatus main body 202 through the path 14c.

  While circulating the carrier liquid through the above route, pollen is collected from the intake hole 9 for a certain period of time. At this time, if necessary, means for forcibly sucking air may be provided. Pollen enters the carrier liquid in the extraction unit 10 of the chip 102 through the intake hole 9, and allergen protein (antigen) contained on the pollen surface or inside is extracted into the carrier liquid. At this time, if necessary, a plurality of magnetic beads having a size of 1 μm to 100 μm are arranged in the extraction unit, and the pollen is moved into the carrier liquid by moving the magnetic beads with a magnetic bead stirring means separately provided on the main body. It is good to stir with. If it does in this way, the antigen extraction efficiency from pollen will improve.

  Since the carrier liquid circulates through the circulation route, the antigen extracted from the pollen is sequentially sent to the reaction unit 11 through the flow path 14b. At this time, pollen shells and the like are insoluble in the carrier solution, and if this pollen mixes into the reaction part, it becomes difficult to accurately detect and measure, so the insoluble substance such as pollen shells and the antigen dissolved in the carrier solution And other soluble substances such as proteins must be separated. This separation may be the same as that shown in the first embodiment. Moreover, it can prevent that a separation part is clogged with the shell of pollen by using together stirring with the above-mentioned magnetic bead.

  The antigen sent to the reaction unit 11 causes an antigen-antibody reaction with the antibody provided in the reaction unit, and the antigen is fixed to the reaction unit. The antigen-antibody reaction depends on the specific reactivity of the antibody to the antigen and the chance that the antigen and the antibody meet each other, and not all antigens react with the antibody in one reaction process. However, in this embodiment, the carrier liquid is collected by the liquid feeding means 3 during pollen collection, and the storage means 2, the pipe 5 a, the introduction part 13 a, the flow path 14 a, the extraction part 10, the flow path 14 b, the reaction part 11, the flow path 14c, adopting a configuration that continuously repeats the circulation of returning to the pipe 5 in the main body 202 through the derivation unit 13b, and increasing the chance that the antigen and the antibody react with each other. As a result, the reproducibility of each measurement can be increased. This circulation is preferably continued for a while after the pollen intake hole 9 is capped and the pollen collection is stopped, in order to react the extracted antigen with the antibody.

  As another method, for example, during pollen collection, the microvalves 15a and 15b are closed, the extraction unit 10 and the reaction unit 11 are made independent, and after a certain period of time, for example, the pollen intake hole 9 is covered, With the collection stopped, the microvalve 15a is opened, and the storage means 2, the pipe 5a, the introduction part 13a, the flow path 14a, the extraction part 10, the flow path 14b, and the reaction are performed by the liquid feeding means 3 in the apparatus main body 202. The cocks 23a, 23b, 23c, 23d and the valves 23a, 23b, 23c in the main body 202 are operated so as to circulate back to the pipe 5 in the main body 202 through the part 11, the flow path 14c, and the lead-out part 13b. In this method, the carrier liquid is circulated. Also in this method, the antigen-antibody reaction can be surely performed by the circulation characteristic of the present invention.

  Immediately after the completion of step 3, the process may proceed to the following step 4. However, in order to wash away soluble substances remaining in the system, the carrier liquid circulated once in step 3 is discharged, and the chip is filled with a new carrier liquid. It is preferable to replace the carrier liquid in the flow channel 14a, the extraction unit 10, the flow channel 14b, the reaction unit 11, and the flow channel 14c. In this case, the liquid feeding means 3 in the apparatus main body 202 passes through the storage means 2 and the pipe 5b, enters the chip from the introduction part 13a, and enters the flow path 14a, the extraction part 10, the flow path 14b, the reaction part 11, and the flow path. 14c, return to the pipe 5 in the main body 202 through the lead-out portion 13b, and enter the waste liquid reservoir 24 through the cock 22d so that the cocks 22a, 22b, 22c, 22d and the valves 23a, 23b, 23c in the apparatus main body 202 are Manipulate.

  By this operation, lysate other than the antigen extracted together with the antigen from the pollen can be removed from the reaction part, and the reactivity between the subsequent antibody-antigen complex and the labeled antibody can be increased more effectively. .

Process 4
After stopping the circulation in step 3, the valve 15a is closed, the valve 15b is opened, the valves 23a and 23c in the main body 202 are closed, and the valve 23b is opened, so that the carrier liquid is fed into the apparatus main body 202. The liquid means 3 passes through the pipes 5a and 5c from the storage means 2 and enters the chip 102 from the second introduction part 13c. The flow path 14d, the labeled substance-recognized substance (labeled antibody) temporary storage part 12, and the flow path 14e Then, it is circulated through a circulation route that returns from the reaction unit 11, the flow path 14c, and the derivation unit 13b to the pipe 5 of the apparatus main body 202. Since the amount of the labeled antibody to be reacted varies depending on the amount of the antigen, the labeled antibody is preferably stored in excess in advance in order to react with all the antibody-antigen complexes.

  The labeled antibody immobilized on the insoluble carrier is sent to the reaction unit 11 by this circulation, and reacts with the antibody-antigen complex formed in step 3 to form an antibody-antigen-labeled antibody complex. To do. Even in this step, in order to efficiently react all the antibody-antigen complexes formed earlier and the labeled antibody, the labeled antibody is circulated through the circulation route as described above to react with the antibody-antigen complex. Since the reaction is performed in a state where the carrier liquid containing the labeled antibody is always flowing, the reproducibility of the reaction efficiency detection result of the antigen-antibody reaction is enhanced.

Process 5
In order to detect the amount of the antibody-antigen-labeled antibody complex formed in the reaction part, it is necessary to remove the unreacted labeled antibody remaining in the reaction part. For this reason, the liquid feeding means 3 in the apparatus main body 202 passes through the storage means 2, the pipes 5 b and 5 c, enters the chip from the introduction part 13 c, and enters the flow path 14 d, the recognition substance temporary storage part 12 with the labeled substance, the flow path 14e, the reaction part 11, the flow path 14c, the lead-out part 13b, return to the pipe 5 in the main body 202, and enter the waste liquid reservoir 24 through the cock 22d so that the cocks 22a, 22b, 22c, 22d in the apparatus main body 202 Then, the valves 23 a, 23 b, and 23 c are operated to flow the unreacted labeled antibody-containing carrier liquid into the waste liquid tank 24 provided in the main body 202.

