WO2020105567A1

WO2020105567A1 - Immuno-chromatography test strip and immuno-chromatography detection kit

Info

Publication number: WO2020105567A1
Application number: PCT/JP2019/044997
Authority: WO
Inventors: 佳菜子伊藤; 景吾河野; 駿酒井; 旭中島; 元喜森田
Original assignee: 積水メディカル株式会社
Priority date: 2018-11-19
Filing date: 2019-11-18
Publication date: 2020-05-28
Also published as: JP2022046827A

Abstract

The present invention addresses the problem of providing an immuno-chromatography test strip having a conjugate held therein which is rapidly eluted due to the passage of a test liquid so as to be able to react with a substance to be detected, wherein the amount of the conjugate contributing to the reaction varies little, and wherein variations of the values measured can be prevented.　The present invention provides an immuno-chromatography test strip having a porous membrane comprising at least a sample supply section, a deployment section, and a detection section. In a portion of the deployment section, a conjugate containing a specific binding substance labeled with a labeling substance is held, along with glucose and/or maltose, in an elutable and dry state.

Description

Immunochromatographic test strip and immunochromatographic detection kit

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

With the widespread use of POCT (point of care), where treatment is performed near the patient, a lateral flow immunochromatography detection method (hereinafter sometimes simply referred to as immunochromatography detection method) using a test piece such as a nitrocellulose membrane is widely used. is doing.
A test piece for an immunochromatographic detection method (hereinafter sometimes simply referred to as an immunochromatographic test piece) generally includes a membrane made of a porous body having at least a sample supply section, a developing section, and a detection section. A labeled antibody (conjugate) that forms a complex with an object to be detected is retained at the expansion start site of the expansion part so that it can be eluted, and the antibody is immobilized on a part of the downstream side of the expansion part to detect the detection part. Is composed of.
Here, when the sample is dropped on the sample supply unit, if the sample contains the detection target, the detection target specifically binds to the labeled antibody at the developing unit to form a complex. The complex binds to the immobilized antibody at the detection part while expanding the development part toward the downstream direction. Thus, in the detection part, a sandwich type complex is formed by the labeled antibody, the detection target and the immobilized antibody, and the detection target can be qualitatively or quantitatively analyzed. ..
An example of the labeled substance that constitutes the labeled antibody is gold colloid particles, and qualitative detection is possible by a color reaction caused by the gold colloid particles. Furthermore, it is also possible to quantitatively detect the detection object in the sample based on the degree of coloration. 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 the immunochromatographic test strip.

By the way, in the immunochromatographic test strip, when the conjugate that has been dried and held in the developing part elutes due to the addition of the sample, conventionally, the dissolution rate of the conjugate varied from test to test, and the amount of conjugate that could react with the detection target fluctuated. There is a problem that there is a large variation in the measured values, probably because it is done.
The following Patent Documents 1 to 3 are known as improved techniques relating to a detection reagent containing such a conjugate.
In Patent Document 1, latex particles, a buffer solution, a protein, and saccharose are used as a latex composition for immunoassay for preventing spontaneous aggregation of latex particles having an antibody bound thereto and enabling highly sensitive immunoassay. , Trehalose, maltose, lactose, sorbitol, D-mannitol, and polyhydric alcohols are disclosed as compositions containing one or more anticoagulants. However, the types of anti-aggregation agents are limited, and among them, only sucrose, trehalose and glycerin have been confirmed to have anti-aggregation effects by tests. In addition, no consideration has been given to variations in measured values.
In Patent Document 2, in order to achieve both high sensitivity and prevention of false positive reaction, a water-soluble polysaccharide having a 1 wt% viscosity of 0.1 cP or more and 300 cP or less is used in the range of 0.001 wt% or more and 20.00 wt% or less. A developing solution for immunochromatography containing a detection reagent labeled with a carrier such as gold colloid particles having an average particle diameter of 1 nm or more and 60 nm or less is disclosed, and CMC is mentioned as a water-soluble polysaccharide having such characteristics. Has been. However, the effect of water-soluble polysaccharides other than CMC is unknown, and no consideration has been given to variations in measured values.
In Patent Document 3, as a solid-phased immunoreagent capable of maintaining high stability in a dry state for a long period of time, an antibody is immobilized on an insoluble carrier such as polystyrene beads and then immersed in a buffer solution containing a specific sugar. After that, a reagent obtained by removing the buffer solution and then freeze-drying is disclosed. However, only trehalose was actually tested and confirmed to have an effect, and the effect on other sugars is unknown. In addition, confirmation of the effect has only been conducted on the stabilizing effect by comparing the residual activity before and after humidification storage, and not on the variation in the measured value.
Patent Document 4 discloses an immunochromatographic test strip containing as a constituent a conjugate pad in which a detection reagent containing a conjugate in which gold colloid is sensitized with an antibody is impregnated together with sucrose. However, there is no particular disclosure about the role of sucrose, and there is no suggestion about other sugars.

JP 2007-315883 Japanese Patent No. 6192374 Japanese Patent Laid-Open No. 2003-215127 International publication 2012/043746 Pamphlet

The present invention is to provide an immunochromatographic test strip capable of preventing the variability of the measured value, in which the conjugate retained in the immunochromatographic test strip is rapidly eluted by the passage of the sample liquid, the amount of the conjugate contributing to the reaction is little changed. And

