JP7137168B2 - Test piece for immunochromatography - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunochromatographic test strip and an immunochromatographic detection method using the test strip.

Along with the popularization of POCT (point of care), which is treatment near the patient, lateral flow type immunochromatographic detection method using test strips such as nitrocellulose membrane is widely used as a simple immunoassay using antigen-antibody reaction. is doing.

A test piece for immunochromatography (hereinafter sometimes simply referred to as an immunochromatographic test piece) is generally a porous membrane comprising a sample supply portion, a developing portion, and a detection portion, and a labeled antibody against a target substance to be detected. is eluted at the development start site of the development section so that it can pass through the development section and reach the detection section after contact with the specimen (sample), and the detection antibody is immobilized on a part of the development section. It has a structure that constitutes a detection unit.

Here, when the sample is dropped onto the sample supply unit, if the sample contains a target substance to be detected, the target substance to be detected specifically binds to the labeled antibody to form a complex, and the complex is , develops the developing portion in the downstream direction, and binds to the immobilized antibody for detection in the detecting portion. Therefore, by detecting the sandwich-type complex of the labeled antibody, the target substance, and the immobilized antibody in the detection unit, the target substance can be qualitatively or quantitatively analyzed.

In the detection part of the immunochromatographic test strip having the above configuration, as a method for detecting the complex of the substance to be detected and the conjugate, there is a method of measuring the intensity of the reflected light derived from the label and calculating the absorbance (reflection absorbance). be. The reflection absorbance is calculated, for example, by measuring the reflection light intensity of two sites near the detection line of the substance to be detected in the detection unit and the vicinity thereof, and using the ratio thereof. When using this method, the complex is detected The intensity of the reflected light near the upstream or downstream side of the detector where the light is detected may increase non-specifically, resulting in disturbance in the measured waveform. In this case, when the concentration of the substance to be detected is low, the disturbance of the measured waveform cannot be ignored and the baseline cannot be drawn. It may become possible.

Since the occurrence frequency and the degree of disturbance in the measured waveform are not constant, the reproducibility of the measurement decreases, and it is necessary to correct the measured value appropriately each time the measurement is performed (in other words, the measured waveform confirmation) was necessary.

In the normal immunochromatographic detection method using a white membrane, the disturbance of the measurement waveform is a phenomenon in which the color of the relevant site on the membrane is relatively whiter than the surrounding color, and the color appears to be missing (so-called white This is visually observed as a dropout phenomenon, hereinafter simply referred to as a white spot), which hinders the visual determination of the coloration derived from the label and causes a lack of accuracy in determination results.

Although the cause of such white spots is not clear, it is assumed that there are differences between production lots of test pieces, differences in storage conditions of test pieces, and differences in manufacturing conditions such as pretreatment conditions for test pieces.
The following literature is known as a method for eliminating such white spot phenomenon.
Patent Document 1 discloses an assay using a sample diluent to which methanol is added in order to prevent white spots that are often observed when immunochromatographic test strips are stored for a long period of time.

Patent Literature 2 discloses a method of treating an antibody-immobilized membrane of an immunochromatography test strip with a specific surfactant such as n-octyl-β-D-glucoside to prevent white spots and disturbance of measured waveforms.

International publication WO2015/080286 pamphlet International publication WO2010/001598 pamphlet

An object of the present invention is to avoid the occurrence of so-called white spots in immunochromatographic test strips by a completely different approach from the above prior art, and to provide test strips that can be detected with better visibility. Another object of the present invention is to provide an immunochromatographic test piece that can prevent variations in the detected values of test lines.

The present inventors have tried to change various elements of the immunochromatographic test strip in order to solve the above problems, and surprisingly, in the immunochromatographic test strip containing a porous membrane, a specific binding substance such as an antibody By providing an opaque base material on the back surface of the porous membrane on which is immobilized on the surface, it is possible to avoid white spots near the test line and to prevent variations in detection values, and have completed the present invention. . That is, the present invention has the following configurations.
(1) An immunochromatographic test strip containing at least a porous membrane and having a sample supply section, a developing section, and a detection section, wherein the detection section is immobilized on the surface of the porous membrane, and the porous membrane is The test piece, wherein an opaque base material having a thickness of 30 μm or more and 200 μm or less is arranged on the back surface.
(2) The test piece according to (1), wherein the opaque substrate is a member for lining a porous membrane.
(3) The immunochromatographic test piece according to (1) or (2), wherein the opaque substrate is a white substrate.
(4) The immunochromatographic test strip according to any one of (1) to (3), including a sample pad serving as the sample supply unit, and a conjugate pad containing a hetero-binding substance labeled with colloidal gold or colored latex. .
(5) The immunochromatographic test strip according to any one of (1) to (4), wherein the specific binding substance is an antibody.
(6) The immunochromatographic test piece according to any one of (1) to (5), wherein a backing sheet having a thickness of 10 μm or more and 600 μm or less is arranged under the porous membrane.
(7) The immunochromatographic test strip according to any one of (1) to (6), which is a test strip for detection by optical means.
(8) An immunochromatographic detection kit comprising the immunochromatographic test piece according to any one of (1) to (7).
(9) An immunochromatographic detection method, characterized by using the following test piece;
An immunochromatography test piece comprising a porous membrane having at least a detection part, and an opaque substrate having a thickness of 30 µm or more and 200 µm or less arranged on the back side of the porous membrane.
(10) The immunochromatographic detection method according to (9), wherein the opaque substrate is a member for lining a porous membrane.
(11) The immunochromatographic detection method according to (9) or (10), wherein the opaque substrate is a white substrate.
(12) The test strip includes a sample pad that serves as the sample supply unit, and a conjugate pad containing a specific binding substance labeled with colloidal gold or colored latex (9) to (11). The described immunochromatographic detection method.
(13) The immunochromatographic detection method according to any one of (9) to (12), wherein the specific binding substance is an antibody.
(14) The immunochromatographic detection method according to any one of (9) to (13), wherein the test piece has a backing sheet having a thickness of 10 μm or more and 600 μm or less under the porous membrane.
(15) The immunochromatographic detection method according to any one of (9) to (14), which comprises detecting an optical parameter derived from the labeling substance by optical means.
(16) The immunochromatographic detection method according to any one of (9) to (15), which comprises a step of determining coloration derived from the labeling substance by visual observation.
(17) A method for reducing variation in measured values in an immunochromatographic detection method, the method comprising using the following test piece;
An immunochromatographic test strip containing at least a porous membrane and having a sample supply section, a developing section, and a detection section, wherein the detection section is immobilized on the surface of the porous membrane, and a thickness of 30 μm is provided on the back side of the porous membrane. A test piece on which an opaque substrate having a thickness of 200 μm or more is arranged.
(18) A method for reducing white spots on a test piece in an immunochromatographic detection method using a test piece, the method characterized by using the following test piece;
An immunochromatographic test strip containing at least a porous membrane and having a sample supply section, a developing section, and a detection section, wherein the detection section is immobilized on the surface of the porous membrane, and a thickness of 30 μm is provided on the back side of the porous membrane. A test piece on which an opaque substrate having a thickness of 200 μm or more is arranged.

