WO2017168506A1

WO2017168506A1 - SPECIFIC IgE INSPECTION METHOD AND SPECIFIC IgE INSPECTION DEVICE

Info

Publication number: WO2017168506A1
Application number: PCT/JP2016/059881
Authority: WO
Inventors: 内田　剛; 仁美清水
Original assignee: 日立化成株式会社
Priority date: 2016-03-28
Filing date: 2016-03-28
Publication date: 2017-10-05
Also published as: JPWO2017168506A1

Abstract

This specific IgE inspection method has a step for causing a specimen to come into contact with a plurality of items of allergens that are each individually fixed to a single inspection chip, a step for imaging the area of the inspection chip to which the plurality of items of allergens have been fixed and optically detecting reactions between the specimen and allergens, a step for selecting items on which to carry out specific IgE inspection from among the information for the plurality of items of allergens subjected to optical detection, and a step for carrying out specific IgE inspection on the selected items.

Description

Specific IgE test method and specific IgE test device

The present invention relates to a specific IgE inspection method and a specific IgE inspection apparatus.

When making a treatment policy for allergies, it is important to identify allergens that the patient is sensitizing from among allergens. Therefore, as shown in

Non-Patent Documents

1 and 2, allergy diagnostic agents and devices capable of simultaneously performing allergy diagnosis related to multiple items of allergens have been studied. In the allergy diagnostic drug and apparatus described in

Non-Patent Documents

1 and 2, specific IgE (immunoglobulin) corresponding to the allergen is measured, and diagnosis for allergy is performed.

When trying to identify allergens without increasing the load on the patient, it is required to test specific IgE corresponding to more allergens at the same time. On the other hand, there is a need in the field where it is desired to limit the items to be tested for specific IgE.

The present invention has been made in view of the above, and an object thereof is to provide a specific IgE inspection method and a specific IgE inspection apparatus capable of changing the number of inspection items in accordance with the needs of the user.

In order to achieve the above object, a specific IgE test method according to an aspect of the present invention includes a step of bringing a specimen into contact with a plurality of allergens individually immobilized on one test chip, Imaging the region where the allergens of the plurality of items on the test chip are immobilized, optically detecting a reaction between the specimen and the allergen, and the optically detected allergens of the plurality of items Of such information, the method includes a step of selecting an item to be tested for specific IgE against the allergen, and a step of performing a test on specific IgE against the allergen for the selected item.

Moreover, the specific IgE test | inspection apparatus which concerns on one form of this invention is the area | region where the said several item allergen was fixed in the test | inspection chip which contacted the specimen with respect to each of the several item allergen fixed individually. An imaging unit that optically detects a reaction between the specimen and the allergen, and specific IgE for the allergen among the information on the allergen of the plurality of items optically detected by the imaging unit A test item setting unit that selects an item to be tested, and a test unit that performs a test related to specific IgE against the allergen for the item selected by the test item setting unit.

According to the specific IgE test method and the specific IgE test apparatus described above, a plurality of allergens immobilized on a test chip are brought into contact with a specimen, and then the plurality of allergens optically detected by imaging are detected. Based on such information, it is possible to set an item to be tested before performing a test related to specific IgE. Therefore, it is possible to perform a test relating to specific IgE only for a preset item. Thus, the number of inspection items can be changed according to the user's needs, and the convenience for the user is improved.

Here, the inspection chip may be a flow-through microarray.

As described above, by adopting a once-through microarray as an inspection chip, a chip capable of inspecting a plurality of items can be manufactured with a simpler configuration. In addition, since the test chip of the once-through microarray can image the reaction results of multiple items of allergen and the sample at a time, the test can be performed more easily. Furthermore, by using the flow-through type, the reaction can be accelerated by increasing the contact opportunities between the allergen and the sample on the surface of the test chip or the complex of the allergen and the sample and the labeled antibody.

Further, the inspection chip may have a plurality of reaction regions in which allergens of the same item are immobilized.