When the substance to be coexisted with the conjugate in order to solve the above-mentioned problems was earnestly studied, the conjugate was coexisted with glucose and / or maltose in the immunochromatographic test piece, whereby the conjugate was rapidly eluted by the passage of the sample liquid. It was found that the amount of conjugate elution was small, and as a result, it was possible to prevent variations in the detected values. Further, they have also found that coagulation of the conjugate itself can be suppressed by allowing the conjugate to coexist with glucose and / or maltose, and have completed the present invention. That is, the present invention has the following configurations.
(1) An immunochromatographic test strip including at least a sample supply unit, a development unit, and a detection unit,
An immunochromatographic test strip in which a conjugate containing a specific binding substance labeled with a labeling substance is held in a dry state so as to be eluted together with glucose and / or maltose in the developing part.
(2) The immunochromatographic test piece according to (1), which includes, as a development part, a conjugate pad in which the conjugate is dried and held so as to be eluted together with glucose and / or maltose.
(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 immunochromatographic test strip according to any one of (1) to (4), wherein the specific binding substance is an antibody.
(6) An immunochromatographic detection kit comprising the immunochromatographic test strip according to any one of (1) to (5).
(7) An immunochromatographic detection method for detecting an object to be detected in a sample using a test piece for immunochromatography,
The immunochromatographic detection method, which comprises a step of bringing a substance to be detected in a sample into contact with a conjugate containing a specific binding substance labeled with a labeling substance in the presence of glucose and / or maltose.
(8) An immunochromatographic detection method for detecting an object to be detected in a sample using an immunochromatographic test strip, the detection method including steps (A) to (C).
(A) Process test piece for supplying a specimen to the sample supply section of the following test piece;
An immunochromatographic test strip comprising at least a sample supply unit, a development unit and a detection unit,
In the development part, a conjugate containing a specific binding substance labeled with a labeling substance is retained in a dry state so that it can be eluted with glucose and / or maltose. (C) a step of forming a complex between the substance to be detected and a conjugate of (C) the step of detecting the complex in a detection part (9), wherein the conjugate can be eluted with glucose and / or maltose The immunochromatographic detection method according to (8), wherein the dried and held conjugate pad is included as a developing portion.
(10) The immunochromatographic detection method according to any one of (9), wherein the conjugate pad is a glass fiber pad.
(11) The immunochromatographic detection method according to any one of (7) to (10), wherein the labeling substance is colloidal gold.
(12) The immunochromatographic detection method 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 for detecting an object to be detected in a sample using a test piece for immunochromatography,
A method for reducing variation in the above-mentioned measured values, which comprises a step of bringing a substance to be detected in a sample into contact with a conjugate containing a specific binding substance labeled with a labeling substance in the presence of glucose and / or maltose.
(14) 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 immunochromatography, the method for reducing variation in measured values including steps (A) to (C). .
(A) Process test piece for supplying a specimen to the sample supply section of the following test piece;
An immunochromatographic test strip comprising at least a sample supply section, a development section and a detection section,
In the development part, a conjugate containing a specific binding substance labeled with a labeling substance is retained in a dry state so that it can be eluted with glucose and / or maltose. (C) a step of forming a complex between the substance to be detected and a conjugate of (C) the step of detecting the complex in a detection part

In the immunochromatographic test strip, by holding the conjugate in the presence of glucose and / or maltose, the dispersibility of the conjugate is secured and aggregation does not occur, and the conjugate is rapidly retained by the passage of the sample liquid. It is possible to provide an immunochromatographic test strip that is more eluted and has a reduced variation in dissolution rate.
Further, since the conjugate is rapidly eluted into the developing portion, the reaction is carried out in the detecting portion within a predetermined reaction time, so that the delay of the test can be prevented.

It is a graph which shows the measured value (sensitivity) at the time of measuring a blank sample by the immunochromatographic 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 a detection target by the immunochromatographic 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 a plasma sample by the immunochromatographic test piece using the conjugate pad containing various concentrations of sucrose or glucose. It is a graph which shows the variation (CV) of the measured value before and after preservation when the immunochromatographic test piece using the conjugate pad containing various sugars is preserve | saved under severe conditions. When measuring a plasma sample (LOW sample)). It is a graph which shows the dispersion | variation (CV) of the measured value before and after preservation | save when the immunochromatographic test piece using the conjugate pad containing various sugars is preserve | saved under severe conditions. When measuring a plasma sample (HIGH sample)). The relative value of the absorbance measurement value before and after storing the immunochromatographic test piece using the conjugate pad containing various sugars under severe 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 immunochromatographic test piece which used the conjugate pad containing various sugars. The relative value of the absorbance measurement value before and after storing the immunochromatographic test piece using the conjugate pad containing various sugars under severe 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 immunochromatographic test piece of this invention.

(Immunochromatographic test piece)
The immunochromatographic test strip of the present invention is an immunochromatographic test strip including at least a sample supply unit, a developing unit, and a detection unit, and a specific binding substance (specific binding substance for detection) is immobilized on the detection unit. Has been converted. The specific binding substance (also referred to as a conjugate) labeled with a labeling substance capable of forming a complex with the detection target is present as described below so that it can pass through the development part and reach the detection part after contact with the sample. Present in modalities (AC).
As an example of embodying these, a sample pad that serves as a sample supply unit, a conjugate pad that holds a conjugate that forms a complex with a detection target together with glucose and / or maltose so as to be eluted, and that serves as a part of a development unit, An example is a test piece including a porous membrane on which a detection antibody is partially immobilized and which serves as a developing portion and a detecting portion. That is, a typical immunochromatographic test strip of the present invention has the following constitution.
(1) A sample pad to which a specimen is supplied (2) A conjugate which is arranged between a sample pad and a membrane including a developing portion, and in which a labeling substance such as gold colloid is sensitized with a first specific binding substance Is placed downstream of the conjugate pad (3) that is retained so that it can be eluted with glucose and / or maltose, and a second specific binding substance that binds to the complex of the conjugate and the detection target is Porous Membrane Immobilized and Having Detecting Portion Here, the sample pad, the conjugate pad, and the porous membrane may each form separate carriers, or two may form one carrier. From the upstream to the downstream of the direction, any aspect is included as long as it is composed of a sample pad, a conjugate pad, and a porous membrane in that order, and a liquid containing a sample is developed.
In addition to the above configuration, the immunochromatographic test piece also includes an immunochromatographic test piece in which any one or more of an absorption pad and 3rd Pad is further arranged and mounted. The test piece can also be usually placed on a solid support such as a plastic pressure-sensitive adhesive sheet. To increase the mechanical strength of the porous membrane with the specific binding substance immobilized, and to prevent the evaporation (drying) of water during the assay, laminate a polyester film etc. on the surface of the test piece. Is also possible. Also, porous membranes are commonly lined with other substrates.
The method for detecting an object to be detected in a sample using the immunochromatographic test strip of the present invention has at least the following steps;
A step of dropping a sample on the sample supply unit,
A step of contacting the detection target in the sample with the conjugate in the presence of glucose and / or maltose, and a step of detecting a complex of the detection target and the conjugate in the sample at the detection unit.

