JP2022046827A - Test piece for immunochromatography and immunochromatographic detection kit - Google Patents

Abstract

To provide an immunochromatographic test piece capable of preventing the measured values from varying since conjugate held in the immunochromatographic test piece can react with an object to be detected by rapid elusion when a sample solution passes therethrough, and fluctuation in the amount of conjugate contributing to the reaction is less.SOLUTION: Provided is an immunochromatographic test piece having a porous membrane which includes at least a sample supply part, a development part and a detection part. In a part of the development part, conjugate containing the specific binding substance labeled with the labeling substance is held in a dry state so as to be capable of being eluted with glucose.SELECTED DRAWING: None

Description

The present invention relates to a test piece for immunochromatography and an immunochromatographic detection kit including the test piece. More specifically, the present invention relates to a test piece for immunochromatography in which a conjugate coexists with a specific sugar, and an immunochromatographic detection kit containing the test piece.

With the spread of POCT (Point of Care) in which treatment is performed near a patient, a lateral flow type laterochromatography detection method using a test piece such as a nitrocellulose membrane (hereinafter, may be simply referred to as an immunochromatographic detection method) has become widespread. is doing.
A test piece for an immunochromatographic detection method (hereinafter, may be simply referred to as an immunochromatographic test piece) generally includes a membrane made of a porous body including at least a sample supply part, a development part, and a detection part. A labeled antibody (conjugate) that forms a complex with the detection target is held in an elutionable manner at the development start site of the development portion, and the antibody is immobilized on a part of the downstream side of the development portion to be detected. Consists of.
Here, when the sample is dropped onto the sample supply section, if the sample contains a detection target, the detection target specifically binds to the labeled antibody at the developing section to form a complex. The complex binds to the immobilized antibody at the detection part while expanding the developing part toward the downstream direction. As described above, in the detection unit, a sandwich type complex is formed by the labeled antibody, the detection target and the immobilized antibody, and by detecting the complex, the detection target can be qualitatively or quantitatively analyzed. ..
An example of a labeled substance constituting a labeled antibody is colloidal gold particles, and qualitative detection is possible by a color reaction by the colloidal gold particles. Furthermore, it is also possible to quantitatively detect the object to be detected in the sample based on the degree of color development. The labeled antibody (conjugate) is generally held in a dried state on a member called a conjugate pad, and the pad constitutes a part of an immunochromatographic test piece.

By the way, in an immunochromatographic test piece, when a conjugate that has been dried and held in a developed portion elutes due to the addition of a sample, conventionally, the dissolution rate of the conjugate varies from test to test, and the amount of conjugate that can react with the object to be detected varies. There was a problem that the measured values varied widely, probably because of this.
As an improved technique for a detection reagent containing such a conjugate, the following techniques of Patent Documents 1 to 3 are known.
Patent Document 1 describes, as an immunoassay latex composition for preventing spontaneous aggregation of antibody-bound latex particles and enabling highly sensitive immunoassay, in addition to latex particles, a buffer solution, and a protein, saccharose. , Trehalose, maltose, lactose, sorbitol, D-mannitol, and polyhydric alcohols are disclosed. However, the types of anti-aggregation agents are limited, and among them, only saccharose, trehalose, and glycerin have been confirmed to have an anti-aggregation effect by tests. In addition, no studies have been conducted on the variation in measured values.
Patent Document 2 describes water-soluble polysaccharides having a 1 wt% viscosity of 0.1 cP or more and 300 cP or less in the range of 0.001 wt% or more and 20.00 wt% or less in order to achieve both high sensitivity and prevention of false positive reactions. A developing solution for immunochromatography containing a detection reagent containing and labeled with a carrier such as colloidal gold particles having an average particle diameter of 1 nm or more and 60 nm or less has been disclosed, and CMC is mentioned as a water-soluble polysaccharide having such characteristics. Has been done. However, the effects of water-soluble polysaccharides other than CMC are unknown, and the variation in measured values has not been investigated at all.
In Patent Document 3, an antibody is immobilized on an insoluble carrier such as polystyrene beads as a solid phase immunoreagent capable of maintaining high stability for a long period of time in a dry state, and then immersed in a buffer solution containing a specific sugar. Then, the reagent obtained by removing the buffer solution and then freeze-drying is disclosed. However, only trehalose has been actually tested and confirmed to be effective, and its effects on other sugars are unknown. In addition, as for the confirmation of the effect, only the stabilizing effect comparing the residual activity before and after the humidified storage has been examined, and the variation in the measured values has not been examined.
Patent Document 4 discloses an immunochromatographic test piece comprising a conjugate pad impregnated with sucrose and a detection reagent containing a conjugate in which an antibody is sensitized to colloidal gold. However, there is no particular disclosure about the role of sucrose, and there is no suggestion for other sugars.

Japanese Unexamined Patent Publication No. 2007-315883 Japanese Patent No. 6192374 Japanese Patent Application Laid-Open No. 2003-215127 International Publication 2012/0437 46 Pamphlet

An object of the present invention is to provide an immunochromatographic test piece in which a conjugate held in an immunochromatographic test piece is rapidly eluted by passing a sample solution, the amount of conjugate contributing to the reaction is small, and variation in measured values can be prevented. And.