According to the present invention, it is possible to provide an immunochromatographic test piece that has excellent visibility during visual detection because white spots near the test line can be avoided and that has less variation in measured values by optical means.

2 is a graph showing variations in measured values when immunochromatographic measurement is performed using immunochromatographic test pieces using porous membranes with a transparent substrate (comparative example) and a white substrate (invention) as backing members. (Test example 2, substance to be detected is on the medium concentration side) 2 is a graph showing variations in measured values when immunochromatographic measurement is performed using immunochromatographic test pieces using porous membranes with a transparent substrate (comparative example) and a white substrate (invention) as backing members. (Test example 3, the substance to be detected is on the low concentration side) 2 is a graph showing variations in measured values when immunochromatographic measurement is performed using immunochromatographic test pieces using porous membranes with a transparent substrate (comparative example) and a white substrate (invention) as backing members. (Test example 4, the substance to be detected is on the high concentration side) 1 is a perspective view showing an immunochromatographic test strip of the present invention; FIG. A is the entire immunochromatographic test strip and B is the porous membrane. 1 is a top view of an immunochromatographic detection device of the present invention; FIG. FIG. 6 shows a cross section of the device shown in FIG. 5 cut along the dotted line.

(Immunochromatographic test piece)
The immunochromatographic test strip of the present invention includes a porous membrane having at least a "sample supply section", a "development section", and a "detection section", and a labeled specific binding substance that binds to a detection target is , so that it can pass through the developing part and reach the detection part after contact with the specimen, it is held in an elutable manner at the development start site of the developing part, and a specific binding substance for detection is immobilized on a part of the developing part and constitutes a detection unit.

As an example of embodying these, a sample pad serving as a sample supply part, a conjugate pad holding a labeled specific binding substance that binds to a detection target so as to be eluted and serving as a part of the developing part, a detection specific binding substance is immobilized on a portion thereof, and includes a porous membrane serving as a developing portion and a detecting portion. That is, a typical immunochromatographic test strip of the present invention has the following configuration.

(1) a sample pad to which a specimen is supplied; (2) a conjugate pad disposed downstream of the sample pad and having a gold colloid surface on which a conjugate sensitized with a first specific binding substance is held so as to be eluted; (3) A porous membrane which is located downstream of the conjugate pad and has on its surface a detection portion on which a second specific binding substance that binds to the complex of the conjugate and the substance to be detected is immobilized. and, on the back side, said porous membrane having an opaque substrate disposed thereon

Here, the sample pad, the conjugate pad, and the porous membrane may each constitute separate carriers, or two may constitute one carrier. Any embodiment is included as long as it is constructed in the order of a pad and a porous membrane.
Immunochromatographic test strips include, in addition to the above configuration, one or more of an absorbent pad and a 3rd pad further arranged and mounted.
The test pieces are usually arranged on a solid phase support such as a plastic adhesive sheet (hereinafter sometimes referred to as a backing sheet).

In addition, 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, the test piece may be laminated with a polyester film or the like. .

The detection method using the immunochromatographic test strip of the present invention is carried out as follows.
When the sample is dropped onto the sample supply unit, if the sample contains the target substance, the target substance specifically binds to the labeled specific binding substance to form a complex. The complex develops through the developing portion in the downstream direction and binds to the specific binding substance immobilized on the detection portion. In the detection unit, a sandwich-type complex is formed by the labeled specific binding substance, the detection target, and the immobilized specific binding substance. Quantitative analysis is possible. Detection includes determination of coloration by visual observation and a method of detecting an optical parameter derived from the label by optical means. When using optical means, it can be manual, automated, and continuous.