Step 6
Next, detection of the antibody-antigen-labeled antibody complex formed in the reaction unit 11 is performed by the detection means 7 provided in the apparatus main body 202. For example, when a fluorescent material is used as the label, the antibody-antigen-labeled antibody complex in the reaction part is irradiated from the ultraviolet light source provided in the detection means 7 through the optical system provided in the detection means 7 and labeled. By detecting the fluorescence emitted from the fluorescent material with a detector provided in the detection means 7, the presence or absence of the antigen and the amount of the antigen can be measured.

Step 7
Thus, one measurement is completed. When the next measurement is performed with a new chip, the chip 102 is replaced.

Step 7-2
On the other hand, when the measurement is subsequently performed using the same chip, the pollen shell remaining in the extraction unit 10 is removed. In addition, when adopting a method of performing a plurality of measurements using the same chip, it is preferable to provide means for removing pollen shells remaining on the chip 102. As this means, for example, a pollen shell removal port having a large opening is attached to the extraction unit 10, and an open / close valve is provided between the extraction unit 40 and the pollen shell removal port, and the carrier liquid for cleaning in the valve open state is used. A method of rinsing the pollen shell from the pollen shell removal port can be exemplified.

Step 7-3
After removing the pollen shell, allergen protein (antigen) and allergen with labeling substance from the recognition substance-allergen protein complex and recognition substance-labeling substance allergen protein complex bound to the reaction part from the introduction port of introduction part 13a A solution having an ion concentration for separating the protein (labeled antibody) is flowed and discharged to the waste liquid reservoir 24 through the outlet 13b. After this operation, the carrier liquid is flowed to wash the extraction unit, the reaction unit, and the associated flow path. The order of the steps 7-2 and 7-3 may be reversed.

  Thereby, all the labeled antibodies used in the reaction are removed. For this reason, in order to perform detection again using the same chip, as shown in FIG. 3, a recognition substance temporary storage unit 12 with a labeled substance, and a flow path 14 e with a microvalve connecting the reaction unit 11 with the recognition substance Introducing / leading parts 13c, 13d that can be connected to the main body, flow paths 14d, 14f that connect the recognizing substance temporary storage part 12 with the labeling substance and a recognizing substance temporary storage part set A with the labeling substance including the microvalve 15b ( In the figure, a configuration in which a plurality of portions surrounded by a broken-line square are provided may be employed.

  In this case, when detection is performed using one set, the microvalve on the chip or the cock of the main body and valves are adjusted so that the other set is not involved. After performing steps 1 to 6 using the storage unit set A, the steps 7-2 and 7-3 described in the first embodiment are performed, and another labeled substance-recognized substance temporary storage unit set A ′. Steps 1 to 6 may be newly performed using

  In the second embodiment, the case where the labeled antibody is immobilized on an insoluble carrier has been described as an example. However, in the case where the labeled antibody is not immobilized on an insoluble carrier, the above step 2 may be replaced with the following step 2 ′. .

Process 2 '
Next, the microvalve 15a is opened, 15b is closed, and the carrier liquid in the storage means 2 is extracted by the liquid feeding means 3 through the introduction parts 13a and 13c, the reaction part, and the recognition substance temporary storage part with the labeling substance. 12 flows in.
At this time, in the apparatus main body 202, the cocks 22a, 22b, 22c, 22d and the valves 23a, 23b are flown so that the carrier liquid flows through the liquid supply route passing through the pipe 5 and the pipe 5b and the routes 5, 5b, 5c, 5d. , 23c. The carrier liquid passes from the storage means 2 in the apparatus main body 202 through the pipe 5b and enters the chip 102 from the introduction part 13a. In the chip, it enters the flow path 14a, the extraction section 10, the flow path 14b, and the reaction section 11, and then flows into the pipe 5 in the main body 202 through the flow path 14c and the lead-out section 13b, and passes through the liquid feeding means 3 and the storage means. Return to 2.

On the other hand, since the cock 22c is controlled so that the carrier liquid also flows through the pipe 5c, the carrier liquid enters from the second introduction part 13c of the chip 102 through the pipe 5c, and flows into the flow path 14d and the recognition substance with the labeling substance. The pipe 5d of the apparatus main body 202 enters the temporary storage unit 12, the flow path 14f, and the lead-out unit 13d. At this time, the valve 23b is adjusted so that the labeled antibody in the recognized substance temporary storage unit 12 with the labeled substance does not flow out of the chip.
This can be controlled by time control calculated from the flow velocity because the volume of the recognition substance temporary storage unit 12 with the labeling substance and the piping is known in advance. When a label that can be detected optically, such as a fluorescent dye, is used as the label, for example, an optical detection means is provided in the vicinity of the lead-out portion 13d of the apparatus main body 202 to detect the labeled antibody in the pipe 5d. This can also be realized by interlocking the operation of the valve 23b. The step of introducing the carrier liquid from the introduction part 13a and the operation of introduction from the second introduction part 13c may be performed simultaneously, or after performing one operation, another operation may be performed.

  In the second embodiment, a competitive reaction method can also be used as in the first embodiment. In this case, instead of the labeled antibody, the labeled substance temporarily storing unit 12 with the labeled substance in the chip 102 is made to have a known amount of labeled antigen in advance to form the labeled substance allergen protein temporary storing unit 17 and fixed to the reaction unit 11. Other chip configurations and main body configurations may be the same except that the amount of antibody produced is known in advance. In the detection method, the steps 4 and 5 in the above embodiment can be omitted, and the step 3 can be replaced with the following step 3 '. In this case, the other steps may be the same as those in the above embodiment, and the steps of Step 1 -Step 2 -Step 3 ′ -Step 6 may be performed. If necessary, Step 7 and Step 8 described in Embodiment 1 are performed after Step 6. Moreover, as shown in FIG. 3, you may provide two or more allergen protein temporary storage part sets A with a labeled substance containing the allergen protein temporary storage part 17 with a labeled substance.

  Next, a detection method when the competition method is used will be described.

  The structure of the detection chip is not significantly different from the sandwich method, but the hole formed as the recognition substance temporary storage unit 12 with the labeling substance contains the allergen protein with the labeling substance whose amount is known in advance. It functions as the allergen protein temporary storage part 17 with a labeled substance. In addition, it is necessary to know in advance the amount of the recognition substance present in the reaction part of the chip.

Step 1, 2
It may be the same as the above sandwich method.