As described above, by having a plurality of reaction regions in which allergens of the same item are immobilized, it is possible to obtain a test result related to specific IgE for allergens based on the results of the plurality of reaction regions. It is possible to prevent an error in the inspection result.

According to the present invention, there are provided a specific IgE inspection method and a specific IgE inspection apparatus capable of changing the number of inspection items according to the user's needs.

It is a figure explaining the specific IgE test | inspection system containing the specific IgE test | inspection apparatus which concerns on embodiment of this invention. It is a figure explaining the structure of a test | inspection chip. It is a figure explaining schematic structure of the device for pretreatment. It is a figure explaining an example of reaction in a test chip. It is a flowchart explaining the specific IgE inspection method which concerns on embodiment of this invention.

Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted.

FIG. 1 is a diagram illustrating a specific IgE inspection system including a specific IgE inspection apparatus according to an embodiment of the present invention. As shown in FIG. 1, the specific IgE test system 1 includes a test chip 2 and a specific IgE test device 3. Further, the specific IgE inspection apparatus 3 includes an imaging unit 31, an inspection item setting unit 32, an inspection unit 33, and an output unit 34.

The specific IgE test system 1 is a system that performs a test related to IgE (specific IgE) specific to a substance (allergen) that causes allergy using a specimen O that is a liquid sample derived from serum in blood or the like. It is. By contacting the allergen immobilized on the test chip 2 and the sample O, specific IgE in the sample O is reacted. Thereafter, it is reacted with an enzyme-labeled secondary antibody to form an allergen-specific IgE-enzyme-labeled antibody complex. Then, the amount of specific IgE reacted with the allergen is determined by measuring the amount of light emitted by reacting the complex with a luminescent reagent (for example, luminol and hydrogen peroxide) by the specific IgE test apparatus 3. Since allergens of a plurality of items are immobilized on the test chip 2, it is possible to evaluate the allergens of the plurality of items by a test using the test chip 2.

Examples of the specimen O used in the specific IgE test system 1 include blood (whole blood), serum in blood, plasma, and liquid obtained by mixing serum or plasma with a diluent or the like. Moreover, the allergen to be examined is not particularly limited.

The inspection chip 2 will be described with reference to FIG. 2A is a plan view of the inspection chip 2, and FIG. 2B is an enlarged view of a part of the inspection chip 2. FIG. 2C is a view of one through-hole 21 in the inspection chip 2 as viewed from above, and FIG. 2D is a view of the through-hole 21 as viewed from side. As shown in FIGS. 1 and 2A, the test chip 2 is a plate-like member having a main surface formed of silicon and having a square shape. Although the material of the test | inspection chip 2 is not specifically limited, Usually used plastics, such as a silicon | silicone and a polystyrene, can be used as a main component. Also, a resin material such as nylon or polypropylene having a network structure can be used as the inspection chip 2. The term “main component” means that the proportion of the component is 50% by mass or more.

The outer shape of the inspection chip 2 is about 3 mm to 15 mm on one side. Further, the thickness of the inspection chip 2 is about 200 μm to 1 mm.

In the inspection chip 2, a plurality of through holes 21 are formed in the thickness direction, and allergens are immobilized on the inner surfaces of the plurality of through holes 21. That is, the test chip 2 is a so-called flow-through microarray, and the through hole 21 is a reaction region between the specimen O and the allergen. Although the shape of the through hole 21 is not particularly limited, for example, as shown in FIGS. 2B and 2C, the cross-sectional shape on the surface orthogonal to the thickness direction can be a square.

Further, it is preferable that the shape (cross-sectional shape and shape in the thickness direction) of the inner surface of the through hole 21 is a shape common to the through holes 21. By making the inner wall shape between the through holes 21 common, it is possible to improve the accuracy of the inspection related to specific IgE.