(sample pad)
In the present invention, the "sample pad" is a part that serves as a sample supply part for receiving a sample, and absorbs a liquid sample in the state of being molded into the pad, allowing the liquid and components of the detection target to pass through. Any substance and form is included.
The sample pads of the present invention can be pretreated to improve water permeability.
Moreover, when whole blood is measured, a erythrocyte aggregating agent can be included. In this case, it may be included in at least a part of the sample pad, and may be included in the entire sample pad.

Examples of suitable materials for sample pads include, but are not limited to, glass fibers, acrylic fibers, hydrophilic polyethylene materials, dry paper, paper pulp, textiles, and the like. Glass fiber pads are preferably used. The sample pad can also have the function of a conjugate pad, which will be described later. In addition, the sample pad may contain a commonly 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)
A conjugate in the present invention refers to a labeled substance immobilized with a specific binding substance that binds to a detection target or a control substance.
The label used in the present invention can constitute a conjugate by sensitizing (immobilizing) a specific binding substance such as an antibody, and is brought into contact with a sample to detect an object (such as an antigen) in the sample. Any material can be used as long as it can play a role as a label in the method, and examples include colloidal gold particles, colloidal platinum particles, color latex particles, magnetic particles, etc. Among them, colloidal gold and colored latex. is preferred, and colloidal gold is even more preferred. These particle diameters may be adjusted according to each type so as to obtain the desired detection sensitivity of the object to be measured. For example, the particle diameter of colloidal gold particles is preferably 20 to 100 nm, more preferably 30 to 100 nm, preferably 60 nm.
The conjugate of the present invention can also block with a blocking agent a region on the surface of colloidal gold or the like to which a specific binding substance is not bound.

The conjugate may exist as a conjugate pad, that is, in a state of being impregnated in a dedicated pad (conjugate pad) other than the sample pad, the third pad, and the porous membrane ( Type A), may be present as a conjugate moiety on a part of the sample pad (type B). Furthermore, it may exist as a separate conjugate reagent to be mixed with the specimen, separate from the test strip (type C).

A typical example of the test piece having the above type A in which the conjugate exists will be described below.
A sample pad, a conjugate pad, a third pad, and a porous membrane are arranged in this order from upstream to downstream in the flow direction of the sample so that at least a portion of each layer overlaps with the upper and lower layers. A test piece with such an arrangement is shown in FIG.

When a sample containing the analyte is applied to the sample pad of such a test strip, the analyte flows through the sample pad to the downstream conjugate pad. In the conjugate pad, the object to be detected and the conjugate come into contact with each other and pass through the pad while forming a complex. The complex then passes through a third pad placed in contact with the underside of the conjugate pad and unrolls into the porous membrane.

Since a specific binding substance that binds to the substance to be detected is immobilized on a portion of the porous membrane, the complex is bound and immobilized here. The immobilized complex is detected by means of detecting absorbance, reflected light, fluorescence, magnetism, etc. derived from the labeling substance.

Next, a test piece in which the mode of existence of the conjugate is 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 pad has a sample supply part and a conjugate part.
The sample supply section is a section that supplies a sample containing a substance to be detected, the conjugate section is a section that contains a conjugate, and the sample supply section is upstream of the conjugate section.

Next, a test piece in which the mode of existence of the conjugate is type C will be described.
The difference from the Type A test strip is that the conjugate pad does not exist as a test strip, and the conjugate exists as a separate conjugate reagent. For example, there is a filter tip in which the conjugate is embedded in the filter. By filtering the specimen using such a filter chip, the conjugate retained in the filter and the substance to be detected bind to form a complex (aggregate). By supplying this to the same test piece as type A except that it does not have a conjugate pad, the detection target can be detected.

(detection reagent)
In the present invention, a "detection reagent" is specifically a solution containing at least a conjugate.
The detection reagent keeps the conjugate stable and facilitates specific binding substances such as antibodies immobilized on the conjugate to specifically react with the analyte when mixed with the sample. Alternatively, it may contain, for example, one or more stabilizers, solubilizers, etc., for the purpose of rapidly and effectively dissolving and fluidizing the conjugate. Examples of the stabilizing agent, solubilizing agent and the like include bovine serum albumin (BSA), sucrose, casein and amino acids.

In addition, the detection reagent may optionally contain known sensitizers and chelating agents such as EDTA and EGTA for the purpose of improving detection sensitivity.
In the present specification, the term "detection" or "measurement" should be interpreted in the broadest sense, including proof of existence and/or quantification of the target to be detected, and should not be construed as restrictive in any sense. must not.

(sample diluent)
In the present invention, if the sample needs to be diluted according to the concentration of the substance to be detected in the sample, a diluent may be used. The diluent significantly inhibits the reaction between the substance to be detected and the specific binding substance, or on the contrary, significantly accelerates the reaction, causing overaggregation of the labeled substance, which causes poor expansion in capillary action, or A diluent having any composition may be used as long as the signal of the reaction between the substance to be detected and the specific binding substance can be detected according to the concentration of the substance.

(Conjugate pad)
In the present invention, the "conjugate pad" is obtained by impregnating a material suitable for a conjugate pad described below with a detection reagent that specifically reacts with a substance to be detected and drying the impregnated material. The conjugate pad has the function of forming a complex between the detection reagent and the substance to be detected when the sample passes through the conjugate pad. The conjugate pad may be placed in contact with the porous membrane on which the specific binding substance is immobilized by itself.
Alternatively, the specimen is placed in contact with the sample pad, receives the specimen that has passed through the sample pad by capillary flow, and subsequently transfers the specimen to a 3rd Pad that contacts with the sample pad on a surface different from the contact surface with the sample pad by capillary flow. can be arranged as follows.