Step 3 '
Next, in a state where the microvalves 15a and 15b are opened, the carrier liquid is stored in the storage means 2, the pipe 5a, the introduction part 13a, the flow path 14a, the extraction parts 10 and 14b, the reaction part 11, the flow by the liquid sending means 3. The route 14c, the route to return to the pipe 5 in the main body 202 through the lead-out part 13b, and the liquid feeding means 3 pass through the storage means 2, the pipes 5a, 5c, the introduction part 13c, the flow path 14d, the allergen protein with a labeling substance. The cocks 22a, 22b, 22c, 22d in the main body 202 are circulated simultaneously through a route that returns to the pipe 5 in the main body 202 through the temporary storage parts 17, 14e, the reaction part 11, the flow path 14c, and the lead-out part 13b. And the valves 23a, 23b, and 23c are operated.

  While circulating the carrier liquid by the above route, pollen is collected from the intake hole 9 provided in the apparatus for a certain period of time. At this time, if necessary, a means for forcibly sucking air may be provided. Pollen enters through the intake hole 9 and enters the carrier liquid in the extraction unit 10, and the pollen surface or internal allergen protein (antigen) is extracted into the carrier liquid. At this time, if necessary, a plurality of magnetic beads having a size of 1 μm to 100 μm are provided in the extraction unit, and the magnetic beads are stirred by the magnetic bead stirring means provided separately in the main body to stir the pollen in the carrier liquid. May be. By this stirring, the antigen extraction efficiency from pollen can be increased.

  Since the carrier liquid circulates through the circulation route, the antigen extracted from the pollen is sequentially sent to the reaction unit 11 through the flow path 14b. At this time, the pollen shell is insoluble in the carrier liquid, and it is necessary to separate the shell from soluble substances such as antigens and other proteins dissolved in the carrier liquid. This separation may be the same as that described in the first embodiment.

  For the antibody provided in the reaction part, the antigen sent to the reaction part 11 is mixed with the labeled antigen sent from the allergen protein with labeling substance (labeled antigen) temporary storage part 17 (mixed carrier liquid) and competes Then, an antigen-antibody reaction is caused, and the antigen and the labeled antigen are fixed to the reaction part according to a certain fraction. However, the competitive reaction depends on the specific reactivity of the antibody to the antigen and the labeled antigen, and the chance that the antigen and the labeled antigen meet the antibody. In one reaction, all antigens and the labeled antigen compete sufficiently with the antibody. Although it is not necessarily fixed to the reaction part with good reproducibility in proportion according to the quantity and reactivity of both, antigen antibodies can be obtained by continuously circulating both (mixed carrier liquid) continuously. The reaction occurs reliably and the reproducibility of the measurement can be increased. For example, the circulation is preferably continued for a while after the pollen intake hole 9 is covered and the collection of the pollen is stopped to react the extracted antigen and the labeled antigen with the antibody.

Process 5
Since it is necessary to remove the unreacted labeled antibody without performing step 4, the solution feeding means 3 in the apparatus main body 202 passes through the storage means 2, the pipes 5b and 5c, enters the chip from the introduction part 13c, It returns to the pipe 5 in the main body 202 through the flow path 14d, the labeled allergen protein temporary storage section 17, the flow path 14e, the reaction section 11, the flow path 14c, and the derivation section 13b, and enters the waste liquid reservoir 24 through the cock 22d. As described above, the cocks 22 a, 22 b, 22 c, 22 d and the valves 23 a, 23 b, 23 c in the apparatus main body 202 are operated so that the carrier liquid containing unreacted labeled antibody flows into the waste liquid tank 24 provided in the main body 202.

Process 5
Steps 6 and 7 are performed in the same manner as the sandwich method.

[Embodiment 3]
Hereinafter, the third embodiment will be described in detail with reference to FIG. As shown in FIG. 4, the allergen detection device of the second embodiment is composed of at least an allergen detection chip 103 and a device main body 203, and as in the first embodiment, the allergen detection chip 103 and the device main body 203. Is configured to be detachable.

  First, the apparatus main body 203 will be described. The apparatus main body 203 includes a storage means 2 for storing a liquid to be injected into the chip, a liquid feeding means 3 for flowing the liquid to the chip, a flow rate adjusting means having valves 23a, 23b, 23c and 23d for adjusting the flow rate, a flow path Channel changing means having cocks 22a, 22b, 22c, 22d for changing the temperature, micro valve driving means (not shown) for operating the opening / closing means (micro valves 15a, 15b) provided on the chip 103, each of the above means Control means 4 for controlling the operation of the above, distribution pipe groups 5, 5a, 5b, 5c, 5d, 5f for circulating the liquid, connection means 6 for connecting the chip and the distribution pipe group, after the antigen-antibody reaction, A detection means 7 for detecting the presence or absence of an antigen and a waste liquid reservoir 24 for discarding the used carrier liquid and washing liquid are provided.

  The storage unit, the liquid supply unit, the flow rate adjustment valve, the cock control unit, the connection, the detection unit, the microvalve driving unit, and the control unit 4 that are components of the apparatus main body 203 are the same as those described in the second embodiment. Components can be used.

Next, the detection chip 103 in the third embodiment will be described.
The detection chip 103 according to the third embodiment has an opening that can collect pollen from the atmosphere, an extraction unit 10 that extracts an antigen (allergen protein) from the incorporated pollen, and an antigen that is a detection target. Specifically reacts with the antigen-antibody complex and the reaction part 11 held in a state where the antibody (recognition substance) that specifically reacts with the antibody does not flow away, and forms an antigen-antibody-labeled antibody complex by the reaction. From the apparatus main body 203, the recognition substance temporary storage section 12 with the labeled substance in which the labeled antibody to be stored, the introduction sections 13a and 13c having the inlet for allowing the carrier liquid to flow into the chip 103 from the apparatus main body 203, and A second introduction part 13e having an introduction port for allowing the carrier liquid to flow into the recognition substance storage part 12 with the labeling substance in the chip 103, and the carrier main body 2 from the chip 103 From the lead-out portions 13b and 13d having lead-out ports for flowing out to the third side, the flow paths 14a, 14b, 14c, 14d, 14e, 14f, 14g, and 14h for flowing the carrier liquid into the chip 103, and the extraction portion 10 A diluting section 18 for diluting the carrier liquid containing the allergen protein to be introduced with the carrier liquid introduced through the flow path 14h is formed on the substrate 40, and the switching means for blocking or passing the flow of the liquid in the chip. The microvalves 15a, 15b and 15c are provided on the substrate 40.