The through-hole 21 of the inspection chip 2 can be, for example, a square with a cross section of 10 μm on a side and a thickness of 500 μm. The wall portion 22 that partitions the through holes 21 has a thickness of about 5 μm. The cross-sectional shape and size of the through hole 21 can be changed as appropriate. However, when the specimen O passes through the through-hole 21, the allergen immobilized on the inner surface of the through-hole 21 comes into contact with the specimen O, and specific IgE in the specimen O reacts. When the contact area between the allergen and the specimen O is large, the reaction between both is promoted. Therefore, it is preferable to design the cross-sectional area of each through-hole 21 to be small so that the total area of the inner surface of each through-hole 21 is large.

The above-described inspection chip 2 can be manufactured by etching using a known semiconductor manufacturing process. By using a method similar to that of the semiconductor manufacturing process, it is possible to provide a very small through-hole 21 adjacently as described above. Moreover, the surface treatment is performed by performing a thermal oxidation treatment after the etching. After the surface treatment is performed, the allergen is immobilized on the inner surface of the through hole 21. The thermal oxidation process is performed as necessary, and is not an essential process.

As shown in FIGS. 2C and 2D, the allergen X is physically adsorbed, electrostatically adsorbed, or chemically immobilized on the entire inner surface of the through hole 21. The method for immobilizing the allergen is not particularly limited as long as it can immobilize the allergen.

In the test chip 2, different types of allergens are not immobilized in the adjacent through-holes 21, but the same allergen is immobilized on the plurality of through-holes 21 included in a specific region. In the case of the test chip 2 shown in FIG. 2, the through-hole 21 in which one type of allergen is immobilized is designed to be a circular spot region R1 having a diameter of about 100 μm. That is, allergens of the same item are fixed to the plurality of through holes 21 included in the spot region R1. In the case of this embodiment, about thirty through holes 21 are included. The test chip 2 is provided with a plurality of spot regions R1 to RN (RN is not shown), and different allergens are immobilized for each spot region. Accordingly, the number of items that can be simultaneously inspected by the inspection chip 2 (the number of allergen types) is determined according to the number of spot regions on the inspection chip 2.

When evaluating the light emission amount, the light emission from the plurality of through holes 21 included in the spot region R1 is evaluated as one light emission. By evaluating the light emission from the plurality of through holes 21 as one light emission, the amount of light emission to be detected can be increased, so that the detection accuracy is improved. Moreover, in the test | inspection chip 2, you may provide multiple spot area | regions comprised from the several through-hole 21 to which the allergen of the same item was fix | immobilized. For example, it is good also as a structure used for the test | inspection by the allergen of the same item of four adjacent (2x2) spot areas. In that case, it is possible to obtain a result related to specific IgE based on the test results in a plurality of spot regions, and it is possible to obtain the test result more accurately and to prevent an error in the test result. It becomes.

In addition, as shown in FIG. 2 (B), the spot region R1 is between the circular spot region R2 formed by the through-hole 21 on which the allergen of a different type from the allergen of the spot region R1 is immobilized. In addition, an interval of about 100 μm is designed. As described above, the spot regions formed by the group of through-holes 21 on which different types of allergens are immobilized are arranged apart from each other, thereby preventing both from mixing. Moreover, the light emission amount for each spot can be accurately measured. The size and interval of the spot area can be changed as appropriate.

In order to bring the allergen immobilized in the through hole 21 of the test chip 2 into contact with the specimen O, the specimen O needs to pass through the through hole 21. A method for allowing the specimen O to pass through the through hole 21 is not particularly limited, but FIG. 3 shows an example of a pretreatment device that can be used when the specimen O is passed through the through hole 21.

The pretreatment device 4 shown in FIG. 3A can be attached to a cylindrical housing 41 having an open top so that the main surface of the inspection chip 2 blocks the cross section of the housing 41. Is. A liquid discharge port 42 is provided below the mounting position of the inspection chip 2 in the housing 41. When such a pretreatment device 4 is used, the sample O is introduced from above the test chip 2 and discharged from the lower discharge port 42, thereby passing the sample O into the through hole 21 of the test chip 2. Can be made. However, it is also conceivable that the contact between the allergen and the specimen O is insufficient only by passing the specimen O once through the through hole 21. Therefore, it is desirable to have a configuration in which the passage is repeated many times.