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. Glass fiber pads are preferably used.

If necessary, the conjugate pad is labeled with a "control reagent" for ensuring the reliability of the immunochromatographic detection method, such as an antibody or label that does not react with the analyte component labeled with the label. It may include highly antigenic proteins such as KLH (keyhole limpet hemocyanin) and the like. These control reagents are components (substances) that are unlikely to exist in the sample and can be selected appropriately.

(3rd pad)
In the present invention, the 3rd Pad means, of the reaction components between the specimen and the detection reagent, the components unnecessary for the detection of the detection target are removed, and the components necessary for the reaction are immobilized with a specific binding substance. It can be placed between the sample pad and the porous membrane for the purpose of allowing smooth deployment of the porous membrane. For example, blood cells and insoluble blood cell debris are desirably removed as unnecessary components for detection. In addition, in the 3rd pad, among the aggregates generated by the reaction between the analyte and the specific binding substance, the aggregate is so large that it cannot move to the membrane on which the specific binding substance is immobilized and spread smoothly. It is also possible to have an additional effect of removing previously formed aggregates.

A 3rd Pad includes any material and form through which the liquid and the component to be detected and the detection reagent can pass. Specific examples include, but are not limited to, glass fibers (glass fibers), acrylic fibers, hydrophilic polyethylene materials, dry paper, paper pulp, textiles, and the like. The 3rd Pad is sometimes called 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.

(hemagglutinating agent)
In the present invention, when whole blood is used as a specimen, it is desirable to use a hemagglutinating agent or a erythrocyte-binding component in combination with the use of the 3rd Pad. Hemagglutinating agents and erythrocyte-binding components to be used in combination are not particularly limited, but known examples include lectins, polyclonal antibodies, monoclonal antibodies, and polycationic hemagglutinating agents. Examples of known polycationic hemagglutinants include polybrene, polylysine, polyacrylamine, and polyalanine, with polybrene being preferred. Polybrene, whose chemical name is hexadimethrine bromide, is a type of cationic polymer given CAS number 28728-55-4.

The hemagglutinating agent and erythrocyte-binding component can be used by adding them to a diluent for diluting the specimen, adding them directly to the specimen, or including them in the sample supply part (sample pad) of the immunochromatographic test strip. can be done. With such a mode of use, red blood cells in whole blood are aggregated.

(porous membrane)
The porous membrane used in the immunochromatographic test strip of the present invention is characterized in that a specific binding substance for detection is immobilized on its surface and an opaque substrate is arranged on its back surface. .

The opaque base material of the present invention, which is arranged on the back surface of the porous membrane, is a member arranged for backing the membrane because the membrane is porous, and a water-impermeable film or the like is used. Such an opaque substrate of the present invention is sometimes called a so-called "backing member". The substrate can be backed by laminating the back surface of the porous membrane, or can be formed by previously laminating the porous membrane on top of a water-impermeable film.
The opaque base material of the present invention may be arranged on the back side of the porous membrane, and may be in contact via an adhesive layer, or may be in contact without an adhesive layer if adhesiveness is ensured. good too. When a backing sheet, which will be described later, is provided, it is placed below the opaque base material (backing member) of the present invention.
In the present invention, the base material on the back surface of the porous membrane, which has conventionally been transparent, is changed to an opaque base material. It was possible to avoid the so-called "white spot phenomenon" (hereinafter sometimes simply referred to as "white spot") that appears to be present.
The thickness of the opaque substrate of the present invention (not including the adhesive layer, etc.) is sufficient as long as it can exhibit the function as a backing member of the porous membrane, and is preferably 30 μm or more and 200 μm or less, and more preferably 60 μm or more and 140 μm or less. The thickness is preferably 80 μm or more and 120 μm or less, most preferably.

The term "opaque" means that the material is not transparent and that the other side cannot be seen through, and although complete opacity is not required, complete opacity is more preferable.
As the opaque base material, a base material similar in color to that of the porous membrane is preferable. When the porous membrane is white, the base material of the present invention is preferably cream-colored or gray, which is close to white, and white is most preferable. preferable.
As the opaque substrate, any water-impermeable and opaque substrate can be used, and any material can be used. Examples include polyethylene, polyethylene terephthalate, polyester, and nylons.

In addition, by providing the opaque substrate of the present invention on the back surface of the porous membrane, it was possible to suppress variations in the measured values on the test line. Although the reason for this is not clear, the matching of the color of the membrane surface and the back surface reduces the appearance non-uniformity of the surface component (nitrocellulose) and optical means of the backing sheet placed under the membrane. It is presumed that simultaneous reproducibility is improved by avoiding the influence of reflected light.

Any material can be used as the porous membrane (hereinafter sometimes simply referred to as membrane). Examples thereof include, but are not limited to, porous membranes of polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, and ceramics. Specific examples include glass fiber filter paper 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 this porous membrane, it is possible to control the flow rate of the immunocomplex of the labeled specific binding substance and the detection target through the membrane. By controlling the flow rate 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 consider and optimize the combination of immunochromatographic test strips with other constituent materials.

(Immobilization of Specific Binding Substance to Porous Membrane)
Immobilization of a specific binding substance such as an antibody against a detection target of the present invention to a porous membrane can be carried out by generally known methods. For example, in the case of the flow-through method, the above specific binding substance is adjusted to a predetermined concentration, and a given amount of the solution is applied to the porous membrane in the form of a dot or a specific symbol such as +. Also, at this time, in order to ensure the reliability of the immunochromatographic detection method, a 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 is used as a "control line". is common. Alternatively, 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 substance to be detected to form a "control line".