In order to solve the above problems, we conducted intensive research on substances that coexist with the conjugate. It was found that the fluctuation of the conjugate elution amount is small, and as a result, the fluctuation of the detected value can be prevented. It was also found that the aggregation of the conjugate itself can be suppressed by coexisting the conjugate with glucose, and the present invention has been completed. That is, the present invention has the following configuration.
(1) An immunochromatographic test piece provided with at least a sample supply unit, a development unit, and a detection unit.
An immunochromatographic test piece in which a conjugate containing a specific binding substance labeled with a labeling substance is held in a dry state so that it can be eluted together with glucose in a developing portion.
(2) The immunochromatographic test piece according to (1), which comprises a conjugate pad in which the conjugate is kept dry so that it can be eluted together with glucose as a developing portion.
(3) The immunochromatographic test piece according to (2), wherein the conjugate pad is a glass fiber pad.
(4) The immunochromatographic test piece according to any one of (1) to (3), wherein the labeling substance is colloidal gold.
(5) The Lateral Flow Test Piece according to any one of (1) to (4), wherein the specific binding substance is an antibody.
(6) A detection kit for an immunochromatography containing the test piece for an immunochromatography according to any one of (1) to (5).
(7) A method for detecting an immunochromatography, which detects an object to be detected in a sample by using a test piece for an immunochromatography.
The method for detecting immunochromatography, which comprises a step of contacting a substance to be detected in a sample with a conjugate containing a specific binding substance labeled with a labeling substance in the presence of glucose.
(8) An immunochromatographic detection method for detecting an object to be detected in a sample by using a lateral flow test piece, the detection method including the steps (A) to (C).
(A) Process test piece for supplying a sample to the sample supply unit of the following test piece;
An immunochromatographic test piece having at least a sample supply unit, a development unit, and a detection unit.
In the developing portion, a conjugate containing a specific binding substance labeled with a labeling substance is held in a dry state so that it can be eluted together with glucose. Step of forming a complex of and a conjugate (C) Step of detecting the complex in a detection unit (9) The immunochromatographic test piece is a conjugate pad in which the conjugate is dried and held so that it can be eluted together with glucose. The method for detecting an immunochromatography according to (8), which is included as a developing unit.
(10) The method for detecting an immunochromatography according to any one of (9), wherein the conjugate pad is a pad made of glass fiber.
(11) The method for detecting an immunochromatography according to any one of (7) to (10), wherein the labeling substance is colloidal gold.
(12) The method for detecting an immunochromatography according to any one of (7) to (11), wherein the specific binding substance is an antibody.
(13) A method for reducing variation in measured values in a method of detecting an object to be detected in a sample using a test piece for later later chromatography.
A method for reducing variation in measured values, which comprises a step of contacting a substance to be detected in a sample with a conjugate containing a specific binding substance labeled with a labeling substance in the presence of glucose.
(14) A method for reducing the variation in measured values in a method for detecting an object to be detected in a sample using a test piece for laterochromatography, which comprises the steps (A) to (C). ..
(A) Process test piece for supplying a sample to the sample supply unit of the following test piece;
An immunochromatographic test piece having at least a sample supply unit, a development unit, and a detection unit.
A substance to be detected in an immunochromatographic test piece (B) sample, wherein a conjugate containing a specific binding substance labeled with a labeling substance is held in a dry state so that it can be eluted together with glucose in the developing portion. Step of forming a complex of and a conjugate (C) A step of detecting the complex in a detection unit.

By holding the conjugate in coexistence with glucose in the lateral flow test piece, the dispersibility of the conjugate is ensured and aggregation does not occur, and the conjugate elutes quickly from the holding portion when the sample solution passes through. It is possible to provide an immunochromatographic test piece in which the variation in dissolution rate is reduced.
Further, since the conjugate is rapidly eluted into the developing portion, the reaction is carried out in the detection portion within a predetermined reaction time, so that the delay in the inspection can be prevented.

It is a graph which shows the measured value (sensitivity) at the time of measuring a blank sample by a blank sample by the the Lateral flow test piece using the conjugate pad containing various sugars. It is a graph which shows the measured value (sensitivity) at the time of measuring the plasma sample containing the detection object by the immunochromatography test piece using the conjugate pad containing various sugars. It is a graph which shows the variation (CV) of the measured value at the time of measuring the plasma sample by the thing, the plasma chromatograph using the conjugate pad containing various concentrations of sucrose or glucose. It is a graph which shows the variation (C.V.) of the measured value before and after storage when the Lateral flow test piece using the conjugate pad containing various sugars is stored under harsh conditions (including low-concentration detection target). When a plasma sample (LOW sample) is measured). It is a graph which shows the variation (CV) of the measured value before and after the storage when the later-statement test piece using the conjugate pad containing various sugars is stored under harsh conditions (including a high-concentration detection object). When a plasma sample (HIGH sample) is measured). The relative value of the absorbance measurement value before and after the immunochromatographic test piece using the conjugate pad containing various sugars was stored under harsh conditions (the absorbance measurement value after acceleration with respect to the absorbance measurement value before acceleration) is shown. It is a graph which shows the variation (CV) of the measured value at the time of measuring a HIGH sample by the Lateral flow test piece using the conjugate pad containing various sugars. The relative value of the absorbance measurement value before and after the immunochromatographic test piece using the conjugate pad containing various sugars was stored under harsh conditions (the absorbance measurement value after acceleration with respect to the absorbance measurement value before acceleration) is shown. It is a schematic diagram which shows the Lateral flow test piece of this invention.

(Immunochromatographic test piece)
The lateral flow test piece of the present invention is an later lateral flow test piece provided with at least a sample supply unit, a developing unit, and a detection unit, and a specific binding substance (specific binding substance for detection) is fixed in the detection unit. It has been transformed. A specific binding substance (also referred to as a conjugate) labeled with a labeling substance that can form a complex with the detection target exists as described later so that it can pass through the developing portion and reach the detection portion after contact with the sample. It exists in the form (A to C).
As an example of embodying these, a sample pad that plays a role of a sample supply part, a conjugate pad that holds a conjugate forming a complex with a detection target together with glucose so as to be eluteable and plays a part of a developing part, and an antibody for detection are used. Examples thereof include a test piece containing a porous membrane, which is partially immobilized and serves as a deploying part and a detecting part. That is, the typical immunochromatographic test piece of the present invention has the following configuration.
(1) Sample pad to which the sample is supplied (2) Conjugate placed between the sample pad and the membrane including the developing portion, and the first specific binding substance is sensitized to the labeling substance such as gold colloid. Conjugate pad (3) is placed downstream of the conjugate pad and is immobilized and detected as a second specific binding substance that binds to the complex of the conjugate and the detection target. Porous membrane having a part Here, the sample pad, the conjugate pad, and the porous membrane may each form a separate carrier, or two may form one carrier, and the sample pad, the conjugate pad, and the porous membrane may form one carrier from the upstream in the developing direction. It is composed of a sample pad, a conjugate pad, and a porous membrane in this order toward the downstream, and includes any aspect as long as the liquid containing the sample is developed. In addition to the above configuration, the immunochromatographic test piece also includes one in which one or more of absorption pads and 3rd pads are further arranged and mounted. The test piece can also be usually placed on a solid phase support such as a plastic pressure-sensitive adhesive sheet. A polyester film or the like is laminated on the surface of the test piece in order to increase the mechanical strength of the porous membrane on which the specific binding substance is immobilized and to prevent evaporation (drying) of water during the assay. Is also possible. Also, the porous membrane is generally lined with another substrate.
The method for detecting an object to be detected in a sample using the Lateral Flow Test Piece of the present invention comprises at least the following steps;
The process of dropping a sample into the sample supply unit,
A step of bringing the detection target in the sample into contact with the conjugate in the presence of glucose, and a step of detecting the complex of the detection target and the conjugate in the sample in the detection unit.