FIG. 3B shows an example of a pretreatment device that allows the specimen O to pass through the through hole 21 a plurality of times. The preprocessing device 5 can be attached to a cylindrical casing 51 whose end is closed so that the main surface of the test chip 2 blocks the cross section of the casing 51. A pump P is connected to an end on one side (upper side in the figure), and an inlet 52 and an outlet 53 for the specimen O are provided on the other side (lower side in the figure). The introduction port 52 and the discharge port 53 can be opened and closed by a valve or the like. In such a pretreatment device 5, the sample O is introduced into the housing 51 from the introduction port 52 and then the introduction port 52 is closed, and the suction and discharge in the housing 51 by the pump P are repeated, thereby penetrating. The specimen O reciprocates between one side (upper side in the figure) and the other side (lower side in the figure) in the casing 51 through the hole 21. Thereby, the specimen O can be passed through the through hole 21 of the test chip 2.

The

pretreatment devices

4 and 5 shown in FIG. 3 can be used when the specimen O is passed through the through-hole 21 and also when the enzyme-labeled secondary antibody and the luminescent reagent are sequentially introduced into the through-hole 21. Can also be used. Note that the

pretreatment devices

4 and 5 shown in FIG. 3 are merely examples, and a mechanism different from the

pretreatment devices

4 and 5 when the specimen O and other reagents are allowed to pass through the through-hole 21. You may use the apparatus which has.

Referring back to FIG. 1, the specific IgE inspection apparatus 3 will be described. The imaging unit 31 of the specific IgE test apparatus 3 images the test chip 2 that has been processed with the sample O and other reagents as described above, and detects the emission intensity in each spot region. As the imaging unit 31, a weak light emission measuring device (luminometer), a CCD (Charge-Coupled Device) image sensor, a CMOS (Complementary MOS) image sensor, or the like can be used. In the imaging part 31, the light from the test | inspection chip 2 is light-received in the light-receiving surface where a light-receiving pixel is arrange | positioned two-dimensionally. Thereafter, charges generated by light reception on the light receiving surface are digitally converted by an A / D converter, and digital information indicating a distribution of light reception intensity of light in each pixel is generated. Thereby, the information regarding the light emission amount in each position of the test | inspection chip 2 is acquired.

FIG. 4 is a diagram schematically showing information related to the light emission amount for each spot area detected by imaging by the imaging unit 31. In FIG. 4, the spot regions R1 to RN are shown to become black as the emission intensity increases. For spot areas where no light emission is detected, the areas corresponding to the spots are indicated by broken lines. In this way, a result in which the light emission amount in the spot region is different is obtained.

The test item setting unit 32 has a function of presetting an item (allergen) to be evaluated for the amount of specific IgE based on the amount of luminescence. The item setting may be performed in advance by a user of the specific IgE inspection device 3 before imaging the inspection chip 2, or may be configured by the user setting the item after imaging.

The inspection unit 33 calculates the amount of specific IgE based on the amount of luminescence for each item (allergen) set as the inspection target in the inspection item setting unit 32 using the result captured by the imaging unit 31. (Perform quantitative measurement). Furthermore, in the test | inspection part 33, it is good also as a structure which performs the process of applying a rank etc. according to the quantity of the specific IgE which concerns on the calculated allergen.

The output unit 34 outputs the result of the examination related to specific IgE in the examination unit 33. Examples of the output method include a method of displaying on a monitor and the like, a method of transmitting to an external device such as a printer, and the like.

The inspection item setting unit 32, the inspection unit 33, and the output unit 34 of the specific IgE inspection device 3 are a CPU (Central Processing Unit), a RAM (Random Access Memory) and a ROM (Read Only Memory) that are main storage devices, and others. A computer including a communication module that performs communication with other devices and hardware such as an auxiliary storage device such as a hard disk can be configured. When these components operate, functions as the test item setting unit 32, the test unit 33, and the output unit 34 of the specific IgE test apparatus 3 are exhibited.