  Of the constituent elements of the chip, the elements other than the dilution section 18 may be the same as those described in the second embodiment. The diluting unit 18 can use a conventionally proposed configuration for uniformly mixing liquids, for example, a long channel as shown in FIG. 8A, or a diameter as shown in FIG. 8B. An example is a configuration in which a hole having a depth of 100 μm or more and a depth of 50 μm to 1 mm is provided, and magnetic beads are provided on the bottom thereof. The configuration shown in FIG. 8 (a) utilizes the fact that two liquids are uniformly mixed while flowing through a long flow path. In FIG. 8 (b), the device main body 203 is provided. The two liquids are stirred and mixed by randomly moving the magnetic particles 8 with a magnet (not shown). However, it is not limited to the said structure.

The antigen detection method using the allergen detection apparatus of Embodiment 3 is demonstrated in detail using FIG.
However, in the following description, it is assumed that the labeled antibody in the recognizing substance temporary storage unit 12 with a labeled substance is fixed to an insoluble carrier. When not being fixed to the insoluble carrier, the following step 2 may be performed in the same manner as the step 2 ′ in the second embodiment.

Process 1
When detecting the pollen antigen, the chip 103 is first mounted on the apparatus main body (main body) 203. At the time of mounting, the pipes 5 provided in the main body are connected by the connecting / disconnecting means 6 of the main body 203 corresponding to the introduction / lead-out portions 13a, 13b, 13c, 13d, 13e provided in the chip 103.

Process 2
Next, the microvalves 15a and 15c are opened, 15b is closed, and the carrier liquid in the storage means 2 is extracted by the liquid feeding means 3 through the introduction / derivation parts 13a, 13e, and 13c, the reaction part, and the labeling substance. It flows into the attached recognition substance temporary storage unit 12.

  At this time, in the main body 203, the carrier liquid enters the chip 103 from the introduction part 13a through the pipes 5b and 5f from the storage means 2 in the main body 203, and enters the flow path 14a, the extraction part 10, the flow path 14g, the dilution part 18, A flow that flows into the pipe 5 in the main body 203 through the flow path 14b, the reaction section 11, and the flow path 14c, through the outlet section 13b, and from the storage section 2 to the chip 103 through the pipes 5b and 5e. The flow rate in the main body 203 is adjusted so that a flow flows into the pipe 5 in the main body 203 through the outlet portion 13b through the flow path 14h, the dilution section 18, the flow path 14b, the reaction section 11, and the flow path 14c. The valves 23a, 23b, 23c, 23d and the cocks 22a, 22b, 22c, 22d are operated. Further, since the cock 22c is controlled so that the carrier liquid also flows in the pipe 5c, the carrier liquid enters from the pipe 5c through the second introduction part 13c of the chip 103, and flows into the flow path 14d and the recognition substance with the labeling substance. The pipe 5d of the main body 203 enters the temporary storage section 12, the flow path 14f, and the outlet section 13d. The step of introducing the carrier liquid from the introducing portions 13a and 13e and the operation of adding from the second introducing portion 13c may be performed simultaneously, or one operation may be performed and then another operation may be performed. Since the volumes of the extraction unit 10, the reaction unit 11, the labeled substance-attached recognition substance temporary storage unit 12 and the like are known in advance, the control unit 4 flows in a necessary amount and stops the inflow.

  By the above operation, each flow path in the chip, the extraction unit 10, the reaction unit 11, and the labeling substance-attached recognizing substance temporary storage unit 12 can be filled with the carrier liquid. Gas can be prevented from mixing, and the reaction can be performed reliably.

  Here, steps 3 to 6 can be performed in the same manner as in the second embodiment, but the configuration for diluting the carrier liquid containing the allergen protein using the dilution unit 18 will be described below.

Step 3-1
The micro valve 15a is opened by the micro valve driving means of the main body 203, and the micro valves 15c and 15b are closed. In this state, the liquid feeding means 3 causes the carrier liquid to enter from the storage means 2 in the apparatus main body 203 through the pipes 5a and 5e, enter the introduction part 13e, pass through the flow path 14h, and pass through the dilution part 18 and the flow path 14b. Then, the reaction part 11 passes through the flow path 14c, passes through the lead-out part 13b, flows into the pipe 5 in the main body 203, and circulates. The flow rate adjusting valves 23a, 23b, 23c, 23d and the cocks 22a, 22b, 22c, 22d are operated. While circulating the carrier liquid by the above route, pollen is collected from the intake hole 9 provided in the chip for a certain period of time. After a certain time, a lid (not shown) is put on the intake hole to prevent pollen from entering.

Step 3-2
Next, the microvalve 15c is opened, and the carrier liquid enters from the introduction part 13a through the pipes 5a and 5f from the storage means 2 in the main body 203, and enters the flow path 14a, the extraction part 10, the dilution part 18, and the flow path. The flow rate adjusting valves 23a, 23b, 23c, 23d and the cocks 22a, 22b, 22c, 22d in the apparatus main body 203 are operated so as to enter the dilution section 18 through 14g. In the dilution section 18, the carrier liquid from the introduction section 13e and the carrier liquid from the extraction section 10 containing the antigen are mixed, and the allergen protein concentration of the carrier liquid flowing into the reaction section 11 is reduced. Thereafter, the fluid passes through the flow path 14b, the reaction section 11, and the flow path 14c, passes through the outlet section 13b, flows into the pipe 5 in the main body 203, and is circulated. Separation of an insoluble substance such as a pollen shell and a dissolved substance dissolved in a carrier liquid such as an antigen can be performed by the method described in Embodiment 1.

  The antigen sent to the reaction unit 11 causes an antigen-antibody reaction with the antibody provided in the reaction unit, and the antigen is fixed to the reaction unit. In the present embodiment, as described above, the carrier liquid enters from the introduction part 13a, enters the dilution part 18 through the flow path 14a, the extraction part 10, and the flow path 14g, and enters from the introduction part 13e to enter the flow path 14h. The liquid passing through the diluting unit 18 is uniformly mixed by the diluting unit 18, and this mixed solution passes through the flow path 14 b, the reaction unit 11, the flow path 14 c, passes through the outlet unit 13 b, and is connected to the pipe in the main body 203. Since the unreacted antigen reacts with the antibody a plurality of times due to the circulation of flowing into 5, the unreacted antigen can be reduced, and as a result, the reproducibility of each measurement can be increased.

  The reactivity of the antigen-antibody reaction is related to the number of contact between the antigen and the antibody, and the number of times is also related to the concentration of the antigen. When the antigen flowing from the extraction unit 10 has a high concentration, it may be difficult to cause diffusion in the reaction unit even if the antigen is flowed to the reaction unit 11 as it is, and the reaction efficiency may be lowered. In the present embodiment, in the dilution unit 18 Since the structure of diluting the antigen-containing carrier liquid from the extraction unit 10 is adopted, after flowing into the reaction unit 11, it can be diffused in a short time to improve the reactivity.