(Sample pad)
In the present invention, the "sample pad" is a part that bears a sample supply unit that receives a sample, and can absorb a liquid sample in a state of being molded on the pad and allow the liquid and the component of the detection target to pass through. Includes any substance and form.
The sample pad of the present invention can be hydrophilized so that the sample can be smoothly developed, and the treatment may be applied to at least the sample supply section to which the sample is dropped. It may be applied wider in the development direction than the sample supply unit, and it is desirable that it is applied to the entire sample pad. The hydrophilization treatment can be performed with a saccharide, a surfactant or the like.
Further, it is desirable that the sample pad of the present invention also retains glucose in order to promote the elution of the conjugate, similarly to the conjugate pad.
Furthermore, the sample pad of the present invention may contain an hemagglutinin agent in case the sample is whole blood or the like. In this case, it suffices to be included in at least a part of the sample pad, and may be included in the whole.
Specific examples of materials suitable for the sample pad include, but are not limited to, glass fiber (glass fiber), acrylic fiber, hydrophilic polyethylene material, dried paper, paper pulp, and woven fabric. Preferably, a fiberglass pad is used. The sample pad can also have the function of a conjugate pad described later. Further, the sample pad may contain a normally used blocking reagent, if necessary, as long as it does not deviate from the object of the present invention and does not affect the reaction system.

(Conjugate)
In the present invention, the conjugate means a substance in which a specific binding substance (or an antigen) for control or a specific binding substance for a detection target is immobilized on a labeling substance.
The labeling substance used in the present invention can be immobilized (also referred to as sensitization) of a specific binding substance to form a conjugate, and is brought into contact with a sample to detect a detection target (antigen) in the sample. Any substance that can play a role as a labeling substance in the method may be used, and examples thereof include metal colloid, polystyrene, polyethylene, porous glass, glass beads, magnetic particles, and the like, among which gold colloid particles are used. preferable.
The particle size of the gold colloidal particles is preferably 20 to 100 nm, more preferably 30 to 100 nm, and particularly preferably 30 to 70 nm.
The conjugate of the present invention can also block a region on the surface of colloidal gold particles or the like to which a specific binding substance is not bound with a blocking agent.
In the present invention, the conjugate is preferably present together with glucose as described later so that the conjugate can be more dispersible and can be rapidly eluted by passing a sample, a developing solution, or the like.

As the mode of existence of the conjugate, it may exist in a state of being impregnated with a mode existing as a conjugate pad, that is, a dedicated pad (conjugate pad) other than the sample pad, the 3rd pad, and the porous membrane (conjugate pad). Type A), it may be present as a detection reagent holding part in a part of the sample pad (type B). Furthermore, it may exist as a separate detection reagent separately from the test piece so as to be mixed with the sample (type C).

Hereinafter, a typical example of a test piece having a conjugate existence mode of the above type A will be described.
The sample pad, the conjugate pad, the 3rd pad, and the porous membrane are arranged in this order from the upstream to the downstream in the flow direction of the sample, and are arranged so as to overlap at least a part with the upper and lower layers. A test piece of such an arrangement example is shown in FIG.
When a sample containing a detection target is supplied to the sample pad of such a test piece, the detection target passes through the sample pad and flows to the conjugate pad on the downstream side. In the conjugate pad, the object to be detected and the conjugate come into contact with each other to form a complex and pass through the pad. The complex then passes through a 3rd Pad placed in contact with the lower surface of the conjugate pad and unfolds into a porous membrane. In the present invention, glucose is dried and held in such a conjugate pad together with the conjugate.
Since a specific binding substance or antigen that immunologically reacts to the substance to be detected is immobilized on a part of the porous membrane, the complex is here bound and fixed by an immune reaction. Will be transformed into. The immobilized complex is detected by means for detecting the absorbance, reflected light, fluorescence, magnetism, etc. derived from the labeling substance. If the labeling substance is a visible labeling substance such as colloidal gold or colored latex, it can also be detected visually.

Next, a test piece having a conjugate existence mode of type B will be described.
The difference from the above-mentioned type A test piece is that the sample pad and the conjugate pad are integrated, that is, a sample supply unit and a detection reagent holding unit are configured in a part of the sample pad. ..
The sample supply unit is a site for supplying a sample containing a detection target, the detection reagent holding unit is a site containing a conjugate, and the sample supply unit is on the upstream side of the detection reagent holding unit. In the present invention, glucose is dry-held in such a detection reagent holding portion.

Next, a test piece having a conjugate existence mode of type C will be described.
The difference from the above-mentioned type A test piece is that the conjugate pad does not exist as a test piece, and the conjugate exists as an individual detection reagent. For example, a filter chip in which a conjugate is held in the filter can be mentioned. By filtering the sample using such a filter chip, the conjugate held in the filter and the detection target are combined to form a complex. By supplying this to the same test piece as Type A except that it does not have a conjugate pad, the object to be detected can be detected. In the present invention, glucose is kept dry in such a filter chip together with the conjugate.

(Detection reagent)
In the present invention, the "detection reagent" is specifically a solution containing at least a conjugate.
In the present invention, it is desirable that the conjugate is maintained in a dry state together with glucose in a state in which it can be eluted into the immunochromatographic test piece. Therefore, the detection reagent of the present invention contains glucose in addition to the conjugate. If the conjugate coexists with glucose, it suppresses the aggregation of the conjugate and disperses it even when it is applied to the conjugate pad, and its dispersibility is maintained even when it is dried. Further, when the dried-held conjugate elutes together with glucose when it is eluted by passing a sample solution or the like, the elution rate of the conjugate becomes constant, and as a result, the amount of the conjugate eluted within a predetermined time becomes constant. , It brings about the action effect that the variation of the measured value is reduced.

Assuming that the standard area of the detection reagent holding part is 0.78 cm ² and the detection reagent application amount is 52.2 μL, the concentration of glucose contained in the detection reagent is preferably 0.5 to 10.0%. 1.0-5.0% is more desirable, 2.0-3.0% is even more desirable. Needless to say, the concentration of glucose contained in the detection reagent can be adjusted in consideration of the amount of glucose retained per area of the detection reagent holding unit when the glucose is dried and held in the detection reagent holding unit.

In addition, the detection reagent keeps the conjugate in a stable state and promotes the specific reaction of the specific binding substance immobilized on the conjugate with the detection target when mixed with the sample. Further, it may contain a stabilizer such as bovine serum albumin (BSA) and amino acids.
Furthermore, the detection reagent may also contain known sensitizers and chelating agents such as EDTA and EGTA, if necessary, for the purpose of improving the detection sensitivity.
In addition, in this specification, the term "detection" or "measurement" needs to be interpreted in the broadest sense including proof and / or quantification of the existence of a detection target, and is interpreted in a limited manner in any sense. must not.