The specific IgE test method using the specific IgE test system 1 described above includes a step of bringing a specimen into contact with a plurality of allergens individually immobilized on one test chip, and a plurality of test chip chips. Imaging the region where the allergen of the item is immobilized, optically detecting the reaction between the specimen and the allergen, and among the information on the optically detected allergen of the plurality of items, specific IgE for the allergen The method includes a step of selecting an item to be tested, and a step of performing a test on specific IgE against allergen for the selected item. A specific procedure will be described with reference to FIG.

First, in the specific IgE inspection apparatus 3, an item to be inspected is selected by a user or the like (S01). Next, the specimen O and the test chip 2 are brought into contact with each other using the pretreatment device 4 or the like, and the specimen O is introduced into the through hole 21 (S02). Thereby, the sample O and the allergen in the through-hole 21 contact. Thereafter, the secondary antibody and the luminescent reagent are sequentially passed through the through-hole 21 after the specimen O has passed, thereby performing a process of emitting specific IgE reacted with the allergen (S03).

Thereafter, the test chip 2 in which the light emission amount changes for each spot region is imaged by the imaging unit 31 of the specific IgE inspection device 3, and the light emission amount for each spot region is detected (S04). Then, in the inspection unit 33, for the item (allergen) set as the inspection object in the inspection item setting unit 32, an inspection related to specific IgE including calculation of the amount of specific IgE based on the amount of luminescence is performed (S05). ). Thereafter, the inspection result is output from the output unit 34 (S06). Thereby, a series of operations related to the examination of specific IgE is completed.

Thus, according to the specific IgE test apparatus according to the present embodiment, the test item setting unit 32 sets the items to be tested in advance before the test unit 33 performs the test related to the specific IgE against the allergen. And in the test | inspection part 33, it test | inspects only about the specific IgE about the item set in the test | inspection item setting part 32, It is characterized by the above-mentioned.

Conventionally, allergy diagnostic agents and devices capable of simultaneously performing allergy diagnosis related to multiple items of allergens have been studied. A diagnostic method using such a diagnostic agent is useful for screening causal allergens of patients with allergic diseases because it can perform tests related to allergens of multiple items at once.

On the other hand, there is a need to perform inspection by limiting the items for specific IgE inspection. For example, a patient whose antigen allergen candidates are specified to some extent may be examined only for the specified allergen. In addition, allergens that can be antigens vary from region to region. In this way, there is an increasing need to limit the items to be subjected to specific IgE inspection depending on the situation.

However, preparing individual inspection chips according to the various needs of users leads to higher costs for the inspection chips. Moreover, since it is still impossible to set the examination items individually as in each patient, it has been difficult to sufficiently satisfy the user's needs.

On the other hand, according to the specific IgE test system 1 including the specific IgE test apparatus 3 and the specific IgE test method according to the present embodiment, before the test of the specific IgE against the allergen by the test unit 33, The items to be inspected can be set in advance in the inspection item setting unit 32. In addition, the inspection unit 33 can perform an inspection related to specific IgE for allergens only for the items set in the inspection item setting unit 32. Thus, the number of inspection items can be changed according to the user's needs, and the convenience for the user is improved. Even when the inspection item changes, the target item can be selected on the side of the specific IgE inspection apparatus 3, so that the inspection can be performed using the same inspection chip 2. Therefore, it is not necessary to manufacture the inspection chip 2 according to the user, and a configuration for changing the number of inspection items or the inspection items according to the user's needs can be realized at a lower cost.

Further, by adopting a through-flow type microarray provided with the through-holes 21 in the thickness direction as described above as the inspection chip 2, a chip capable of performing inspections related to a plurality of items is manufactured with a simpler configuration. can do. In addition, the cross-flow configuration increases the chance of contact between the allergen on the surface of the test chip and the sample, or between the allergen-sample complex and the labeled antibody, thereby accelerating the reaction. can do. In addition, since the flow-through microarray test chip 2 can capture the reaction results of multiple items of allergens and the sample O at once by the imaging unit 31 of the specific IgE test apparatus 3, the test is performed more easily. be able to.