Step 3-3
After this circulation for a certain time, the process returns to step 3-1.
The steps from step 3-1 to step 3-3 are repeated several times.

In the third embodiment, a means for dividing and connecting them is provided between the diluting part and the extracting part, and the polling collecting and antigen extracting step and the storing means 2 in the main body 203 are obtained by dividing the parts for a certain time. From the introduction part 13e through the pipes 5a and 5e, through the flow path 14h, through the dilution part 18, the flow path 14b, the reaction part 11, and the flow path 14c, through the lead-out part 13b, and into the main body 203. This is characterized in that the antigen-antibody reaction by circulation entering the pipe 5 can be made independent.
According to the configuration of the third embodiment, the antigen-antibody reaction can be performed with a relatively small number of pollen, and the antigen-antibody reaction can be performed many times during the pollen collection. This can be done more reliably.

  In addition, since the antigen-antibody reaction can be performed during pollen collection, the time from pollen collection to completion of the antigen-antibody reaction can be shortened.

Process 4
After completing the repetition of steps 3-1 to 3-3, the microvalve 15a is closed, the microvalve 15b is opened, and the carrier liquid is supplied from the storage means 2 to the pipe 5a by the liquid feeding means 3 in the main body 203. 5c, enters the chip 103 from the second introduction part 13c, and flows from the flow path 14d, the recognition substance temporary storage part 12 with the labeled substance, the flow path 14e, the reaction part 11, the flow path 14c, and the lead-out part 13b to the apparatus main body 203. The flow rate adjusting valves 23a, 23b, 23c, 23d and the cocks 22a, 22b, 22c, 22d in the apparatus main body 203 are operated so as to circulate through the circulation route returning to the pipe 5.

  The labeled antibody fixed to the insoluble carrier is sent to the reaction unit 11 by this circulation, and reacts with the antigen reacted with the antibody in the reaction unit 11 to form an antibody-antigen-labeled antibody complex. Even in this step, in order to ensure that all the antibody-antigen complexes formed earlier and the labeled antibody react, the labeled antibody is circulated through the circulation route, thereby increasing the chance of reacting with the antibody-antigen complex. ing.

  Steps 5 and 6 may be performed in the same manner as the method described in the first embodiment. If necessary, Step 7 shown in Embodiment Mode 1 may be performed. The competition method can be used in the same manner as in the second embodiment. Moreover, you may provide the some recognition substance temporary storage part set A with a labeled substance. As a detection method in these cases, the method shown in the second embodiment may be adopted.

  Although Embodiment 3 has been described with reference to the device and chip shown in FIG. 4, the present invention is not limited to the above-described device, chip configuration, and method.

[Embodiment 4]
Hereinafter, the fourth embodiment will be described in detail with reference to FIG.

  First, in the case of using a method of capturing and detecting an antigen by a so-called sandwich method in which the antigen is sandwiched between an immobilized antibody and a labeled antibody, a diluting part is provided between the extraction part 10 and the reaction part 11 in FIG. Since the configuration is the same except that is provided, the antigen can be detected in the same manner as the method described in the first embodiment.

  In Embodiment 4, a case where a competitive reaction is used will be described.

  As shown in FIG. 5, the structure of the chip 104 is such that the allergen protein-labeled substance (labeled antigen) temporary storage unit 17 is connected to the mixing unit 16 via the flow path 14e provided with the microvalve 15b. Except that the part 13e and the flow path 14h are not provided, and that the labeled antigen-attached allergen protein temporary storage part 17 is provided with a labeled antigen whose amount is known in advance, and the amount of the immobilized antibody is known in advance. The configuration is the same as that described in the third embodiment.

  The mixing unit can employ the configuration of the dilution unit of the third embodiment as it is.

  As shown in FIG. 5, the configuration of the apparatus main body 204 is provided with a connecting means for connecting the pipe 5e, the valve 23c, and the introduction portion 13e of the chip 104 in accordance with the change in the configuration of the chip 103 of the third embodiment. Except for the absence, it may be the same as the configuration described in the third embodiment.

The antigen detection method using the allergen detection apparatus of Embodiment 4 is demonstrated in detail using FIG.
However, in the following description, it is assumed that the labeled antigen in the allergen protein temporary storage unit 17 with a labeled substance is fixed to an insoluble carrier. If it is not fixed to an insoluble carrier, it may be performed in the same manner as in step 2 ′ of the second embodiment.

  In the allergen detection apparatus according to the fourth embodiment, the allergen detection chip 104 and the apparatus main body 204 can be attached and detached as in the inventions described in the second and third embodiments. Further, when pollen detection is not performed, it is preferable that the chip is refrigerated or frozen in a state where the antigen / antibody is dissolved in the carrier solution or in a solid state so that the antigen / antibody is not inactivated.

Process 1
Except not connecting with 13e, it carries out similarly to the said Embodiment 3. FIG.

Process 2
Next, the microvalves 15a and 15c are opened, 15b is closed, and the carrier liquid in the storage means 2 is extracted by the liquid feeding means 3 through the introduction parts 13a and 13c, the extraction part 10, the reaction part 11, and the labeling substance allergen protein. It flows into the temporary storage unit 17.

  At this time, in the apparatus main body 204, the carrier liquid enters the chip 104 from the introduction section 13a through the pipes 5b and 5f from the storage means 2 in the main body 204, and enters the flow path 14a, the extraction section 10, the flow path 14g, and the mixing section. 16, flow rate adjusting valves 23a, 23b, 23d in the main body 204 so as to flow into the pipe 5 in the main body 204 through the outlet portion 13b through the flow path 14b, the reaction section 11, and the flow path 14c. Operate 22a, 22b, 22c, and 22d. Further, since the cock 22c is controlled so that the carrier liquid also flows through the pipe 5c, the carrier liquid enters from the pipe 5c through the second introduction part 13c of the chip 104, and flows into the channel 14d and the allergen protein with the labeling substance. The pipe 5d of the main body 204 enters the temporary storage part 17, the flow path 14f, and the outlet part 13d. The step of introducing the carrier liquid from the introduction part 13a and the operation of introduction from the second introduction part 13c may be performed simultaneously, or after performing one operation, another operation may be performed. Since the volumes of the extraction unit 10, the reaction unit 11, the labeled substance-attached recognition substance temporary storage unit 12 and the like are known in advance, the control unit 4 flows in a necessary amount and stops the inflow.