(Sample pad)
In the present invention, the “sample pad” is a part that serves as a sample supply unit that receives a specimen, absorbs a liquid sample in a state where the sample is molded into the pad, and allows the liquid and the components of the detection target to pass through. It includes any substance and form.
The sample pad of the present invention can be subjected to a hydrophilic treatment so that the development of the sample can be performed smoothly, and the treatment may be performed on at least the sample supply portion of the sample pad where the sample is dropped, It may be wider than the sample supply portion in the developing direction, and is preferably applied to the entire sample pad. The hydrophilic treatment can be performed with sugars, surfactants and the like.
Further, it is desirable that the sample pad of the present invention holds glucose and / or maltose in order to accelerate the elution of the conjugate, like the conjugate pad.
Furthermore, in case the sample is whole blood or the like, the sample pad of the present invention may contain a hemagglutination agent. In this case, it may be contained in at least a part of the sample pad, or may be contained in the entire sample pad.
Specific examples of materials suitable for the sample pad include, but are not limited to, glass fiber, acrylic fiber, hydrophilic polyethylene material, dry paper, paper pulp, woven fabric, and the like. A glass fiber pad is preferably used. The sample pad can also have the function of a conjugate pad described later. Further, the sample pad may contain a commonly used blocking reagent, if necessary, within a range that does not deviate from the object of the present invention and does not affect the reaction system.

(Conjugate)
In the present invention, the conjugate refers to a substance in which a specific binding substance for a target substance to be detected or a specific binding substance for control (or antigen) is immobilized on a labeling substance.
The labeling substance used in the present invention can form a conjugate by immobilizing (also referred to as sensitization) a specific binding substance, and detects a target substance (antigen) in a sample by contacting with a sample. Any method may be used as long as it can play a role as a labeling substance in the method, and examples thereof include metal colloid, polystyrene, polyethylene, porous glass, glass beads, and magnetic particles. preferable.
The particle diameter of the gold colloid 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 gold colloid 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 and / or maltose so as to enhance dispersibility and allow rapid elution by allowing a sample, a developing solution, or the like to pass through.

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

Hereinafter, a typical example of the test piece of the type A in which the conjugate is present will be described.
The sample pad, the conjugate pad, 3rd Pad, and the porous membrane are arranged in this order from upstream to downstream in the flow direction of the sample, and are arranged such that at least a part of each overlaps with the upper and lower layers. A test piece having such an arrangement example is shown in FIG.
When the sample containing the 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 pass through the pad while forming a complex by contacting the object. The complex is then passed through a 3rd Pad placed in contact with the lower surface of the conjugate pad and developed into a porous membrane. In the present invention, such a conjugate pad holds glucose and / or maltose together with the conjugate in a dry state.
A specific binding substance or antigen that immunologically reacts with the detection target is immobilized on a part of the porous membrane, so that the complex binds and is immobilized by the immunoreaction here. Will be realized. The immobilized complex is detected by means of detecting absorbance, reflected light, fluorescence, magnetism and the like derived from the labeling substance. When the labeling substance is a labeling substance having visibility such as colloidal gold or colored latex, it can be detected by visual observation.

Next, the test piece whose conjugate is of the type B will be described.
The difference from the type A test piece is that the sample pad and the conjugate pad are integrated, that is, the sample supply part and the detection reagent holding part are formed in a part of the sample pad. ..
The sample supply section is a section that supplies a sample containing a detection target, the detection reagent holding section is a section that contains a conjugate, and the sample supply section is on the upstream side of the detection reagent holding section. In the present invention, glucose and / or maltose are dried and held in such a detection reagent holding unit.

Next, a test piece whose conjugate is of type C will be described.
The difference from the above type A test strip is that the conjugate pad does not exist as a test strip, but the conjugate does exist as an individual detection reagent. For example, a filter chip in which the conjugate is retained in the filter can be mentioned. By using such a filter chip to filter the sample, the conjugate retained in the filter and the target substance to be detected bind to each other to form a complex. An object to be detected can be detected by supplying this to the same test piece as that of type A except that it has no conjugate pad. In the present invention, glucose and / or maltose are dried and held together with the conjugate in such a filter chip.

(Detection reagent)
In the present invention, the “detection reagent” is specifically a solution containing at least the conjugate.
In the present invention, the conjugate is preferably retained in a dry state together with glucose and / or maltose in a state capable of being eluted in the immunochromatographic test strip. Therefore, the detection reagent of the present invention contains glucose and / or maltose in addition to the conjugate. When the conjugate coexists with glucose and / or maltose, the aggregation of the conjugate is suppressed and dispersed even when applied to the conjugate pad, and the dispersibility is maintained even when dried. In addition, when the conjugate that is dried and retained is eluted together with glucose and / or maltose when the conjugate is eluted by passage of a sample solution or the like, the conjugate elution rate becomes constant, and as a result, the conjugate that elutes within a predetermined time The amount becomes constant, which brings about an effect that variation in measured values is reduced.