In the case of the lateral flow method, the specific binding substance is adjusted to a predetermined concentration, and a device having a mechanism capable of moving the liquid in the horizontal direction while ejecting the liquid from the nozzle at a constant speed is used. , by applying it to the porous membrane in a line. At this time, the concentration of the specific binding substance is preferably 0.1-5 mg/mL, more preferably 0.5-3 mg/mL. In addition, the immobilization amount of the specific binding substance on the porous membrane can be optimized by adjusting the coating amount dropped onto the porous membrane in the case of the flow-through type, and in the case of the lateral flow type It can be optimized by adjusting the ejection speed from the nozzles of the above devices. 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 liquid or the like is developed so as to pass through the porous membrane perpendicularly, and the term "lateral flow membrane assay" Refers to a method in which the specimen liquid or the like is developed so as to move in parallel to the porous membrane.

Further, in the present invention, regarding the position at which the specific binding substance that binds to the object is applied to the porous membrane, in the case of the lateral flow method, the detection reagent develops from the conjugate pad by capillary action, and each They may be arranged so as to sequentially pass the lines on which the specific binding substances are applied. Preferably, 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 preferably located downstream. At this time, it is desirable that the distance between each line is sufficient to enable signal detection of the label. Even in the case of the flow-through type, the position where the specific binding substance that binds to the object is applied should be arranged so that the signal of the label can be detected.

A specific binding substance solution to be applied to the above-mentioned porous membrane can usually be prepared using a predetermined buffer solution. Examples of the buffer include commonly used buffers such as phosphate buffer, Tris buffer and Good's buffer. 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 pH 8.0 buffer 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, preservatives such as proclin, and the like. Salts include those such as NaCl that are included for adjusting the ionic strength, as well as those such as sodium hydroxide that are added in the process of adjusting the pH of the buffer solution. After the specific binding substance has been immobilized on the porous membrane, blocking can be carried out by applying a commonly used blocking agent in the form of a solution or vapor to cover areas other than the specific binding substance immobilization site. In this specification, a porous membrane on which a specific binding substance is immobilized as described above is sometimes referred to as a "specific binding substance-immobilized membrane".

(absorbent pad)
In the present invention, the absorbent pad is a liquid-absorbent part that controls the development of the specimen by absorbing the specimen that has moved and passed through the porous membrane. In the lateral flow type, it may be provided at the most downstream side of the test strip, and in the flow-through type, it may be provided, for example, below the specific binding substance-immobilized membrane. As the absorbent pad, for example, filter paper can be used, but it is not limited to this.

(backing sheet)
In the present invention, the backing sheet is a solid phase for supporting the test piece, and a plastic sheet with an adhesive adhered thereto is usually used. Materials for the backing sheet include, but are not limited to, polystyrene, polyester, polypropylene, and vinyl chloride. Although the color of the backing sheet is not particularly limited, it is preferably the same color as the membrane, and if the membrane is white, the backing sheet is also preferably white.
The thickness of the above-mentioned backing sheet of the present invention (not including the adhesive layer, etc.) is sufficient as long as it can exhibit the function as a supporting member for the test piece, and is preferably 10 μm or more and 600 μm or less, more preferably 150 μm or more and 350 μm or less, and 200 μm. Larger and 320 micrometers or less are most preferable.

(top film)
In the present invention, the top film refers to a sheet-like member covering the uppermost surface of the test piece, which increases the mechanical strength of the porous membrane on which the specific binding substance is immobilized, It is used for the purpose of preventing evaporation (drying) of water. The material of the top film may be any material that can achieve the above-mentioned purpose, and examples thereof include polyester, and the test piece can be coated with an adhesive material or laminated. In the present invention, it has been found that the void phenomenon, which is enhanced by coating the test piece with a top film, can be weakened by making the backing member of the membrane opaque. Therefore, the effect of the present invention is more pronounced with the test piece provided with the top film.
The thickness of the top film of the present invention (not including the adhesive layer, etc.) is sufficient as long as it can protect the membrane and prevent moisture from evaporating during the assay, and is preferably 10 μm or more and 150 μm or less, and 20 μm or more and 80 μm or less. is much more preferred.

(detection device)
The test strip for immunochromatography of the present invention is an appropriate one in consideration of the size of the test strip, the addition method and position of the sample, the immobilization position of the specific binding substance on the specific binding substance immobilized membrane, the signal detection method, etc. It can be stored and mounted in a suitable container (housing) for use, and such a stored and mounted state is called a "device". An example of the device of the present invention is shown in FIGS. 5 and 6 as a top view and a cross-sectional view.

(others)
In the present specification, the "porous membrane" is referred to as the "solid phase," and the physical or chemical support or support state of a specific binding substance such as an antigen or antibody on the porous membrane is referred to as "immobilization." It is sometimes expressed as "immobilization", "immobilization", "sensitization" and "adsorption".

(specimen)
In the detection method of the present invention, the "specimen" including the object to be detected refers to biological samples such as blood, urine, sputum, saliva, nasal discharge, nasal swab, pharyngeal swab, other bodily fluids, feces, and the like. A biological sample may be used as a specimen as it is, and the specimen of the present invention also includes a sample diluted with a diluent, extracted and/or filtered. Blood samples include whole blood, erythrocytes, plasma, serum and the like.
Blood specimens also include specimens collected using a blood collection tube to which an anticoagulant such as EDTA or heparin is added at the time of blood collection.