In the operation of inserting from the second introduction portion 13c, as a chip configuration, a separation portion having a width or thickness smaller than the size of the carrier on which the labeled antibody is immobilized is provided between the allergen protein 12 with a labeled substance and the flow path 14f. By providing, it is possible to stop the labeled antibody from flowing into the apparatus main body 204.
By the above operation, each flow path, extraction part, reaction part, labeled substance-added allergen protein temporary storage part 17 in the chip can be filled with the carrier liquid, and in the subsequent steps, gas such as air is contained in the carrier liquid. Mixing can be prevented and the reaction can be performed reliably.

Process 3
Next, the microvalve driving means of the main body closes the microvalve 15c and the valve 23a in the apparatus main body 204, isolates the extraction unit 10, and collects pollen and extracts antigens from pollen for a certain period of time. At this time, magnetic particles may be provided in the extraction unit 10 and the magnetic particles may be stirred by a magnet or the like provided in the apparatus main body 204 separately. By doing so, there is an effect that the extraction efficiency can be increased. After a certain time, the lid provided on the apparatus main body 204 is closed, and the pollen collection is finished.

Process 4
Next, the microvalves 15b and 15c are opened. In this state, the liquid feeding means 3 causes the carrier liquid to enter the chip 104 from the introduction part 13a through the pipes 5a and 5f from the storage means 2 in the apparatus main body 204, and to obtain the flow path 14a, the extraction part 10, the flow path 14g, A route that flows through the mixing section 16, the flow path 14b, the reaction section 11, and the flow path 14c through the derivation section 13b and into the pipe 5 in the main body 204, and the storage section 2 through the pipes 5a and 5c, and the introduction section 13c. From the flow path 14d, the labeled allergen protein temporary storage section 17, the flow path 14e, the mixing section 16, the flow path 14b, the reaction section 11, the flow path 14c, and the outlet section 13b to the piping of the apparatus main body 204. The flow rate adjusting valves 23a, 23b, and 23d and the cocks 22a, 22b, 22c, and 22d in the apparatus main body 204 are operated so as to flow along the route back to 5.

  By simultaneously circulating the carrier liquid through the above two routes, the carrier liquid containing the antigen that flows into the mixing section 16 from the extraction section 10 and the labeled antigen that flows into the mixing section 16 from the allergen protein temporary storage section 17 with the labeled substance. The contained carrier liquid is mixed in the mixing section 16 and flows into the reaction section 11 in a state where the antigen and labeled antigen are uniformly mixed in the carrier liquid (mixed carrier liquid). The antigen and the labeled antigen react with each other in competition with the antibody provided in the reaction unit 11, and are immobilized on the immobilized antibody in accordance with the ratio of the antigen to the labeled antigen in the carrier solution.

  Even in a competitive reaction, there is a problem that all antigens and labeled antigens do not react with the antibody in one reaction, but the reaction can be repeated many times by continuously circulating the mixed carrier liquid containing both antigens for a certain period of time. This will ensure competitive reactions and improve the reproducibility and efficiency of the reactions.

  The feature of the configuration of the fourth embodiment is that the antigen and the labeled antibody are sent to the reaction unit 11 in a state where they are uniformly mixed at substantially the same ratio, the amount flowing from the extraction unit 10 to the mixing unit 16 and the allergen protein with the labeling substance The ratio of the antigen and the labeled antibody in the carrier liquid sent to the reaction unit 11 can be changed by adjusting the amount flowing into the mixing unit 16 from the temporary storage unit 17. From this feature, the competitive reaction in the reaction unit 12 As a result, the reproducibility and efficiency of the reaction can be further improved.

Process 5
In order to remove unreacted antigen / labeled antigen from the system, the cock 22a, 22b, 22d and valves 23a, 23b in the apparatus main body 204 are arranged so that the carrier liquid enters the waste liquid reservoir 24 through the cock 22d in the apparatus main body 204. , 23 c and the unreacted labeled antigen-containing carrier liquid is caused to flow into a waste liquid tank 24 provided in the main body 204.

Step 6
What is necessary is just to carry out similarly to the method of the said Embodiment 1. FIG.

  In the case where measurement is performed a plurality of times using the same chip, the mixing unit 16 is used instead of the configuration in which the chip 102 shown in the second embodiment is provided with a plurality of recognizing substance temporary storage unit sets A with a labeled substance around the reaction unit 11. A configuration in which a plurality of allergen protein temporary storage unit sets A with a labeling substance are provided around the substrate may be used.

  Although the fourth embodiment has been described with reference to the apparatus and chip of FIG. 5, the present invention ensures that the reaction is performed by performing the antigen-antibody reaction many times by the circulation process including the reaction part. As long as the effect of obtaining reproducibility of the reaction is obtained, and the same effect can be obtained by the circulation process, the apparatus, the chip configuration, and the method described in the fourth embodiment are not limited. .

(Example 1)
An allergen detection apparatus according to Example 1 was manufactured and its performance was confirmed. Since the basic configuration of Example 1 is the same as that of the first embodiment, the contents described in the first embodiment are omitted, and only more specific contents will be described below.

  Chip 101 was fabricated as follows. A 52 mm square glass substrate was ultrasonically cleaned with isopropyl alcohol. After a resist pattern is formed on this glass substrate by photolithography, the introduction part 41, the lead-out part 42, the extraction part 43, the reaction part 44, and these are connected by wet etching with hydrogen fluoride and ammonium fluoride aqueous solution. The flow path 45,46,47 and the separation part 49 which consists of a some cylinder were produced. The introduction part 41 and the lead-out part 42 were circular holes with a diameter of 3 mm and a depth of 0.1 mm, and the extraction part 43 and the reaction part 44 were circular holes with a diameter of 5 mm and a depth of 0.1 mm. The channels 45 and 46 are grooves having a width of 0.6 mm, a depth of 0.1 mm, and a length of 5 mm, and the channel 47 is a groove having a width of 0.04 mm, a depth of 0.1 mm, and a length of 5 mm. .

Further, a separation portion 49 in which four cylinders 49 having a diameter of about 100 microns were formed between the extraction portion 43 and the flow path 46 with a gap of about 20 microns was formed by a photolithography method and a wet etching method. Furthermore, 5000 plastic beads 50 having a diameter of about 0.05 mm (50 μm) on which a recognition substance (anti-Cry j-1 monochrome antibody: immobilized antibody) having been subjected to a blocking treatment is immobilized are arranged in the reaction portion 44. . Thereafter, the measurement chip was covered with a plastic plate with holes formed at locations corresponding to the introduction / extraction unit and extraction unit 43.
Since the diameter of the plastic bead is larger than the width (0.04 mm) of the flow path 47, the beads do not flow out to the flow path 47 side. Moreover, since the bead diameter is smaller than the length in the depth direction (0.1 mm) of the channel 47, the channel 47 is not blocked.