(Conjugate pad)
In the present invention, the "conjugate pad" is obtained by impregnating a material suitable for the conjugate pad with a detection reagent that specifically reacts with the object to be detected and drying it. The conjugate pad has a function of forming a complex between the detection reagent and the detection target as the sample passes through the conjugate pad. The conjugate pad may be arranged by itself so as to be in contact with the specific binding substance-immobilized membrane. Alternatively, a sample that is placed in contact with the sample pad and has passed through the sample pad by capillary flow is received, and the sample is subsequently transferred by capillary flow to a 3rd pad that is in contact with the sample pad on a surface different from the contact surface. It may be arranged as follows. The selection of one or more sites of the sample pad and the conjugate pad and how the selected site is arranged on the specific binding substance-immobilized membrane can be appropriately changed.

Suitable materials for the conjugate pad include, but are not limited to, paper, cellulose mixtures, nitrocellulose, polyesters, acrylonitrile copolymers, glass fibers or non-woven fibers such as rayon. Preferably, a fiberglass pad is used.
The conjugate pad of the present invention is obtained by immersing a coating liquid containing glucose and a conjugate in the conjugate pad and drying it. Therefore, the conjugate will be kept dry in the conjugate pad along with glucose.

As a method of applying the detection reagent to the conjugate pad, it has a mechanism capable of preparing a detection reagent solution containing glucose at a predetermined concentration and moving the solution in the horizontal direction while discharging the solution from the nozzle at a constant speed. By applying the above liquid to the conjugate pad in a line shape or the like using an apparatus or the like and drying it, the solution can be retained so that it can be eluted. Further, a certain amount of the detection reagent solution having a predetermined concentration may be collected with a pipette or the like and applied to a part or the entire surface of the conjugate pad. Further, the conjugate pad can be dipped in a container containing a detection reagent solution having a predetermined concentration and applied to the entire surface.

If necessary, the conjugate pad may contain a "control reagent" for ensuring the reliability of the detection result by the immunochromatographic detection method, for example, a specific binding substance that does not react with the sample component labeled with the labeling substance. And high antigenic proteins such as KLH (keyhole limpet hemocyanin) labeled with a labeling substance and the like. These control reagents are components (substances) that are unlikely to be present in the sample, and can be appropriately selected.
When the conjugate is not present on the conjugate pad but on a part of the porous membrane having the detection part as the detection reagent holding part, the coating liquid containing the above-mentioned conjugate is similarly applied to the position upstream from the detection part. The detection reagent holding portion can be formed by drying.

The holding amount of glucose coexisting with the conjugate in the detection reagent holding portion is preferably in the range of 0.335 to 6.69 mg / cm ² , and more preferably 0.669 to 3.35 mg / cm ² . Even more preferably, it is 1.34 to 2.01 mg / cm ² . If it is less than 0.335 mg / cm ² , the dispersibility of the conjugate may be inferior when it is kept dry, and if it is higher than 6.69 mg / cm ² , the dissolution rate of glucose is lowered and the elution of the conjugate is delayed. Because.
In order to obtain the retention amount, the concentration of glucose in the detection reagent may be adjusted to the above-mentioned preferable retention amount so that the concentration of glucose in the detection reagent solution may be adjusted, and the area of the standard detection reagent holding portion may be adjusted. Assuming 0.78 cm ² and a coating amount of 52.2 μL, the glucose concentration (C) in the detection reagent solution is 0.5% to 10% according to the following formula. More preferably, it is 1.0 to 5.0%. Even more preferably, it is 2.0 to 3.0%.
A (mg / cm ² ) = BμL × (C% / 100) / Dcm ²
A: Amount of glucose retained per unit area of the detection reagent holding part B: Amount of the detection reagent solution to be impregnated in the detection reagent holding part C:% concentration of glucose in the detection reagent solution (w / v)
D: Area of detection reagent holding part

(Red blood cell flocculant)
In the present invention, when whole blood is used as a sample, it is desirable to use an hemagglutinin agent in combination with the above-mentioned 3rd Pad. As the hemagglutinin agent, a known polycationic hemagglutinin agent can be used. Of these, polybrene is preferable. Polybrene, whose chemical name is hexadimethrin bromide, is a type of cationic polymer to which CAS No. 28728-55-4 is assigned.
As the mode of use of the hemagglutinin agent such as polybrene, in addition to the mode of adding to the sample diluent or directly to the sample, the hemagglutinant should be contained in the sample supply part (sample pad) of the immunochromatographic test piece. Can be done. According to such a usage mode, the erythrocyte flocculant comes into contact with the whole blood, and the erythrocytes in the whole blood are aggregated.

(Immobilization of specific binding substances on a porous membrane)
Immobilization of a specific binding substance such as an antibody to a detection target in the immunochromatographic test piece of the present invention on a porous membrane can be carried out by a generally known method. For example, in the case of the flow-through type, the above-mentioned specific binding substance is prepared at a predetermined concentration, and the liquid is applied to a porous membrane in a fixed amount, in a specific symbol shape such as a point or +. At this time, in order to ensure the reliability of the detection result by the immunochromatographic detection method, the protein or compound that can bind to the conjugate is immobilized at a position different from the specific binding substance that binds to the detection target, and "control detection" is performed. It is common to say "part". Further, the specific binding substance that binds to the control reagent can be immobilized at a position different from that of the specific binding substance that binds to the detection target object to form a “control detection unit”.

In the case of the lateral flow type, a device having a mechanism capable of adjusting the above-mentioned specific binding substance to a predetermined concentration and moving the liquid horizontally while discharging the liquid from the nozzle at a constant speed is used. , By applying to a porous membrane in a line shape. At this time, the concentration of the specific binding substance is preferably 0.1 to 5 mg / mL, more preferably 0.5 to 3 mg / mL. Further, the immobilization amount of the porous membrane of the specific binding substance can be optimized by adjusting the amount of application dropped on the porous membrane in the case of the flow-through type, and the above in the case of the lateral flow type. It can be optimized by adjusting the discharge rate from the nozzle of the device. In particular, in the case of the lateral flow type, 0.5 to 2 μL / cm is suitable. In the present invention, the term "flow-through membrane assay" refers to a method in which the sample solution or the like is developed so as to pass perpendicular to the porous membrane, and the term "lateral flow membrane assay" refers to the method. It refers to a method in which a sample solution or the like is developed so as to move in a parallel direction with respect to a porous membrane.

Further, in the present invention, regarding the application position of the specific binding substance that binds to the object to the porous membrane, in the case of the lateral flow type, the detection reagent is developed from the detection reagent holding portion by the capillary phenomenon. Each specific binding substance may be arranged so as to pass through the coated line in order. It is preferable that the line coated with the specific binding substance that binds to the detection target is located upstream, and the line coated with the control-specific binding substance is located downstream thereof. At this time, it is desirable that the distance between the lines is sufficient so that the signal of the labeling substance can be detected. Even in the case of the flow-through type, the application position of the specific binding substance that binds to the object may be arranged so that the signal of the labeling substance can be detected.