(object to be detected)
Objects to be detected in the present invention include substances present in biological specimens such as blood (whole blood), red blood cells, serum, plasma, urine, saliva, or sputum, such as CRP (C-reactive protein), IgA, and 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 and fibrinolysis markers, oxidized LDL, BNP (brain natriuretic peptide), H-FABP (cardiac fatty acid binding protein), circulation-related markers such as cardiac troponin I (cTnI), metabolism-related markers such as adiponectin, CEA (carcinoembryonic antigen), AFP (α- fetoprotein), CA19-9, CA125, PSA (prostate-specific antigen) and other tumor markers, HBV (hepatitis B virus), HCV (hepatitis C virus), Chlamydia trachomatis, Neisseria gonorrhoeae and other infectious disease-related markers, allergen-specific Examples include IgE (immunoglobulin E), hormones, and drugs. Among these, D-dimer, CRP, BNP, H-FABP, cTnI and the like are more preferable because there is a high demand for using whole blood as a sample.

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

(Antibodies used in the present invention)
Antibodies against the detection target used in the present invention are not limited at all by the method of production as long as they are antibodies that specifically react with the detection target, and may be polyclonal antibodies or monoclonal antibodies. may Monoclonal antibodies are more preferred. In general, the hybridoma that produces the antibody is a myeloma of the same kind as the spleen cells of an animal immunized with an object to be detected 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, in addition to the whole antibody molecule, it is also possible to use a functional fragment of an antibody having antigen-antibody reaction activity. In addition to those obtained through the immunization process of general animals (mouse, goat, sheep, etc.), genetic recombination technology etc. has changed the amino acid sequence of an animal species different from the animal immunized with the immunogen (substance to be measured). antibodies (such as chimeric specific binding agents, humanized antibodies, or fully humanized antibodies). Functional fragments of antibodies include F(ab') ₂ , Fab', single-chain antibodies (scFv), etc., which are fragments having antigen-antibody reaction activity. These functional fragments of antibodies can be produced by treating the antibody obtained as described above with a proteolytic enzyme (eg, pepsin, papain, etc.).

Here, when the antibody used in the measurement method for detecting the target substance by forming a so-called sandwich is a monoclonal antibody, the antibody for immobilizing the labeled substance (first antibody) and the antibody for immobilizing the porous membrane (second antibody) are used. Antibody) relationship is that if the epitope of the first antibody is monovalent, the epitope of the second antibody is different from that of the first antibody, and if the epitope of the first antibody is multivalent, the second antibody is used. The epitope of the antibody may be the same as that of the first antibody or may be different.

(kit)
A detection kit using an immunochromatographic test strip of the present invention is an immunochromatographic test strip comprising at least a porous membrane, a sample supply part, a developing part, and a detection part on which an antibody is immobilized, wherein the detection part is a porous membrane and an opaque substrate disposed on the back surface of the porous membrane.
This detection kit also includes reagents necessary for detection (e.g., detection reagent containing conjugate), sample diluent, test tube, swab for stool collection, instruction manual, housing for storing test strip, etc. It's okay.
Specific examples of the present invention will be described below, but the present invention is not limited to these examples.

[Test Example 1] Confirmation test for effect of avoiding white spot phenomenon Comparison of the effect of avoiding white spot phenomenon when using the antibody-immobilized membrane lined with the white base material of the present invention with that when lined with a conventional transparent base material did.

1. Preparation of immunochromatographic detection device of the present invention 1) Preparation of colloidal gold-labeled anti-cTnI monoclonal antibody (anti-cTnI antibody conjugate) (i) Preparation of colloidal gold solution (60 nm)
To 5000 mL of purified water heated to 93° C., 10 mL of 7% (w/v) triammonium citrate aqueous solution was added and mixed with stirring. Subsequently, 10 mL of a 5% (w/v) tetrachloroauric (III) acid aqueous solution was added, and the mixture was reacted for 10 minutes while stirring, and then the reaction liquid was boiled. After that, it was cooled in ice water to prepare a gold colloid solution having an average particle size of 60 nm.
This solution of colloidal gold having an average particle size of 60 nm was adjusted with purified water so that the absorbance at the maximum absorption wavelength of the colloidal gold was 1 OD/mL.

(ii) Preparation of anti-cTnI antibody conjugate Anti-cTnI diluted to 69.3 μg/mL with 2 mM Tris-HCl buffer (pH 7.0) for 200 mL of the 1 OD/mL 60 nm colloidal gold solution (pH 8.0) 10 mL of monoclonal antibody was added and stirred for 10 minutes at room temperature. 10 mL of purified water containing 0.5% (w/v) Neo Protein Saver (Toyobo Co., Ltd., No. NPS-301) was added to the mixture of the colloidal gold and the antibody, and the mixture was stirred at room temperature for 5 minutes. . After that, it was centrifuged at 11900×g for 45 minutes at 10°C. After removing the supernatant, 10 mL of 0.2% (w/v) Neo Protein Saver aqueous solution was added to the resulting sediment to suspend the conjugate and obtain an anti-cTnI antibody conjugate.