As the concentration of glucose contained in the detection reagent, 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, 0.5 to 10.0% is desirable, 1.0 to 5.0% is more desirable, and 2.0 to 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 retaining unit when glucose is dried and retained in the detection reagent retaining unit.
The concentration of maltose contained in the detection reagent is preferably 0.5 to 10.0%, more preferably 1.0 to 5.0%, and more preferably 2.0 to 3.0% based on the same assumption as that of glucose. Is even more desirable.
When the detection reagent contains both glucose and maltose, the total concentration is preferably within the above range.

Further, the detection reagent is for the purpose of keeping the conjugate in a stable state and promoting the specific reaction of the specific binding substance immobilized on the conjugate with the detection target when mixed with the sample. Further, it may further contain stabilizers 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 for the purpose of improving detection sensitivity, if necessary.
In this specification, the term “detection” or “measurement” needs to be interpreted in the broadest sense including proof of the presence of a detection target and / or quantification, and is not limited to any meaning. must not.

(Conjugate pad)
In the present invention, the “conjugate pad” is a material obtained by impregnating a material suitable for the conjugate pad with a detection reagent that specifically reacts with an object to be detected and drying the material. The conjugate pad has a function of forming a complex between the detection reagent and the detection target when the sample passes through the conjugate pad. The conjugate pad may be arranged so as to contact the specific binding substance-immobilized membrane by itself. Alternatively, it is placed in contact with the sample pad, receives the sample that has passed through the sample pad by capillary flow, and then transfers the sample by capillary flow to a 3rd Pad that is in contact with a surface different from the contact surface with the sample pad. You may arrange so. It should be noted that selection of one or more sites of the sample pad and the conjugate pad and how to arrange the selected sites 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 blends, nitrocellulose, polyesters, acrylonitrile copolymers, glass fibers or non-woven fibers such as rayon. A glass fiber pad is preferably used.
The conjugate pad of the present invention can be obtained by immersing the coating solution containing glucose and / or maltose and the conjugate in the conjugate pad and drying. Thus, the conjugate will be kept dry in the conjugate pad with glucose and / or maltose.

As a method of applying the detection reagent to the conjugate pad, a detection reagent solution containing glucose and / or maltose at a predetermined concentration is prepared, and the solution is horizontally moved while discharging the solution from the nozzle at a constant speed. It is possible to hold the solution so that it can be eluted by applying it to the conjugate pad in a line or the like and drying it using an apparatus having a mechanism capable of doing so. Alternatively, a predetermined amount of the detection reagent solution having a predetermined concentration may be sampled with a pipette or the like and applied to a part or the whole surface of the conjugate pad. Further, the conjugate pad may be dipped in a container containing a detection reagent solution having a predetermined concentration and applied on the entire surface.

The conjugate pad, if necessary, is 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 a sample component labeled with a labeling substance. Or a highly antigenic protein such as KLH (Keyhole mussel hemocyanin) labeled with a labeling substance. 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 a conjugate pad but is present as a detection reagent holding part in a part of the porous membrane having a detection part, the coating solution containing the above conjugate is similarly applied to a position upstream from the detection part. Then, the detection reagent holding part can be formed by drying.

The lower limit of the amount of glucose coexisting with the conjugate in the detection reagent holding part is preferably 0.335 mg / cm ² or more, and more preferably 0.669 mg / cm ² or more. Even more preferably, it is 1.34 mg / cm ² or more. The upper limit is preferably 6.69 mg / cm ² or less, more preferably 3.35 mg / cm ² or less. Even more preferably, it is 2.01 mg / cm ² or less. Further, the range of the retained amount is preferably 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 poor when kept dry, and if it is higher than 6.69 mg / cm ² , the dissolution rate of glucose decreases and the elution of the conjugate becomes slow. Because.
In order to obtain the retention amount, the concentration of glucose in the detection reagent may be adjusted by adjusting the glucose concentration in the detection reagent solution so as to have the preferable retention amount, and the area of the standard detection reagent retention unit. Is 0.78 cm ² and the coating amount is 52.2 μL, the lower limit of the glucose concentration (C) in the detection reagent solution is 0.5% or more, and more preferably 1.0% or more from the following formula. And even more preferably 2.0 or more. The upper limit is 10% or less, more preferably 5.0% or less, and even more preferably 3.0% or less. The range of glucose concentration (C) in the detection reagent solution is 0.5% to 10%, more preferably 1.0 to 5.0%, and even more preferably 2.0 to It is 3.0%.
A (mg / cm ² ) = BμL × (C% / 100) / Dcm ²
A: Amount of glucose retained per unit area of the detection reagent holding unit B: Amount of detection reagent solution to be impregnated in the detection reagent holding unit C:% concentration of glucose in the detection reagent solution (w / v)
D: Area of detection reagent holding unit It is desirable to adjust the amount of maltose to be coexisted with the conjugate in the detection reagent holding unit as in the case of glucose.
Furthermore, when the sugar coexisting with the conjugate contains both glucose and maltose, it is desirable to adjust the total amount of both to be in the above range.

(Hemagglutination agent)
In the present invention, when whole blood is used as a sample, it is desirable to use a hemagglutination agent in addition to the above 3rd Pad. A known polycationic hemagglutination agent can be used as the hemagglutination agent. Of these, polybrene is preferred. Polybrene is a kind of a cationic polymer which has a chemical name of hexadimethrin bromide and is assigned CAS number 28728-55-4.
As the usage mode of the hemagglutination agent such as polybrene, in addition to the mode of adding it to the sample diluent or directly to the specimen, the hemagglutination agent should be contained in the sample supply section (sample pad) of the immunochromatographic test strip. You can According to such a use mode, the hemagglutination agent comes into contact with whole blood, and the red blood cells in the whole blood are aggregated.