The specific binding substance solution to be applied to the above-mentioned porous membrane can usually be prepared by using a predetermined buffer solution. Examples of the type of the buffer solution include commonly used buffer solutions such as a phosphate buffer solution, a Tris buffer solution, and a Good's buffer solution. The pH of the buffer solution is preferably in the range of 6.0 to 9.5, and may be appropriately set according to the properties of the specific binding substance to be used. For example, a buffer solution having a pH of 8.0 can be used for the anti-cTnI antibody described later. The buffer solution may further contain salts such as NaCl, stabilizers such as sucrose, preservatives, preservatives such as proclin, and the like. Salts include those added for adjusting the ionic strength such as NaCl, and those added in the step of adjusting the pH of the buffer solution such as sodium hydroxide. After immobilizing the specific binding substance on the porous membrane, it is also possible to further cover a part other than the specific binding substance immobilization site with a normally used blocking agent in the form of a solution or vapor to perform blocking. In the present specification, the porous membrane on which the specific binding substance is immobilized as described above is referred to as “specific binding substance-immobilized membrane”, and among them, the porous membrane on which the antibody is immobilized is referred to as “antibody-immobilized”. Sometimes called "membrane".

(Porosity membrane)
In the present invention, as the porous membrane (hereinafter, may be simply referred to as a membrane), any porous material can be used. Examples thereof include, but are not limited to, polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, and ceramics. Specific examples thereof include glass fiber filter paper and nitrocellulose membrane sold by Merck Millipore, Toyo Filter Paper, GE Healthcare and the like. In addition, by appropriately selecting the pore size and structure of the porous membrane made of this porous body, the rate at which the immune complex between the gold colloid-labeled specific binding substance (conjugate) and the detection target flows through the membrane can be determined. It is possible to control. Since the amount of labeled specific binding substance bound to the specific binding substance immobilized on the membrane can be adjusted by controlling the flow rate in the membrane, the pore size and structure of the membrane are the present invention. It is desirable to optimize the immunochromatographic test piece in consideration of the combination with other constituent materials.

(3rd Pad)
In the present invention, the 3rd Pad is a reaction component of a sample and a detection reagent from which a component unnecessary for detection of a detection target is removed, and a component necessary for the reaction is immobilized with a specific binding substance. The porous membrane can be placed for the purpose of allowing smooth deployment. For example, it is desirable that blood cells and insoluble crushed blood cells are removed as components unnecessary for detection. Further, in this 3rd Pad, among the aggregates generated by the antigen-specific binding substance reaction, the aggregates that have moved to the specific binding substance-immobilized membrane and have grown to the extent that they cannot be smoothly developed are removed in advance. It is also possible to have the additional effect of.
The 3rd Pad includes the liquid, the component to be detected, and any substance and form through which the detection reagent can pass. Specific examples include, but are not limited to, glass fiber (glass fiber), acrylic fiber, hydrophilic polyethylene material, dried paper, paper pulp, and woven fabric. The 3rd Pad is sometimes referred to as a blood cell separation membrane when used for the purpose of separating blood cells. In the present invention, when whole blood is used as a sample, it is desirable to use a blood cell separation membrane in order to more reliably separate and remove blood cells that could not be completely removed by the sample pad alone.

(Absorption pad)
In the present invention, the absorption pad is a portion having liquid absorbability that controls the development of the sample by absorbing the sample that has moved and passed through the porous membrane. In the lateral flow type, it may be provided at the most downstream of the strip configuration, and in the flow-through type, it may be provided, for example, under the specific binding substance-immobilized membrane. As the absorbing pad, for example, filter paper can be used, but the absorption pad is not limited thereto.

(Detection device)
The immunochromatographic test piece of the present invention is appropriate in consideration of the size of the test piece, the method and position of addition of the sample, the position of immobilization of the specific binding substance on the specific binding substance-immobilized membrane, the method of detecting a signal, and the like. It can be stored and mounted in a container (housing) for use, and the state of being stored and mounted in this way is called a "detection device".
In the detection device of the present invention, in addition to the sample supply unit, a developing liquid supply unit that separately supplies the developing liquid may be provided on the upstream side of the sample supply unit.

(others)
In the present specification, the "porous membrane" is "solid phase", and the state in which a specific binding substance such as an antigen or an antibody is physically or chemically retained or retained in the porous membrane is "fixed". It may be expressed as "immobilization", "immobilization", "sensitization", or "adsorption".

(Sample)
In the detection method of the present invention, the "specimen" including the object to be detected refers to a biological sample such as blood, urine, sputum, saliva, nasal discharge, nasal swab, pharyngeal swab, other body fluid, and feces. The biological sample may be used as a sample as it is, and a sample obtained by appropriately diluting with a diluted solution, extracting and / or filtering and extracting is also included in the sample of the present invention. Examples of blood samples include whole blood, red blood cells, plasma, serum and the like.
The blood sample also includes a plasma sample collected by a blood collection tube to which an anticoagulant such as EDTA or heparin is added at the time of blood collection.

(Sample diluent)
In the present invention, when it is necessary to dilute the sample according to the concentration of the object to be detected in the sample, a diluted solution may be used. The diluted solution significantly inhibits the reaction between the substance to be detected and the specific binding substance, or conversely, significantly promotes the reaction and causes the labeling substance to overaggregate, resulting in poor deployment in the capillary phenomenon, or is specific. A diluted solution having any composition may be used as long as the signal detection of the reaction between the specific binding substance and the specific binding substance according to the concentration of the binding substance is not impossible. The sample diluent may also be referred to as a sample extract or a developing solution.

(Detection target)
The detection target of the present invention is a substance present in a biological sample such as blood (whole blood), erythrocyte, serum, plasma, urine, saliva or sputum, for example, CRP (C-reactive protein), IgA, IgG. , IgM and other inflammatory markers, fibrin degradation products (eg D-dimer), soluble fibrin, TAT (thrombin-antithrombin complex), PIC (plasmin-plasmin inhibitor complex) and other coagulation / fibrinolysis markers, oxidized LDL, BNP (cerebral sodium diuretic peptide), H-FABP (cardiac fatty acid binding protein), circulation-related markers such as myocardial troponin I (cTnI), metabolism-related markers such as adiponectin, CEA (carcinoembryonic antigen), AFP (α-) Fetprotein), CA19-9, CA125, PSA (prostatic specific antigen) and other tumor markers, HBV (hepatitis B virus), HCV (hepatitis C virus), chlamydia trachomatis, gonococcal and other infectious disease-related markers, allergen-specific Examples thereof include IgE (immunoglobulin E), hormones, and drugs.