(iii) Preparation of colloidal gold solution (40 nm)
To 5000 mL of purified water heated to 73° C., 10 mL of 5% (w/v) triammonium citrate aqueous solution was added and mixed with stirring. Subsequently, 10 mL of a 5% (w/v) tetrachloroauric (III) acid aqueous solution was added, and the mixture was reacted for 10 minutes while stirring, and then the reaction liquid was boiled. After that, it was cooled in ice water to prepare a gold colloid solution having an average particle size of 40 nm.
This gold colloid solution with an average particle size of 40 nm was adjusted with purified water to have an absorbance of 1 OD/mL at the maximum absorption wavelength of the gold colloid.

(iv) Preparation of colloidal gold-labeled KLH (KLH conjugate) for control line For 200 mL of the 1 OD/mL 40 nm colloidal gold solution (pH 6.1), add 2 mM phosphate buffer (pH 6.1) to 375 µg/mL. 2.67 mL of KLH (manufactured by Sigma) dissolved so as to obtain a solution was added, and the mixture was stirred at room temperature for 10 minutes. 20 mL of 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. Thereafter, the mixture was centrifuged at 10°C for 45 minutes, the supernatant was removed, and 10.7 mL of Conjugate Dilution Buffer (Scripps) was added to the resulting sediment to suspend the conjugate to obtain the KLH conjugate. rice field.

2) Preparation of conjugate pads Anti-cTnI antibody conjugate 3 OD, KLH conjugate 0.75 OD, 0.5% LipidureBL-1301, 0.25 mg/ml Heteroblock, 2.4% lactose, 2.0% NPS , 20 mM MOPS (pH 7.2), and 1/6 of the total amount of Conjugate Dilution Buffer to prepare a conjugate solution. .2 times the volume was impregnated. It was dried by heating at 70° C. for 45 minutes in a dry oven to obtain a conjugate pad.

3) Preparation of anti-cTnI monoclonal antibody-immobilized membrane (antibody-immobilized membrane) For the test line, anti-cTnI monoclonal antibody was added in 10 mM PB containing 2.5% sucrose (containing 0.09% NaN ₃ ) (pH 8.0). It was diluted and prepared to 3 mg/mL.
For the control line, a rabbit anti-KLH polyclonal antibody (manufactured by Bethyl) was diluted to 1 mg/mL with 10 mM PB ₍ containing 0.09% NaN3) (pH 7.2) containing 2.5% sucrose.
The above anti-cTnI monoclonal antibody was applied to the position inside one end of the short side of the nitrocellulose membrane lined with a transparent or white base material shown in Table 1 using an immunochromatographic dispenser "XYZ3050" (BIO DOT) at 1 µL/cm. and applied in a line to form a test line. An 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. It was dried in a dry oven at 70°C for 45 minutes 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 prepared as a sample pad pretreatment solution.
A glass fiber pad (Lydall) was appropriately cut into a required size, impregnated with a sample pad pretreatment solution 1.5 times the volume of the pad, and dried in a dry oven at 70°C for 45 minutes. was used as a sample pad.

5) Preparation of immunochromatographic test piece An antibody-immobilized porous membrane (b-1) lined with the above base material (b-2) is attached to the backing sheet (a), and the anti-cTnI antibody (c ), then anti-KLH antibody (d), and a blood cell separation membrane (3rd Pad) (e) was mounted on the membrane. Next, the conjugate pad (f) prepared in 2) above is arranged and mounted, and the sample pad (g) prepared in 4) above is arranged and mounted so as to overlap with this conjugate pad, and at the opposite end An absorbent pad (h) was placed and worn.
Finally, the surfaces of the absorbent pad and the antibody-immobilized membrane were covered with a top film (i) so that part of the antibody-immobilized membrane was exposed. An immunochromatography test piece was prepared by cutting into a structure in which each component was superimposed in this manner. FIG. 4 shows a schematic configuration diagram of the immunochromatographic test piece.
Alternatively, the test piece may be stored and mounted in a special plastic housing to form an immunochromatographic test detection device. 5 and 6 show a schematic top view and a schematic cross-sectional view of the immunochromatographic detection device (k). (n) is a sample addition window, (o) is a detection window, (L) and (m) are housings, and (j) is an immunochromatographic test strip.

2. Detection or measurement by immunochromatography 120 μL of a plasma specimen solution containing any concentration of cardiac troponin I (cTnI) within the range of 0 to 10 ng/mL is added to the sample pad window of the immunochromatographic test detection device prepared above, After 15 minutes, the vicinity of the test line was visually observed to evaluate the presence or absence of white spots.
Table 1 shows the results.
<White spot evaluation criteria>
12 or 20 samples were visually evaluated, and when even one white spot was found, it was judged as "presence" of white spot, and when there was no white spot, it was judged as "absent".

3. Results When a nitrocellulose membrane lined with a transparent base material was used as an antibody-immobilized membrane, a phenomenon of white spots was observed slightly before the test line. When the nitrocellulose membrane was used as the antibody-immobilized membrane, the phenomenon of white spots was not observed. Therefore, by improving the visibility in the visual evaluation of the immunochromatographic test, more accurate evaluation becomes possible.
In addition, this time, white spots were determined and evaluated by visual observation, but if optical measurement is performed by an apparatus, disturbance of the waveform can be avoided and the baseline can be set correctly.

[Test Example 2] Confirmation test of CV reduction effect Comparison of variation in measured values when optical measurement is performed using an antibody-immobilized membrane lined with a white substrate of the present invention and when lined with a conventional transparent substrate did.