(Immobilization of specific binding substance on porous membrane)
Immobilization of a specific binding substance such as an antibody against an object to be detected in the immunochromatographic test strip of the present invention onto a porous membrane can be carried out by a generally known method. For example, in the case of the flow-through method, the above-mentioned specific binding substance is prepared at a predetermined concentration, and the solution is applied to the porous membrane in a specific symbol shape such as a dot or +. In addition, 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 the “control detection is performed. It is general that it is defined as “part”. Further, it is also possible to immobilize the specific binding substance that binds to the control reagent at a position different from the position of the specific binding substance that binds to the detection target to form a “control detection unit”.

In the case of the lateral flow type, a device having a mechanism capable of moving the above-mentioned specific binding substance to a predetermined concentration and horizontally moving it while discharging the liquid from the nozzle at a constant speed is used. , Is applied in a line on the porous membrane. 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 immobilized amount of the specific binding substance on the porous membrane can be optimized by adjusting the coating amount 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 speed from the nozzle of the device. Particularly, in the case of the lateral flow type, 0.5 to 2 μL / cm is suitable. In the present invention, the term "flow-through type membrane assay" refers to a method in which a sample solution or the like is developed so as to pass perpendicularly to the porous membrane, and the term "lateral flow type membrane assay" refers to It refers to the method of developing so that the sample liquid and the like move in parallel to the porous membrane.

Further, in the present invention, the coating position on the porous membrane of the specific binding substance that binds to the target, in the case of a lateral flow type, from the detection reagent holding unit, the detection reagent is developed by capillary action, It may be arranged so as to sequentially pass through the lines coated with the respective specific binding substances. 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 be sufficiently large so that the signal of the labeling substance can be detected. Also in the case of the flow-through type, the application position of the specific binding substance that binds to the target 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 be usually prepared using a predetermined buffer solution. Examples of the buffer solution include a commonly used buffer solution 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 set appropriately according to the properties of the specific binding substance used.
For example, a buffer solution having a pH of 8.0 can be used for the anti-cTnI antibody described below. The buffer solution may further contain salts such as NaCl, stabilizers and preservatives such as sucrose, and preservatives such as proclin. The salts include not only salts such as NaCl that are added for adjusting the ionic strength, but also salts such as sodium hydroxide that are added in the step of adjusting the pH of the buffer solution. After immobilizing the specific binding substance on the porous membrane, blocking can also be performed by further applying a commonly used blocking agent in the form of a solution or vapor to cover other than the specific binding substance immobilization site. In the present specification, the specific binding substance-immobilized porous membrane as described above is referred to as “specific binding substance-immobilized membrane”, and the antibody-immobilized porous membrane is referred to as “antibody-immobilized”. Sometimes called a "membrane."

(Porous membrane)
In the present invention, as the porous membrane (hereinafter sometimes 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 papers and nitrocellulose membranes sold by Merck Millipore, Toyo Roshi Kaisha, GE Healthcare, and the like. In addition, by appropriately selecting the pore size and structure of the porous membrane made of this porous material, the speed at which the immune complex of the gold colloid-labeled specific binding substance (conjugate) and the detection target flows through the membrane can be determined. It is possible to control. By controlling the rate of flow through the membrane, the amount of the labeled specific binding substance that binds to the specific binding substance immobilized on the membrane can be adjusted. It is desirable to optimize the immunochromatographic test piece in consideration of the combination with other constituent materials.

(3rd Pad)
In the present invention, 3rd Pad means that among the reaction components of the sample and the detection reagent, components unnecessary for the detection of the detection target are removed, and the components required for the reaction have a specific binding substance immobilized thereon. The porous membrane can be arranged for the purpose of smoothly expanding it. For example, it is desirable that blood cells, insoluble blood cell lysates, and the like be removed as unnecessary components for detection. In addition, in the 3rd Pad, among the aggregates generated by the reaction of the antigen-specific binding substance, the aggregates that have moved to the specific binding substance-immobilized membrane and have become large enough to prevent smooth development are removed in advance. It is also possible to have the additional effect of.
The 3rd Pad includes any substance and form through which a liquid, a component to be detected, and a detection reagent can pass. Specific examples include, but are not limited to, glass fiber, glass fiber, acrylic fiber, hydrophilic polyethylene material, polysulfone, dry paper, paper pulp, woven fabric, and the like. When used for the purpose of separating blood cells, 3rd Pad is sometimes called a blood cell separation membrane. In the present invention, when whole blood is used as a sample, it is preferable 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 absorbent pad is a portion having liquid absorbency that controls the development of the sample by absorbing the sample that has moved / passed through the porous membrane. In the lateral flow type, it may be provided at the most downstream side of the strip structure, and in the flow through type, it may be provided, for example, below the specific binding substance-immobilized membrane. The absorbent pad may be, for example, filter paper, but is not limited to this.

(Detection device)
The immunochromatographic test strip of the present invention is suitable in consideration of the size of the test strip, the method and position of addition of the sample, the immobilization position of the specific binding substance on the specific binding substance immobilization membrane, the detection method of the signal, etc. It can be stored and installed in a container (housing) for use, and the state in which it is stored and installed is called a "detection device."
In the detection device of the present invention, in addition to the sample supply section, a developing solution supply section for separately supplying a developing solution can be provided on the upstream side of the sample supply section.

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

(Sample)
In the detection method of the present invention, the “specimen” containing the detection target refers to blood, urine, sputum, saliva, nasal discharge, nasal swab, pharyngeal swab, other body fluids, biological samples such as feces, and the like. The biological sample may be used as a sample as it is, and a sample obtained by appropriately diluting with a diluting solution, extracting and / or extracting by filtration is also included in the sample of the present invention. 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, a diluent may be used when the sample needs to be diluted depending on the concentration of the detection target in the sample. The diluent significantly inhibits the reaction between the target substance and the specific binding substance, or conversely significantly accelerates the reaction to cause over-aggregation of the labeling substance, resulting in poor expansion in capillary phenomenon, and Diluents of any composition may be used as long as signal detection of the reaction between the specific binding substance and the specific binding substance depending on the concentration of the binding substance is not impossible. The sample diluting solution may also be called a sample extract or developing solution.