(Specific binding substance)
In the present invention, examples of the specific binding substance to the substance to be measured carried on the insoluble carrier particles such as latex include proteins, peptides, amino acids, lipids, sugars, DNA, RNA, receptors, haptens and the like. The origin is not particularly limited, such as high or low molecular weight and natural or synthetic, but examples thereof include antibodies or antigens that can be used for immunological measurement methods utilizing an immune response.

(Antibody used in the present invention)
The antibody against the detection target used in the present invention is not limited by the method of producing as long as it is an antibody that specifically reacts with the detection target, and even if it is a polyclonal antibody, it is a monoclonal antibody. May be. More preferably, it is a monoclonal antibody. In general, hybridomas that produce the antibody are mieroma of the same species as the spleen cells of animals immunized with the detection target as an immunogen according to the method of Kohler and Milstein (see Nature, Vol. 256, p. 495 (1975)). It can be produced by cell fusion with cells (myeloma cells).

As the antibody of the present invention, it is also possible to use a functional fragment of an antibody having an antigen-antibody reaction activity in addition to the entire antibody molecule. In addition to those obtained through the process of immunizing general animals (mouse, goat, sheep, etc.), the amino acid sequence of an animal species different from that of animals that immunize the immunogen (measurement target substance) is changed by gene recombination technology, etc. The antibody (chimera-specific binding substance, humanized antibody, fully humanized antibody, etc.) may be used. Examples of the functional fragment of the antibody include F (ab') ₂ , Fab', which is a fragment having an antigen-antibody reaction activity, and a single-chain antibody (scFv). Functional fragments of these antibodies can be produced by treating the antibodies obtained as described above with a proteolytic enzyme (eg, pepsin, papain, etc.).
Here, when the antibody used in the measurement method for detecting an object to be detected by forming a so-called sandwich is a monoclonal antibody, a labeled antibody for immobilization (first antibody) and an antibody for porous membrane immobilization (second). Regarding the relationship (antibody), when the epitope of the first antibody is monovalent, the epitope of the second antibody is different from that of the first antibody, and when the epitope of the first antibody is polyvalent, the second antibody is used. The epitope of the antibody in the above may be the same as that of the first antibody or may be different.

(kit)
The detection kit using the immunochromatography of the present invention is an immunochromatographic test piece provided with at least a sample supply part, a development part and a detection part, and a part of the development part is the first labeled substance. It suffices as long as it contains an immunochromatographic test piece in which a conjugate (detection reagent) containing a specific binding substance is retained eluteably together with glucose.
The detection kit may also include reagents necessary for detection, a diluted solution of a sample, a test tube, a filtration filter, a cotton swab for collecting a sample, an instruction manual, a housing for storing a test piece, and the like.

The method for detecting an object to be detected in a sample using the immunochromatographic test piece of the present invention is a method including the following steps (A) to (C).
(A) Process test piece for supplying a sample to the sample supply unit of the following test piece;
An immunochromatographic test piece having at least a sample supply unit, a development unit, and a detection unit.
In the developing portion, a conjugate containing a specific binding substance labeled with a labeling substance is held in a dry state so that it can be eluted together with glucose. The substance to be detected in the immunochromatographic test piece (B) sample. Step of forming a complex of and a conjugate (C) Step of detecting the complex in a detection unit Here, in the detection of the complex, qualitative detection by visual observation and quantitative measurement by optical detection means are performed. Both are included. Further, in the quantitative measurement by the optical detection means of the present invention, in addition to the method of measuring the test pieces one by one, the method of using a device for continuously and automatically measuring a plurality of test pieces, or a method of using a plurality of test pieces. Various embodiments are included, such as a method using a device for simultaneously measuring the above.
Specific examples of the present invention will be described below, but the present invention is not limited thereto.

[Example 1] Screening of saccharides applied to the conjugate pad of the present invention As saccharides applied to the conjugate pad, the blank value is reduced and the sample measurement sensitivity is higher than that of lactose conventionally used. Screening was performed on the condition of.
1. 1. Preparation of detection device of the present invention 1) Preparation of gold colloid-labeled anti-cTnI monoclonal antibody (anti-cTnI antibody conjugate) (i) Preparation of gold colloid solution 7% (w) of 500 mL of purified water heated to 93 ° C. / V) 1 mL of a triammonium citrate aqueous solution was added, and the mixture was stirred and mixed. Subsequently, 1 mL of a 5% (w / v) aqueous solution of tetrachloroauric acid (III) was added, and the mixture was reacted for 10 minutes with stirring, and then the reaction solution was boiled. Then, it was cooled in ice water to prepare a solution of colloidal gold having an average particle size of 60 nm. This solution of colloidal gold having an average particle size of 60 nm was adjusted to 1 OD / mL by the absorbance of the colloidal gold at the maximum absorption wavelength with purified water.

(Ii) Preparation of anti-cTnI antibody conjugate Anti-cTnI monoclonal antibody diluted to 20 μg / mL with 2 mM Tris-hydrochloride buffer (pH 7.0) against the above 1 OD / mL colloidal gold solution (pH 8.0). It was added and stirred at room temperature for 10 minutes. Purified water containing 0.5% (w / v) of NeoProtein Saver (Toyobo Co., Ltd., No. NPS-301) was added to the mixed solution of the gold colloid and the antibody, and the mixture was stirred at room temperature for 5 minutes. Then, it was centrifuged at 11900 × g for 45 minutes at 10 ° C. After removing the supernatant, 1 mL of a 0.2% (w / v) NeoProtein Saver aqueous solution was added to the obtained sediment to suspend the conjugate to obtain an anti-cTnI antibody conjugate.

(Iii) Preparation of colloidal gold labeled KLH (KLH conjugate) for control line KLH (Sigma Co., Ltd.) dissolved in 20 mL of the above 1 OD / mL colloidal gold solution to 620 μg / mL with 2 mmol / L phosphate buffer. 1 mL was added, and the mixture was stirred at room temperature for 10 minutes. 1 mL of a 10% bovine serum albumin (BSA) aqueous solution was added to the mixed solution of the gold colloid and KLH, and the mixture was stirred at room temperature for 5 minutes. Then, the mixture was centrifuged at 10 ° C. for 45 minutes to remove the supernatant, and then 1 mL of the conjugate diluted solution was added to the obtained sediment to suspend the conjugate to obtain a KLH conjugate.