Four chips 101 were prepared, each of which was incorporated into the apparatus main body 204, and a test was conducted to confirm the reproducibility of the measurement by circulating the carrier liquid. The test conditions are as follows.
<Antigen for detection>
An antigen solution in which Cry j-1 was dissolved in a carrier solution at 1.5 micromolar concentration was prepared, and 2 μl of this solution was put in the extraction unit 43.
<Fixed antibody>
An anti-Cry j-1 monoclonal antibody fixed and held on the plastic beads described above was used. The number of antibodies arranged in the reaction part 44 was made sufficiently larger than the number of antigens to be detected.
<Carrier liquid>
A phosphate buffer solution having a phosphate concentration of 0.01 mol and a pH of 7.4 was used.
<Labeled antibody>
An anti-Cry j-1 monoclonal antibody with a fluorescent dye dissolved at 3 molar concentration in a carrier solution was used.

<Circulation conditions, etc.>
The carrier liquid was circulated at a rate of 2 μl / second for 10 minutes (approximately 4 times). After the extraction reaction step was completed, a washing operation was performed for 3 to 5 minutes with a fresh carrier liquid (see step 3 in Embodiment 1). Thereafter, the anti-Cry j-1 monoclonal antibody solution with the fluorescent dye contained in the recognition substance tank 54 with the labeling substance is introduced into the chip from the introduction part 41, and circulates at 2 μl / second for 10 minutes (approximately 4 circulations). Thereafter, the washing operation for 3 to 5 minutes with the fresh carrier liquid was performed in the same manner as described above (see steps 4 and 5 in the first embodiment). Thereafter, the fluorescence intensity was measured by irradiating the reaction unit 44 with ultraviolet light from the detection means 55 (see step 6 in the first embodiment). Note that matters not described here are the same as those in the first embodiment.

  As a result of the allergen detection test performed using each of the four chips, the measured value between the chips is in the range of 92 to 102% with respect to the theoretical value (the amount of allergen protein put in the extraction unit), and the minimum value It was confirmed that the maximum value error was within 10%.

(Comparative example)
Using the same apparatus as in Example 1 above, in the extraction reaction step (Step 3 in Embodiment 1), the valve 48c is set so that the carrier liquid does not flow out in the state where the antigen and the immobilized antibody are present in the reaction part 43. The reaction was carried out for 10 minutes with the carrier liquid kept stationary in the extraction section 43. In addition, a labeled antibody is introduced in the reaction step with the labeled substance-recognized substance (step 4 in the first embodiment), and the valve 48c is adjusted so that the anti-Cry j-1 monoclonal antibody solution does not flow out. The anti-Cry j-1 monoclonal antibody and the allergen protein complex were reacted for 10 minutes. Except for these, the allergen protein detection test was performed 4 times in the same manner as in Example 1.

  As a result, the measured value between the chips is in the range of 79 to 91% with respect to the theoretical value (the amount of allergen protein put in the extraction part), and the error between the minimum value and the maximum value is 15%. Detection accuracy and reproducibility were poor compared to

  In Example 1, the monoclonal antibody was used for both the antibody immobilized in the reaction part and the labeled antibody reacted with the antigen-antibody complex produced in the reaction part, but either one was a monoclonal antibody and the other was a polyclonal antibody. Of course, it may be an antibody. The antibody to be immobilized is preferably subjected to a blocking treatment so as not to react with other substances.

  As described above, according to the present invention, it is possible to provide a method for detecting allergen that can reproducibly measure the amount and / or type of allergen protein contained in allergen protein particles, and according to the present invention, such a detection method. And a device using this chip can be provided at low cost. Therefore, its industrial significance is great.

1 is a conceptual diagram of an allergen detection device according to Embodiment 1. FIG. FIG. 4 is a conceptual diagram of an allergen detection device according to Embodiment 2. The conceptual diagram of the chip | tip containing a some label | marker part substance storage part set. FIG. 5 is a conceptual diagram of an allergen detection device according to Embodiment 3. FIG. 6 is a conceptual diagram of an allergen detection device according to a fourth embodiment. The schematic fragmentary sectional view of the extraction part which concerns on this invention. The schematic fragmentary sectional view of a microvalve. The schematic fragmentary figure of a dilution part and a mixing part. The conceptual diagram of the allergen detection apparatus which concerns on patent document 6. FIG. The conceptual diagram of the chip for allergen detection which concerns on patent document 7. FIG.

Explanation of symbols

101, 102, 103, 104 Chip for allergen detection 2 Storage means 3 Liquid feeding means 4 Control means 5, 5a, 5b, 5c, 5d, 5f Piping 6a, 6b, 6c, 6d, 6e Connecting means 7, 55 Detection means 8 Magnetic particle 9 Intake hole 10, 43 Extraction unit 11, 44 Reaction unit 12 Recognized substance temporary storage unit with labeling substance 13a, 13c, 13e, 41 Introduction unit (second introduction unit)
13b, 13d, 13f, 42 Deriving part 14a, 14b, 14c, 14d, 14e, 14f, 14g,
14h, 45, 46, 47 Flow path 15a, 15b, 15c Micro valve 16 Mixing unit 17 Temporary allergen protein storage unit 18 with labeling substance 18 Dilution unit 201, 202, 203, 204 Main unit 22a, 22b, 22c, 22d, 48 Cock 23a, 23b, 23c, 23d Valve 24 Waste liquid reservoir 31 Lid 40 Substrate 49 Separation part 51 Distribution pipe 52 Pump 53 Carrier liquid tank 54 Recognized substance solution tank with labeling substance 56 Waste liquid tank 57 Allergen protein solution tank with labeling substance

Claims (20)