(Detection target)
The detection target of the present invention is a substance present in a biological sample such as blood (whole blood), red blood cells, serum, plasma, urine, saliva or sputum, for example, CRP (C-reactive protein), IgA, IgG. , Inflammation-related markers such as IgM, fibrin degradation products (eg D-dimer), soluble fibrin, TAT (thrombin-antithrombin complex), PIC (plasmin-plasmin inhibitor complex) and other coagulation / fibrinolytic markers, oxidized LDL, Circulation-related markers such as BNP (brain natriuretic peptide), H-FABP (heart-type fatty acid binding protein), cardiac troponin I (cTnI), metabolism-related markers such as adiponectin, CEA (carcinoembryonic antigen), AFP (α-) Tumor markers such as fetoprotein), CA19-9, CA125, PSA (prostate specific antigen), HBV (hepatitis B virus), HCV (hepatitis C virus), chlamydia trachomatis, gonorrhea-related markers, allergen specific Examples include IgE (immunoglobulin E), hormones, drugs and the like.

(Specific binding substance)
In the present invention, specific binding substances for the substance to be measured carried on insoluble carrier particles such as latex include proteins, peptides, amino acids, lipids, carbohydrates, DNA, RNA, receptors, haptens, and the like. There are no particular restrictions on the origin of the molecular weight, such as high or low, natural or synthetic, and examples include antibodies or antigens that can be used in immunoassays that utilize immune reactions.

(Antibody used in the present invention)
The antibody against the detection target used in the present invention is not limited by the method for producing it as long as it is an antibody that specifically reacts with the detection target, and may be a polyclonal antibody or a monoclonal antibody. May be. More preferably, it is a monoclonal antibody. Generally, a hybridoma producing the antibody is a myeloma of the same species as the spleen cells of an animal immunized with the detection target as an immunogen according to the method of Kohler and Milstein (see Nature, Vol. 256, page 495 (1975)). It can be produced by cell fusion with cells (myeloma cells).

As the antibody of the present invention, in addition to the whole antibody molecule, it is also possible to use a functional fragment of an antibody having an antigen-antibody reaction activity. In addition to those obtained through the process of immunizing general animals (mouse, goat, sheep, etc.), the amino acid sequence of animal species different from the animal immunized with the immunogen (substance to be measured) by genetic recombination technology etc. Antibodies (chimeric 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 ′ and single chain antibody (scFv), which are fragments having antigen-antibody reaction activity. Functional fragments of these antibodies can be produced by treating the antibodies obtained as described above with a proteolytic enzyme (eg, pepsin or papain).
Here, when the antibody used in the assay method for detecting an object to be detected by the formation of a so-called sandwich is a monoclonal antibody, an antibody for immobilizing a label (first antibody) and an antibody for immobilizing a porous membrane (second Antibody), the epitope of the second antibody is different from that of the first antibody when the epitope of the first antibody is monovalent, and is the second when the epitope of the first antibody is multivalent. The epitope of the antibody may be the same as or different from that of the first antibody.

(kit)
The detection kit utilizing the immunochromatography of the present invention is an immunochromatographic test strip having at least a sample supply unit, a developing unit and a detecting unit, and a part of the developing unit is labeled with a labeling substance. It is sufficient that the conjugate (detection reagent) containing the specific binding substance contains the immunochromatographic test strip retained so as to be eluted together with glucose and / or maltose.
The detection kit may further include reagents necessary for detection, a sample diluent, a test tube, a filtration filter, a swab for collecting a sample, an instruction manual, a housing for storing a test piece, and the like.

The immunochromatographic detection method for detecting a detection target substance in a sample according to the present invention using a test piece for immunochromatography is a specific binding of the detection target substance in the sample labeled with a labeling substance in the presence of glucose and / or maltose. A method comprising contacting with a conjugate containing a volatile substance. More specifically, the method includes the following steps (A) to (C).
(A) Process test piece for supplying a specimen to the sample supply section of the following test piece;
An immunochromatographic test strip comprising at least a sample supply unit, a development unit and a detection unit,
In the developing part, a conjugate containing a specific binding substance labeled with a labeling substance is retained in a dry state so that it can be eluted together with glucose and / or maltose. (C) a step of forming a complex between the substance to be detected and a conjugate of (C) a step of detecting the complex in the detection section, wherein the detection of the complex includes qualitative detection by visual observation and optical detection. Both quantitative measurements by means are included. Further, in the quantitative measurement by the optical detecting means of the present invention, in addition to the method of measuring the test pieces one by one, a method of using an apparatus for continuously and automatically measuring a plurality of test pieces and a plurality of test pieces Various modes are included, such as a method using a device for simultaneously measuring the.

Specific examples of the present invention will be described below, but these are merely examples and the present invention is not limited thereto.

[Example 1] Screening of saccharides applied to the conjugate pad of the present invention As a saccharide applied to a conjugate pad, a blank value is reduced as compared with conventionally used lactose, and sample measurement sensitivity is increased, Was screened under the condition.
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) to 500 mL of purified water heated to 93 ° C. / V) 1 mL of an aqueous solution of triammonium citrate was added and mixed with stirring. Subsequently, 1 mL of a 5% (w / v) tetrachlorogold (III) aqueous solution was added, the mixture was reacted for 10 minutes with stirring, and then the reaction solution was boiled. Then, the solution was cooled in ice water to prepare a gold colloid solution having an average particle size of 60 nm. This gold colloid solution having an average particle diameter of 60 nm was adjusted to 1 OD / mL with purified water by the absorbance at the maximum absorption wavelength of the gold colloid.