2) Preparation of conjugate pad Anti-cTnI antibody conjugate 3OD, KLH conjugate 0.75OD, 0.5% LipidureBL-1301, 0.25mg / mL Ratioblock, various saccharides below, 2.0% NPS, 20 mM MOPS (pH 7.2) is mixed to prepare a detection reagent, and a glass fiber pad (Merck Millipore) cut to an appropriate size is impregnated with a liquid volume ratio of 52.2 μL per 0.78 cm ² . rice field. It was dried by heating in a dry oven at 70 ° C. for 45 minutes to obtain a conjugate pad.
<Tested sugars>
2.4% lactose, 1% lactose, 4% lactose, 2.4% sucrose, 2.4% trehalose, 2.4% maltose, 2.4% glucose, 2.4% fructose

3) Preparation of anti-cTnI monoclonal antibody-immobilized membrane (antibody-immobilized membrane) so that the final concentration is 2.5% and 3 mg / mL at 10 mM PB pH 8.0 containing 0.09% NaN ₃ for the test line. A solution supplemented with sucrose and an anti-cTnI monoclonal antibody was prepared.
In addition, a rabbit anti-KLH polyclonal antibody (manufactured by Bethyl) was diluted and prepared in the same manner as described above for the control line.
The above anti-cTnI monoclonal antibody was applied to the inside of one end of the short side of a nitrocellulose membrane (Hi-Flow plus HF180, Merck Millipore) to 1 μL / cm using an immunochromatographic dispenser “XYZ3050” (BIO DOT). It was set and applied in a line to form a test line. The anti-KLH polyclonal antibody was similarly applied at an interval of about 4 mm from the position of the test line to form a control line. The membrane was dried at 70 ° C. for 45 minutes in a dry oven to obtain an antibody-immobilized membrane.

4) Preparation of sample pad A glass fiber pad (Lydall) obtained by cutting 20 mM MOPS (pH 7.2) containing 0.5% lactose and 2% polybrene to an appropriate size is 1.5 times the volume of the pad. A sample pad was used as a sample pad after being soaked in a volume and dried at 70 ° C. for 45 minutes in a dry oven.

5) Preparation of Lateral Flow Test Piece The antibody-immobilized membrane (b) is attached to the plastic pressure-sensitive adhesive sheet (a), and the anti-cTnI antibody (c) is placed on the upstream side of the development, followed by the anti-KLH antibody (d). A coating portion was arranged, and a blood cell separation membrane (3rd Pad) (e) was further mounted on the membrane. Next, the conjugate pad (f) produced in 2) above is placed and mounted, and the sample pad (g) prepared in 4) above is placed and mounted so as to overlap the conjugate pad, and the sample pad (g) prepared in 4) above is placed and mounted on the opposite end. The absorption pad (h) was arranged and attached. In this way, each component was cut into a superposed structure to prepare an immunochromatographic test piece. The test piece may be stored and mounted in a special plastic housing (having a sample addition window and a detection window, not shown in FIG. 9) during the assay, and may be in the form of a detection device. FIG. 9 shows a schematic configuration diagram of the lateral flow test piece.

2. 2. Screening method 120 μL of plasma sample containing 1000 pg / mL myocardial troponin I (cTnI) was added to the sample pad window of the detection device prepared above, and after 15 minutes, an immunochromatographic reader Rapid Pier (registered trademark, Sekisui Medical Co., Ltd.) was used. The coloration amount (absorbance) of the test line was measured. 120 μL of plasma sample containing no cTnI as a blank was measured in the same manner as above. Each measurement was performed 3 times (n = 3), and the average value was obtained. For screening, saccharides with lower measured values for blanks and higher measured values for plasma samples containing cTnI were selected than with conventional 2.4% lactose. The results are shown in FIGS. 1 and 2.

3. 3. Results According to this figure, it was found that sucrose, trehalose, maltose, and glucose suppress the blank value and increase the measurement sensitivity of the sample as compared with the conventional lactose.

[Test Example 2] Confirmation test of CV reduction effect (1)
Among the saccharides selected by the screening of Test Example 1, the CV reducing effect was confirmed for various concentrations of sucrose and glucose. For comparison, the CV reduction effect was also confirmed for the conventionally used lactose.
1. 1. Test method The sample is a plasma sample containing 150 pg / mL myocardial troponin I (cTnI), and 1.0%, 2.4%, 4.0% sucrose or glucose, 2.4% as saccharides for adjusting the detection reagent. The coloration amount (absorbance) of the test line was measured and their CV values (variations) were calculated in the same manner as in Test Example 1 except that the number of n was set to 5. CV is the Coefficient of Variation and can be obtained by dividing the standard deviation by the mean value. The results are shown in FIG.

2. 2. Test Results According to this figure, when sucrose or glucose is added to the conjugate coating solution, the measured values are less variable and the simultaneous reproducibility is excellent at any concentration as compared with the case where conventional lactose is added. It could be confirmed.

[Test Example 3] Simultaneous reproducibility and accelerated stability confirmation test after storage under harsh conditions (1)
The test piece obtained by adding glucose, lactose, and sucrose to the conjugate coating solution was stored at 37 ° C. for 10 days under severe conditions, and a test was conducted to confirm the variation and stability of the measured values.
1. 1. Test method A test piece of the present invention was prepared in the same manner as in Test Example 1 except that 2.4% lactose, 4.0% sucrose, and 2.4% glucose were used as saccharides for adjusting the detection reagent. The test was performed except that the n number was 4 and the samples were LOW sample (plasma sample containing 150 pg / mL myocardial troponin I (cTnI)) and HIGH sample (plasma sample containing 500 pg / mL myocardial troponin I (cTnI)). CV was calculated in the same manner as in Example 2. In addition, the same sample was measured for the test piece stored at 37 ° C. for 10 days, and the CV was calculated. FIG. 4 shows the results of variations in the measured values of the LOW sample, and FIG. 5 shows the results of the variations in the measured values of the HIGH sample. Before storage under harsh conditions is referred to as "before acceleration", and after storage under harsh conditions is referred to as "after acceleration".
Further, as an evaluation of stability, FIG. 6 shows a relative value of the absorbance measurement values before and after storage under harsh conditions (the absorbance measurement value “after acceleration” with respect to the absorbance measurement value “before acceleration”).

2. 2. Test Results According to FIGS. 4 and 5, when sucrose or glucose was added to the conjugate coating solution, the measured values were higher than when lactose was added even after storage under harsh conditions, regardless of the concentration of the sample. It was confirmed that there was little variation and excellent simultaneous reproducibility.
Further, according to FIG. 6, it was confirmed that when glucose was added to the conjugate coating liquid, the change in the measured value was small and the stability was excellent as compared with the case where sucrose was added even after storage under harsh conditions. did it.
When the above test results were put together, it was confirmed that when glucose was added to the conjugate coating solution, both the simultaneous reproducibility and the stability were excellent.