1. Manufacture of immunochromatographic device An immunochromatographic test was performed in the same manner as in Test Example 1 except that two types of membranes (HF180, CN140) lined with a transparent substrate and a porous membrane (CN150) lined with a white substrate of the present invention were used. A piece was produced.

2. Detection or measurement by immunochromatography 120 μL of plasma sample liquid containing 40 or 60 pg/mL of cardiac troponin I (cTnI) was added to the sample pad window of the immunochromatography test device prepared above, and after 15 minutes, immunochromatography reader Rapid Pier (Sekisui Medical Co.) was used to measure the amount of coloration (absorbance) of the test line, and the CV value (variation) was calculated. CV is a coefficient of variation, and can be obtained by dividing the standard deviation by the average value. The results are shown in FIG.

3. Results When the porous membrane lined with the white substrate of the present invention was used as the antibody-immobilized membrane, the CV value decreased compared to the case where the membrane lined with the transparent substrate was used as the antibody-immobilized membrane. Improved reproducibility of measurements.

[Test Example 3] Confirmation test of CV reduction effect (low concentration side)
A test was conducted to see if the variation reduction effect of Test Example 2 could be obtained even when the concentration of the substance to be detected was low.

1. Manufacture of immunochromatographic device An immunochromatographic test was performed in the same manner as in Test Example 1 except that two lots of a membrane (CN150) lined with a transparent substrate and a porous membrane (CN150) lined with a white substrate of the present invention were used. A piece was produced.

2. Detection or measurement by immunochromatography Measurement was performed in the same manner as in Test Example 2 except that a plasma sample solution containing 15 pg/mL of cardiac troponin I (cTnI) was used, and CV values (variation) were calculated. The results are shown in FIG.

3. Results When the porous membrane lined with the white substrate of the present invention was used as the antibody-immobilized membrane, even if the concentration of the target to be detected was low, the antibody was immobilized on the membrane lined with the transparent substrate. The CV value decreased and the reproducibility of measurement improved compared to the case of using a membrane. Also, similar results were obtained when two different lots were used.

[Test Example 4] Confirmation test of CV reduction effect (high concentration side)
A test was conducted to see if the variation reduction effect of Test Example 2 could be obtained even when the concentration of the substance to be detected was high.

1. Manufacture of immunochromatographic device An immunochromatographic test piece was manufactured in the same manner as in Test Example 1 except that a membrane (CN150) lined with a transparent substrate and a porous membrane (CN150) lined with a white substrate of the present invention were used. did.

2. Detection or measurement by immunochromatography Measurement was performed in the same manner as in Test Example 2 except that a plasma sample solution containing 400 pg/mL of cardiac troponin I (cTnI) was used, and CV values (variation) were calculated. The results are shown in FIG.

3. Results When the porous membrane lined with the white substrate of the present invention was used as an antibody-immobilized membrane, even if the concentration of the target to be detected was high, the porous membrane lined with the transparent substrate was able to The CV value was greatly reduced compared to the immobilized membrane, and the reproducibility of measurement was greatly improved.

An immunochromatographic test strip comprising at least the porous membrane of the present invention and having a sample supply portion, a developing portion, and a detection portion on which a specific binding substance is immobilized, wherein the detection portion is immobilized on the surface of the porous membrane Thus, a test piece in which an opaque base material is arranged on the back surface of the porous membrane can avoid white spots near the test line.

In addition, an immunochromatographic test strip with little variation in measured values can be provided.
According to the present invention, not only is the visibility in visual judgment improved by avoiding white spots on the test piece, but also the variation in measured values in optical measurement is reduced, so that any detection method can achieve more accurate measurement. can be realized.

(a) backing sheet (b) antibody-immobilized membrane (b-1) porous membrane (b-2) backing substrate (c) anti-cTnI antibody (test line)
(d) anti-KLH antibody (control line)
(e) Blood cell separation membrane (3rd Pad)
(f) conjugate pad (g) sample pad (h) absorbent pad (i) top film (j) immunochromatographic test strip (k) immunochromatographic detection device (L) upper housing (m) lower housing (n) sample addition window (o) detection window

Claims (4)

An immunochromatographic test strip containing at least a porous membrane and having a sample supply part, a developing part, and a detection part, wherein the detection part is immobilized on the surface of the porous membrane, and the back surface of the porous membrane has a thickness is 30 μm or more and 200 μm or less , and a backing sheet is further arranged on the back surface of the opaque substrate. An immunochromatographic detection method characterized by using the following test piece;
An immunochromatography test piece comprising a porous membrane having at least a detection part, an opaque substrate having a thickness of 30 μm or more and 200 μm or less on the back side of the porous membrane , and a backing sheet further arranged on the back side of the opaque substrate. A method for reducing variation in measured values in an immunochromatographic detection method, characterized by using the following test piece;
An immunochromatographic test strip containing at least a porous membrane and having a sample supply section, a developing section, and a detection section, wherein the detection section is immobilized on the surface of the porous membrane, and the back side of the porous membrane has a thickness of 30 μm. A test piece comprising an opaque substrate having a thickness of 200 μm or more and a backing sheet further disposed on the back surface of the opaque substrate. A method for reducing white spots on a test piece in an immunochromatographic detection method using a test piece, wherein the method is characterized by using the following test piece;
An immunochromatographic test strip containing at least a porous membrane and having a sample supply section, a developing section, and a detection section, wherein the detection section is immobilized on the surface of the porous membrane, and a thickness of 30 μm is provided on the back side of the porous membrane. A test piece on which an opaque substrate having a thickness of 200 μm or more is arranged.

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