(Ii) Preparation of Anti-cTnI Antibody Conjugate An anti-cTnI monoclonal antibody diluted to 20 μg / mL with 2 mM Tris-hydrochloric acid buffer (pH 7.0) was added to the 1 OD / mL gold colloid solution (pH 8.0). Add and stir at room temperature for 10 minutes. Purified water containing 0.5% (w / v) of NeoProtein Saver (Toyobo Co., 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 xg for 45 minutes at 10 ° C. After removing the supernatant, 1 mL of 0.2% (w / v) NeoProteinSaver aqueous solution was added to the obtained precipitate to suspend the conjugate, and thus an anti-cTnI antibody conjugate was obtained.

(Iii) Preparation of gold colloid labeled KLH for control line (KLH conjugate) 20 mL of the gold colloid solution of 1 OD / mL was dissolved in 2 mmol / L phosphate buffer to 620 μg / mL (KLH (Sigma Co.)). (Manufactured by K.K.) was added and 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, after centrifugation at 10 ° C. for 45 minutes to remove the supernatant, 1 mL of the conjugate diluent was added to the obtained precipitate to suspend the conjugate, to obtain a KLH conjugate.

2) Preparation of conjugate pad 3 OD / mL of anti-cTnI antibody conjugate, 0.75 OD / mL of KLH conjugate, 0.5% Lipidure BL-1301, 0.25 mg / mL Heteroblock, various saccharides described below, 2 A detection reagent was prepared by mixing 0.0% NPS and 20 mM MOPS (pH 7.2), and 52.2 μL of the solution was added per 0.78 cm ² to a glass fiber pad (Merck Millipore) that was appropriately cut into a necessary size. It was permeated in a volume ratio. It was dried by heating in a dry oven at 70 ° C. for 45 minutes to obtain a conjugate pad.
<Tested saccharides>
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) 10 mM PB pH 8.0 containing 0.09% NaN ₃ for the test line so that the final concentrations were 2.5% and 3 mg / mL. A solution containing sucrose and anti-cTnI monoclonal antibody was prepared.
In addition, a rabbit anti-KLH polyclonal antibody (manufactured by Bethyl) was diluted and prepared for the control line in the same manner as described above.
At a position inside one end of the short side of a nitrocellulose membrane (Hi-Flow plus HF180, Merck Millipore), the anti-cTnI monoclonal antibody was adjusted to 1 μL / cm using an immunochromatographic dispenser “XYZ3050” (BIO DOT). It was set and applied in a line to form a test line. An anti-KLH polyclonal antibody was similarly applied at intervals of about 4 mm from the position of the test line to form a control line. It was dried at 70 ° C. for 45 minutes in a dry oven to obtain an antibody-immobilized membrane.

4) Preparation of sample pad 20 mM MOPS (pH 7.2) containing 0.5% lactose and 2% polybrene was cut into a glass fiber pad (Lydall) 1.5 times the volume of the pad. What was soaked in a volume and dried in a dry oven at 70 ° C. for 45 minutes was used as a sample pad.

5) Preparation of immunochromatographic test piece The above-mentioned antibody-immobilized membrane (b) was attached to a plastic pressure-sensitive adhesive sheet (a), and the anti-cTnI antibody (c) and then the anti-KLH antibody (d) were arranged in this order on the upstream side of development. An application part was arranged, and a blood cell separation membrane (3rd Pad) (e) was mounted on the membrane. Then, the conjugate pad (f) produced in 2) above is placed and mounted, and further the sample pad (g) produced in 4) above is placed and attached so as to overlap the conjugate pad, and at the opposite end, An absorbent pad (h) was placed and mounted. Thus, an immunochromatographic test piece was produced by cutting into a structure in which the respective constituent elements were superposed. During the assay, the test strip may be stored and mounted in a dedicated plastic housing (having a sample addition window and a detection window, not shown in FIG. 9) to form a detection device. FIG. 9 shows a schematic configuration diagram of the immunochromatographic test piece.

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

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

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

2. Test results According to this figure, when sucrose or glucose is added to the conjugate coating solution, there is little variation in measured values and excellent simultaneous reproducibility at any concentration as compared to the case where conventional lactose is added. It could be confirmed.

[Test Example 3] Simultaneous reproducibility and accelerated stability confirmation test after storage under severe conditions (1)
A test piece in which glucose, lactose, and sucrose were added to the conjugate coating solution was stored at 37 ° C. for 10 days to be stored under severe conditions, and a test for confirming variation variation and stability of measured values was performed.
1. Test Method A test piece of the present invention was produced 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. Test except that the number of n was 4 and the samples were LOW samples (plasma samples containing 150 pg / mL cardiac troponin I (cTnI)) and HIGH samples (plasma samples containing 500 pg / mL cardiac troponin I (cTnI)) CV was calculated in the same manner as in Example 2. Also, with respect to the test piece stored at 37 ° C. for 10 days, the same sample was measured and the CV was calculated. The result of variation in the measured value of the LOW sample is shown in FIG. 4, and the result of the variation in the measured value of the HIGH sample is shown in FIG. Before being stored under severe conditions, it is referred to as “before acceleration”, and after being stored under severe conditions as “after acceleration”.
Further, as stability evaluation, FIG. 6 shows the relative values of the absorbance measurement values before and after storage under severe conditions (the absorbance measurement value after “acceleration” with respect to the absorbance measurement value before “acceleration”).

2. Test results According to FIGS. 4 and 5, when sucrose or glucose was added to the conjugate coating solution, the measured value was higher than that when lactose was added after storage under severe conditions regardless of the concentration of the sample. It was confirmed that there was little variation and the simultaneous reproducibility was excellent.
Further, according to FIG. 6, it was confirmed that when glucose was added to the conjugate coating solution, 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 severe conditions. did it.
From the above test results, it was confirmed that when glucose was added to the conjugate coating solution, both simultaneous reproducibility and stability were excellent.