[Test Example 4] Confirmation test of CV reduction effect (2)
Among the saccharides selected by the screening of Test Example 1, glucose, maltose, and trehalose were confirmed to have a CV reducing effect. For comparison, the CV reduction effect was also confirmed for lactose.
1. 1. Test method The sample was a HIGH sample (a plasma sample containing 500 pg / mL myocardial troponin I (cTnI)), and 2.4% glucose, maltose, trehalose, and lactose were used as saccharides to adjust the detection reagent, and nitro. The color development amount (absorbance) of the test line was measured in the same manner as in Test Example 2 except that (UniSart CN150 white backed, Saltorius Stedim Biotech, model number 1UN15WR1000025NT) was used for the cellulose membrane, and their CV values (variations) were measured. ) Was calculated respectively. The results are shown in FIG.

2. 2. Test Results According to this figure, it was confirmed that when glucose and maltose were added to the conjugate coating solution, the measured values did not vary much and the simultaneous reproducibility was excellent as compared with the case where the conventional lactose was added.

[Test Example 5] Accelerated stability confirmation test after storage under harsh conditions (2)
A test piece obtained by adding glucose, maltose, and trehalose to the conjugate coating solution was stored at 37 ° C. for 10 days under severe conditions, and a test was conducted to confirm the stability.
1. 1. Test method A test piece of the present invention was prepared in the same manner as in Test Example 1 except that 2.4% lactose, 2.4% glucose, 2.4% maltose, and 2.4% trehalose were used as saccharides for adjusting the detection reagent. did. The n number was 5, and a HIGH sample (a plasma sample containing 1000 pg / mL myocardial troponin I (cTnI)) was used, and a nitrocellulose membrane (UniSart CN150 white backed, Sartorius Stedim Biotech, model number 1UN15WR100025NT) was used. The test was carried out in the same manner as in Test Example 3 except that the test was performed, and the stability was evaluated. Relative value of the absorbance measurement value before and after storage under harsh conditions FIG. 8 shows the absorbance measurement value “after acceleration” with respect to the absorbance measurement value “before acceleration”.

2. 2. Test Results According to this figure, it can be confirmed that when glucose is added to the conjugate coating solution, the measured value does not change much and the stability is excellent compared to the case where maltose is added even after storage under harsh conditions. rice field.
From the results of Test Examples 1 and 4 above, it was confirmed that when glucose or maltose was added to the conjugate coating solution, the simultaneous reproducibility was excellent as compared with the case where conventional lactose or other sugars were added. ..
In addition, when the results of Test Examples 1 to 5 are combined, it can be reconfirmed that when glucose is added to the conjugate coating solution, it is superior in both simultaneous reproducibility and stability as compared with conventional lactose and other sugars. rice field.

According to the immunochromatographic test piece of the present invention, since the conjugate is retained in coexistence with glucose, the dispersibility of the conjugate is ensured, and the conjugate promptly retains the detection reagent of the test piece upon passage of the sample solution. It is possible to provide an immunochromatographic test piece that elutes from the portion and reduces the variation in the elution rate.
Further, according to the immunochromatographic test piece of the present invention, the conjugate is rapidly eluted into the test piece developing part, so that the detection part surely develops color within a predetermined reaction time and the inspection is prevented from being delayed. Can be done.

(A) Plastic adhesive sheet (b) Antibody-immobilized membrane (c) Anti-cTnI antibody (test line)
(D) Anti-KLH antibody (control line)
(E) 3rd Pad
(F) Conjugate pad (g) Sample pad (h) Absorption pad

Claims (14)

An immunochromatographic test piece having at least a sample supply unit, a development unit, and a detection unit.
An immunochromatographic test piece in which a conjugate containing a specific binding substance labeled with a labeling substance is held in a dry state so that it can be eluted together with glucose in a developing portion. The immunochromatographic test piece according to claim 1, further comprising a conjugate pad in which the conjugate is dried and held so that it can be eluted together with glucose as a developing portion. The immunochromatographic test piece according to claim 2, wherein the conjugate pad is a pad made of glass fiber. The immunochromatographic test piece according to any one of claims 1 to 3, wherein the labeling substance is colloidal gold. The immunochromatographic test piece according to any one of claims 1 to 4, wherein the specific binding substance is an antibody. A detection kit for an immunochromatography, which comprises the test piece for an immunochromatographic test according to any one of claims 1 to 5. This is an immunochromatographic detection method that detects an object to be detected in a sample using a test piece for later later chromatograph.
The method for detecting immunochromatography, which comprises a step of contacting a substance to be detected in a sample with a conjugate containing a specific binding substance labeled with a labeling substance in the presence of glucose. A method for detecting an immunochromatographic test in which an object to be detected in a sample is detected by using a lateral flow test piece, and the above-mentioned detection method including the steps (A) to (C).
(A) Process test piece for supplying a sample to the sample supply unit of the following test piece;
An immunochromatographic test piece having at least a sample supply unit, a development unit, and a detection unit.
A substance to be detected in an immunochromatographic test piece (B) sample, wherein a conjugate containing a specific binding substance labeled with a labeling substance is held in a dry state so that it can be eluted together with glucose in the developing portion. Step of forming a complex of and a conjugate (C) A step of detecting the complex in a detection unit. The method for detecting an immunochromatography according to claim 8, wherein the immunochromatographic test piece includes a conjugate pad in which the conjugate is dried and held so that it can be eluted together with glucose as a developing portion. The method for detecting an immunochromatography according to any one of claims 9, wherein the conjugate pad is a pad made of glass fiber. The method for detecting an immunochromatography according to any one of claims 7 to 10, wherein the labeling substance is colloidal gold. The method for detecting an immunochromatography according to any one of claims 7 to 11, wherein the specific binding substance is an antibody. It is a method for reducing the variation in measured values in the method of detecting an object to be detected in a sample using a test piece for later later chromatography.
A method for reducing variation in measured values, which comprises a step of contacting a substance to be detected in a sample with a conjugate containing a specific binding substance labeled with a labeling substance in the presence of glucose. A method for reducing variation in measured values in a method for detecting an object to be detected in a sample using a test piece for later later chromatography, which comprises the steps (A) to (C).
(A) Process test piece for supplying a sample to the sample supply unit of the following test piece;
An immunochromatographic test piece having at least a sample supply unit, a development unit, and a detection unit.
A substance to be detected in an immunochromatographic test piece (B) sample, wherein a conjugate containing a specific binding substance labeled with a labeling substance is held in a dry state so that it can be eluted together with glucose in the developing portion. Step of forming a complex of and a conjugate (C) A step of detecting the complex in a detection unit.

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