An extraction step for extracting the allergen protein into the carrier liquid from the allergen substance taken into the extraction unit from the external environment;
The extraction carrier liquid containing the allergen protein is introduced into a reaction part that is held in a state where a recognition substance that specifically reacts with the allergen protein is not washed away, and the allergen protein and the recognition substance are reacted to be recognized. A complex generating step for generating a substance-allergen protein complex;
An allergen protein detection method comprising an allergen protein detection pretreatment step comprising:
The method for introducing an extraction carrier liquid in the complex generation step is a method in which the extraction carrier liquid containing the allergen protein is circulated and flowed into the reaction section twice or more. The method for detecting an allergen protein. An extraction step for extracting the allergen protein into the carrier liquid from the allergen substance taken into the extraction unit from the external environment;
The extraction carrier liquid containing the allergen protein is introduced into a reaction part that is held in a state where a recognition substance that specifically reacts with the allergen protein is not washed away, and the allergen protein and the recognition substance are reacted to be recognized. A complex generating step for generating a substance-allergen protein complex;
An allergen protein detection method comprising an allergen protein detection pretreatment step comprising:
The carrier liquid is circulated between the extraction part and the reaction part, whereby the allergen protein extraction and the reaction between the allergen protein and the recognition substance are repeated twice or more with the same carrier liquid. To detect allergen protein. After the complex generation step, instead of the carrier solution containing the allergen protein, the carrier solution containing a recognition substance with a labeling substance is circulated twice or more to the reaction part, whereby the recognition with the labeling substance is performed in the reaction part. The method for detecting an allergen protein according to claim 1 or 2, further comprising a labeling substance reaction step in which a substance reacts with the recognition substance-allergen protein complex. The method for detecting an allergen protein according to claim 3, further comprising a washing step of washing the reaction part by flowing a carrier solution between the complex generation step and the labeling substance reaction step. An extraction step for extracting the allergen protein into the carrier liquid from the allergen substance taken into the extraction unit from the external environment;
A mixing step of mixing the extraction carrier liquid containing the allergen protein and the carrier liquid containing the allergen protein with a labeling substance whose content is known in advance to form a mixed carrier liquid;
It is a recognition substance that specifically reacts with the allergen protein, and the mixed carrier liquid is introduced into a reaction part that is fixed in a state where the recognition substance whose amount is known in advance is not washed away, A complex generating step of generating a recognition substance-allergen protein complex and a recognition substance-labeling substance allergen protein complex by competitively reacting the allergen protein and the labeling substance-containing allergen protein;
An allergen protein detection method comprising an allergen protein detection pretreatment step comprising:
The method for detecting an allergen protein, wherein the method for introducing the mixed carrier liquid in the complex generation step is a method in which the mixed carrier liquid is circulated and flowed into the reaction section twice or more. Mixing in the said reaction part is performed in the said reaction part, and the mixed carrier liquid mixed in the said reaction part is made to circulate and flow into the said reaction part 2 times or more, The allergen protein detection method characterized by the above-mentioned. An introduction part having an introduction port for introducing a carrier liquid into the chip from outside the chip;
An extraction unit that communicates with the introduction unit so that a carrier liquid can flow in from the introduction unit and has an opening for taking in allergen-containing particles, and extracts allergen protein from the allergen-containing particles into the carrier solution;
A reaction part that is communicated with the extraction part so that a carrier liquid can flow in from the extraction part, and is held in a state in which a recognition substance that specifically reacts with an allergen protein does not flow out;
A lead-out part that is located downstream of the reaction part and has a lead-out port for leading the carrier liquid out of the chip;
Is an allergen detection chip formed on a single substrate. The allergen detection chip further has a circulation channel for circulating a carrier liquid between the introduction part and the lead-out part.
The allergen detection chip according to claim 7. The allergen detection chip further includes a second introduction part and a labeling substance temporary storage part for temporarily storing a recognition substance with a labeling substance, and the labeling substance temporary storage part is located inside the chip from outside the chip. Communicated with the second introduction part for introducing the carrier liquid, and also communicated with the reaction part so that the carrier liquid introduced from the second introduction part can flow into the reaction part,
The allergen detection chip according to claim 7. The allergen detection chip further includes a second introduction part and a labeled substance allergen protein temporary storage part for temporarily storing a known amount of the allergen protein with a labeling substance, and the allergen with the labeling substance The protein temporary storage unit communicates with a second introduction unit that introduces a carrier liquid into the chip from the outside of the chip, and the carrier liquid introduced from the second introduction unit can flow into the reaction unit. It communicates with the reaction part,
The amount of the recognition substance present in the reaction part is known in advance;
The allergen detection chip according to claim 7. The allergen detection chip further includes a mixing unit that mixes the carrier liquid containing the allergen protein flowing out from the extraction unit and the allergen protein flowing out from the allergen protein temporary storage unit with the labeled substance, the extraction unit and the reaction Between the reaction part and the allergen protein temporary storage part with the labeling substance,
The chip for allergen detection according to claim 10. The allergen detection chip further includes a carrier liquid that has flowed out of the extraction unit, and a dilution unit that is diluted with a carrier liquid introduced from other than the extraction unit, between the extraction unit and the reaction unit.
The allergen detection chip according to claim 7. The allergen detection chip further has an opening / closing means for blocking or passing the flow of the carrier liquid between the mixing unit and the reaction unit.
The allergen detection chip according to claim 11. The allergen detection chip further has an opening / closing means for blocking or passing the flow of the carrier liquid between the dilution section and the reaction section.
The allergen detection chip according to claim 12. The allergen detection chip is provided with separation means for removing insoluble substances in the carrier liquid between the extraction unit and the reaction unit.
The allergen detection chip according to claim 7, 9, 10, 11, 12, 13 or 14. The separation means is a flow path configured such that the minimum diameter of the flow path existing between the extraction section and the reaction section is smaller than the maximum diameter of the insoluble substance existing in the carrier liquid.
The allergen detection chip according to claim 15. An allergen detection chip according to claim 7, 9, 10, 11, 12, 13, 14, 15 or 16,
A carrier liquid storage section for storing the carrier liquid;
A distribution piping group for distributing the carrier liquid;
Liquid feeding means for circulating the carrier liquid in the distribution pipe group;
Detection means for detecting the amount and / or type of allergen protein present in the reaction part of the allergen detection chip;
With
The carrier liquid storage unit, the liquid feeding means, the introduction part of the allergen detection chip, and the lead-out part of the allergen detection chip are connected by the distribution pipe group, and the carrier liquid can circulate and circulate. An allergen detection device in which a path is formed. The allergen detection apparatus further includes a recognition substance-temporary storage unit with a labeling substance that stores a recognition substance with a labeling substance that specifically reacts with the allergen protein.
The allergen detection device according to claim 17. The allergen detection device further has a recognition substance temporary storage unit with a labeling substance for storing a known amount of the allergen protein with a labeling substance in advance.
The allergen detection device according to claim 17. The distribution pipe group includes connection means that can be detachably connected to the introduction part and the lead-out part of the allergen detection device,
The allergen detection device according to claim 17, 18 or 19.

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