[Test Example 4] CV reduction effect confirmation test (2)
Among the sugars selected by the screening in Test Example 1, glucose, maltose, and trehalose were confirmed to have a CV reducing effect. For comparison, lactose was similarly confirmed to have a CV reducing effect.
1. Test method A HIGH sample (a plasma sample containing 500 pg / mL cardiac troponin I (cTnI)) was used, and 2.4% glucose, maltose, trehalose, and lactose were used as saccharides for adjusting the detection reagent. Color amounts (absorbance) of the test lines were measured in the same manner as in Test Example 2 except that (UniSart CN150 white backed, Sartorius Stedim Biotech, model number 1UN15WR100025NT) was used as the cellulose membrane, and their CV values (variation) were measured. ) Was calculated respectively. The results are shown in Fig. 7.

2. Test Results According to this figure, it was confirmed that when glucose or maltose was added to the conjugate coating solution, there were few variations in measured values and the simultaneous reproducibility was excellent as compared with the case where conventional lactose was added.

[Test Example 5] Accelerated stability confirmation test after storage under severe conditions (2)
A test piece prepared by adding glucose, maltose, and trehalose to the conjugate coating solution was stored at 37 ° C. for 10 days to be stored under severe conditions, and a test for confirming stability was conducted.
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 number of n was set to 5, and a HIGH sample (a plasma sample containing 1000 pg / mL cardiac troponin I (cTnI)) was used, and a nitrocellulose membrane (UniSart CN150 white backed, Sartorius Stedim Biotech, model number 1UN15WR100025NT) was used. Tests were performed in the same manner as in Test Example 3 except that the stability was evaluated. The relative values of the absorbance measurement values before and after storage under severe conditions are shown in FIG. 8 with respect to the “after acceleration” absorbance measurement values with respect to the “before acceleration” absorbance measurement values.

2. Test results According to this figure, when glucose was added to the conjugate coating solution, it was confirmed that the stability of the measured values was small and the stability was excellent compared to the case where maltose was added even after storage under severe conditions. It was
From the results of Test Examples 1 and 4 described 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 another sugar was added. ..
Moreover, when the results of Test Examples 1 to 5 are summed up, it can be reconfirmed that when glucose is added to the conjugate coating solution, both simultaneous reproducibility and stability are superior to conventional lactose and other sugars. It was

According to the immunochromatographic test strip of the present invention, since the conjugate is allowed to coexist with glucose and / or maltose to be retained, the dispersibility of the conjugate is ensured, and the conjugate is promptly passed by the passage of the sample liquid. It is possible to provide an immunochromatographic test strip which is eluted from the detection reagent holding part of (3) and has a reduced variation in the elution rate.
Further, according to the immunochromatographic test strip of the present invention, the conjugate is rapidly eluted to the test strip developing portion, so that the detection portion surely develops color within a predetermined reaction time and prevents the delay of the test. You can

(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

An immunochromatographic test strip comprising at least a sample supply unit, a development unit and a detection unit,
An immunochromatographic test strip in which a conjugate containing a specific binding substance labeled with a labeling substance is held in a dry state so as to be eluted together with glucose and / or maltose in the developing part.
The immunochromatographic test strip according to claim 1, wherein the conjugate comprises a conjugate pad, which is dried and held so as to be eluted together with glucose and / or maltose, as a developing portion.
The immunochromatographic test piece according to claim 2, wherein the conjugate pad is a glass fiber pad.
The immunochromatographic test strip according to any one of claims 1 to 3, wherein the labeling substance is colloidal gold.
The immunochromatographic test strip according to any one of claims 1 to 4, wherein the specific binding substance is an antibody.
An immunochromatographic detection kit comprising the immunochromatographic test strip according to any one of claims 1 to 5.
An immunochromatographic detection method for detecting an object to be detected in a specimen using a test piece for immunochromatography,
The immunochromatographic detection method, which comprises a step of bringing a substance to be detected in a sample into contact with a conjugate containing a specific binding substance labeled with a labeling substance in the presence of glucose and / or maltose.
An immunochromatographic detection method for detecting an object to be detected in a sample using an immunochromatographic test strip, comprising the steps (A) to (C).
(A) Process test piece for supplying a specimen to the sample supply section of the following test piece;
An immunochromatographic test strip comprising at least a sample supply unit, a development unit and a detection unit,
In the developing part, a conjugate containing a specific binding substance labeled with a labeling substance is retained in a dry state so that it can be eluted together with glucose and / or maltose. (C) a step of forming a complex of the substance to be detected and a conjugate of (C) the step of detecting the complex in a detection part
9. The immunochromatographic detection method according to claim 8, wherein the immunochromatographic test strip includes, as a developing part, a conjugate pad in which the conjugate is dried and held so as to be eluted together with glucose and / or maltose.
The immunochromatographic detection method according to claim 9, wherein the conjugate pad is a glass fiber pad.
The immunochromatographic detection method according to claim 7, wherein the labeling substance is gold colloid.
The immunochromatographic detection method according to claim 7, wherein the specific binding substance is an antibody.
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 immunochromatography,
A method for reducing variation in the above-mentioned measured values, which comprises a step of bringing a substance to be detected in a sample into contact with a conjugate containing a specific binding substance labeled with a labeling substance in the presence of glucose and / or maltose.
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 immunochromatography, the method for reducing variation in measured values including steps (A) to (C).
(A) Process test piece for supplying a specimen to the sample supply section of the following test piece;
An immunochromatographic test strip comprising at least a sample supply unit, a development unit and a detection unit,
In the developing part, a conjugate containing a specific binding substance labeled with a labeling substance is retained in a dry state so that it can be eluted together with glucose and / or maltose. (C) a step of forming a complex of the substance to be detected and a conjugate of (C) the step of detecting the complex in a detection part