Further, the above-described inspection chip 2 has a plurality of spot regions constituted by a plurality of through holes 21 to which allergens of the same item are immobilized. By having a plurality of spot regions by the through-holes 21 to which allergens of the same item are fixed and performing a test related to specific IgE for allergens for each spot region, it is possible to prevent test results from being erroneous. Become.

The present invention has been described in detail above based on the embodiments. However, the present invention is not limited to the above embodiment. The present invention can be modified in various ways as described below without departing from the scope of the invention.

For example, in the above embodiment, the case where the inspection chip 2 is a once-through microarray has been described, but the shape of the inspection chip 2 can be changed as appropriate. The configuration of the test chip 2 can be changed as appropriate as long as at least a plurality of allergens are immobilized and the specimen O and the allergen can contact each other. As a configuration that can be used as an inspection chip in a shape different from that of the once-through microarray, for example, a chip in which a plurality of allergens are immobilized on a flat substrate is used as the inspection chip according to the present embodiment. In addition, a chip in which a plurality of allergens are immobilized on a substrate having an uneven surface can be used as the inspection chip according to the present embodiment.

In the above embodiment, the configuration in which the setting of the inspection item is performed before the imaging of the inspection chip 2 has been described. However, the configuration may be such that the inspection item is set (selected) after the imaging of the inspection chip 2. In addition, after the imaging of the inspection chip 2, when the information related to the light emission amount for each spot region is held by the specific IgE inspection device 3, the inspection related to specific IgE using the information is repeated. Can be done. In such a case, for example, only a representative allergen is evaluated as a screening stage, and at a stage where the field of a substance that is assumed to be an allergen can be narrowed to some extent, the evaluation related to the allergen in the relevant field is made more detailed. It is also possible. In this way, the timing for setting the inspection item can be changed as appropriate.

In the above embodiment, the reaction between the allergen and the specific IgE of the specimen O is measured as the amount of luminescence using a luminescent reagent. However, instead of using a luminescent reagent, the measurement is performed using a fluorescent reagent or the like. It may be changed. That is, the reaction between the allergen and the specific IgE of the specimen O can be appropriately changed within a range that can be detected optically. When a light source is necessary for optical detection, the imaging unit 31 may have a light source.

DESCRIPTION OF SYMBOLS 1 ... Specific IgE test | inspection system, 2 ... Test chip, 21 ... Through-hole, 22 ... Wall part, 3 ... Specific IgE test | inspection apparatus, 31 ... Imaging part, 32 ... Test item setting part, 33 ... Test | inspection part, 34 ... Output part.

Claims

A step of bringing a specimen into contact with a plurality of allergens individually immobilized on one test chip;
Imaging a region where the allergens of the plurality of items in the test chip are immobilized, and optically detecting a reaction between the specimen and the allergen;
A step of selecting an item to be tested for specific IgE against the allergen among the information on the allergen of the plurality of items detected optically;
Performing a test relating to specific IgE against the allergen for the selected item;
A specific IgE test method comprising:
The specific IgE inspection method according to claim 1, wherein the inspection chip is a once-through microarray.
The specific IgE test method according to claim 2, wherein the test chip has a plurality of reaction regions in which allergens of the same item are immobilized.
An area where the plurality of allergens are immobilized on the test chip in which the specimen is brought into contact with each of the plurality of individually immobilized allergens is imaged, and the reaction between the specimen and the allergen is optically detected. An imaging unit to detect;
A test item setting unit for selecting an item to be tested for specific IgE against the allergen among the information on the allergen of the plurality of items optically detected by the imaging unit;
For the item selected by the test item setting unit, a test unit that performs a test related to specific IgE against the allergen;
A specific IgE testing device comprising: