JP2020125994A - Immunochromatographic specimen - Google Patents

Abstract

To avoid the generation of the so-called void phenomenon of an immunochromatographic specimen and provide an immunochromatographic specimen that allows detection with improved visibility and also prevents variation in the detection value in the test line.SOLUTION: An immunochromatographic specimen includes at least a porous membrane and has a sample supply part, a developing part, and a detection part where a peculiar bonding substance is immobilized. The detection part is immobilized on the top surface of the porous membrane, and an opaque base material of 30 to 200 μm thickness is disposed on the reverse surface of the porous membrane.SELECTED DRAWING: Figure 1

Description

The present invention relates to a test piece for immunochromatography and a method for detecting immunochromatography using the test piece.

With the widespread use of POCT (point of care), where treatment is performed near the patient, a lateral flow immunochromatographic detection method using a test piece such as a nitrocellulose membrane is widely used as a simple immunoassay utilizing the antigen-antibody reaction. doing.

A test piece for immunochromatography (hereinafter may be simply referred to as an immunochromatographic test piece) is generally a membrane composed of a porous body having a sample supply part, a developing part, and a detection part, and a labeled antibody for a target substance to be detected. However, after contact with the sample (sample), it is retained so that it can be eluted at the development start site of the development part so that it can pass through the development part and reach the detection part. Furthermore, the antibody for detection is immobilized on part of the development part. In this structure, the detection unit is configured.

Here, when the analyte is dropped into the sample supply unit, and the analyte is contained in the analyte, the analyte specifically binds to the labeled antibody to form a complex, and the complex is , The developing part is developed in the downstream direction, and is bound to the immobilized antibody for detection in the detecting part. Therefore, the detection target can be qualitatively or quantitatively analyzed by detecting the sandwich type complex of the labeled antibody, the detection target, and the immobilized antibody in the detection unit.

In the detection section of the immunochromatographic test strip having the above-mentioned configuration, as a method for detecting the complex of the substance to be detected and the conjugate, there is a method of calculating the absorbance (reflected absorbance) by measuring the intensity of the reflected light originating from the label is there. The reflection absorbance is calculated, for example, by measuring the reflected light intensity of the detection line of the substance to be detected in the detection part and two parts in the vicinity of the detection line, and using the ratio thereof, the complex is detected when the method is used. In some cases, the reflected light intensity near the upstream side or the downstream side of the detection unit increases nonspecifically and the measured waveform is disturbed. In this case, when the concentration of the substance to be detected is low, it is not possible to ignore the disturbance of the measurement waveform and draw the baseline, so it is not possible to accurately detect the specific signal (peak) and the measurement itself becomes May be possible.

Since the frequency of such disturbances in the measurement waveform and the degree of the disturbance in the measurement waveform are not constant, the reproducibility of measurement is reduced, and correction to an appropriate measurement value is made each time measurement is performed (in other words, measurement waveform is Confirmation) was necessary.

In a usual immunochromatographic detection method using a white membrane, the disturbance of the measurement waveform is a phenomenon in which the color of the corresponding part of the membrane is relatively whiter than the color around it and the color appears to be missing (so-called white This is also visually observed as a dropout phenomenon (hereinafter, may be referred to simply as a blank spot), which also hinders the visual determination of the coloration derived from the marker, which is a cause of inaccurate determination results.

Although the cause of such white spots is not clear, differences in production lots of test pieces, differences in storage state of test pieces, differences in manufacturing conditions such as pretreatment conditions for test pieces, etc. are expected.
The following documents are known as methods for eliminating such a white spot phenomenon.
Patent Document 1 discloses an assay using a sample diluent added with methanol in order to prevent white spots that are often observed when an immunochromatographic test piece is stored for a long period of time.

Patent Document 2 discloses a method for preventing white spots and disturbance of measurement waveform by treating an antibody-immobilized membrane of an immunochromatographic test strip with a specific surfactant such as n-octyl-β-D-glucoside.

International publication WO2015/080286 Brochure International publication WO2010/001598 Pamphlet

An object of the present invention is to provide a test piece capable of detecting with higher visibility by avoiding the occurrence of a so-called blank area in an immunochromatographic test piece by an approach completely different from the above-mentioned prior art. Another object of the present invention is to provide an immunochromatographic test strip that can prevent variations in the detection values of test lines.

The present inventors have attempted to change various elements of the immunochromatographic test strip to solve the above problems, and surprisingly, in the immunochromatographic test strip including 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 having the surface immobilized on the surface, it was found that white spots in the vicinity of the test line can be avoided and the variation in the detected value can be prevented, and the present invention has been completed. .. That is, the present invention has the following configurations.
(1) An immunochromatographic test strip including at least a porous membrane, 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, The test piece, wherein an opaque substrate 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), which includes a sample pad serving as the sample supply unit and a conjugate pad containing a heterologous binding substance labeled with gold colloid 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 including the immunochromatographic test strip according to any one of (1) to (7).
(9) An immunochromatographic detection method, which comprises using the following test piece;
An immunochromatographic test strip comprising a porous membrane having at least a detection part, and an opaque base material having a thickness of 30 μm or more and 200 μm or less is 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) In any one of (9) to (11), the test piece includes a sample pad serving as the sample supply section and a conjugate pad containing a specific binding substance labeled with gold colloid or colored latex. The immunochromatographic detection method described.
(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 arranged under the porous membrane.
(15) The immunochromatographic detection method according to any one of (9) to (14), which comprises a step of detecting an optical parameter derived from the labeling substance by an optical means.
(16) The immunochromatographic detection method according to any one of (9) to (15), which includes a step of determining the coloration derived from the labeling substance by visual observation.
(17) A method for reducing variations in measured values in an immunochromatographic detection method, which comprises using the following test piece;
An immunochromatographic test strip including at least a porous membrane, 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 thickness is 30 μm on the back side of the porous membrane. A test piece on which an opaque substrate having a size of 200 μm or less is arranged.
(18) A method for reducing white spots in a test piece in an immunochromatographic detection method using the test piece, which comprises using the following test piece;
An immunochromatographic test strip including at least a porous membrane, 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 thickness is 30 μm on the back side of the porous membrane. A test piece on which an opaque substrate having a size of 200 μm or less is arranged.

According to the present invention, it is possible to provide an immunochromatographic test strip which is excellent in visibility at the time of visual detection because white spots near the test line can be avoided, and in which variations in measured values by optical means are small.

6 is a graph showing variations in measured values when immunochromatographic measurement is performed with an immunochromatographic test piece using a porous membrane having a transparent substrate (comparative example) and a white substrate (present invention) as a backing member. (Test Example 2, detected substance is at medium concentration side) 6 is a graph showing variations in measured values when immunochromatographic measurement is performed with an immunochromatographic test piece using a porous membrane having a transparent substrate (comparative example) and a white substrate (present invention) as a backing member. (Test Example 3, detected substance low concentration side) 6 is a graph showing variations in measured values when immunochromatographic measurement is performed with an immunochromatographic test piece using a porous membrane having a transparent substrate (comparative example) and a white substrate (present invention) as a backing member. (Test Example 4, high concentration of the substance to be detected) It is a perspective view which shows the immunochromatographic test piece of this invention. A is the whole immunochromatographic test piece, and B is a porous membrane. It is a top view of the immunochromatographic detection device of this invention. 6 shows a cut surface when the device shown in FIG. 5 is cut along a dotted line.

(Immunochromatographic test piece)
The immunochromatographic test strip of the present invention includes a porous membrane having at least a “sample supply unit”, a “developing unit”, and a “detection unit”, and a labeled specific binding substance that binds to a detection target is , It is held so that it can be eluted at the deployment start site of the deployment section so that it can reach the detection section through the deployment section after contact with the sample, and the specific binding substance for detection is immobilized on part of the deployment section. And constitutes a detector.

As an example of embodying these, a sample pad that serves as a sample supply unit, a conjugate pad that holds a labeled specific binding substance that binds to a detection target so that it can be eluted, and that serves as a part of the development unit, The test piece includes a porous membrane in which the specific binding substance of (3) is partially immobilized and which serves as a developing portion and a detecting portion. That is, a typical immunochromatographic test piece of the present invention has the following constitution.

(1) A sample pad to which a specimen is supplied (2) A conjugate pad which is arranged downstream of the sample pad and holds a conjugate in which the first specific binding substance is sensitized on the colloidal gold surface so that it can be eluted (3) A porous membrane which is disposed downstream of the conjugate pad and has a detection part on the surface on which a second specific binding substance that binds to the complex of the conjugate and the detection target is immobilized. The porous membrane on the back side of which is an opaque substrate

Here, the sample pad, the conjugate pad, and the porous membrane may each form a separate carrier, or the two may form a single carrier. Any embodiment is included as long as it is composed of a pad and a porous membrane in this order.
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 a 3rd pad is further arranged and mounted.
The test pieces are usually arranged on a solid support such as a plastic pressure-sensitive adhesive sheet (hereinafter sometimes referred to as a backing sheet).

In addition, a polyester film or the like may be laminated on the test piece in order to increase the mechanical strength of the porous membrane on which the specific binding substance is immobilized and prevent evaporation (drying) of water during the assay. ..

The detection method using the immunochromatographic test strip of the present invention is performed as follows.
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 specific binding substance to form a complex. The complex develops the developing part in the downstream direction and binds to the specific binding substance immobilized on the detecting part. In the detection part, a sandwich-type complex is formed by the labeled specific binding substance, the detection target substance, and the immobilized specific binding substance.By detecting this, the detection target substance is qualitatively or Quantitative analysis is possible. Examples of the detection include a method of determining coloration by visual observation and a method of detecting an optical parameter derived from the labeled body by an optical means. When using optical means, it can be automated as well as manual, and continuous measurement is possible.

(Sample pad)
In the present invention, the “sample pad” is a portion that serves as a sample supply unit that receives a sample, absorbs a liquid sample in a state of being 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 may be pretreated to improve water permeability.
When measuring whole blood, a hemagglutination agent can be included. In this case, it may be contained in at least a part of the sample pad, or may be contained in the whole.

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 means a substance in which a specific binding substance or a control substance that binds to a detection target is immobilized on a label.
The label used in the present invention can form a conjugate by sensitizing (immobilizing) a specific binding substance such as an antibody, and contacting with a sample to detect an object (antigen etc.) in the sample. In the method described above, any substance can be used as long as it can play a role as a label, and examples thereof include gold colloid particles, platinum colloid particles, color latex particles, magnetic particles, and the like. Is desirable, and colloidal gold is even more desirable. These particle diameters may be adjusted according to the respective types so that the desired detection sensitivity of the measurement object can be obtained. For example, the particle diameter of the gold colloid particles is preferably 20 to 100 nm, more preferably It is from 30 to 100 nm, and particularly preferably 60 nm.
The conjugate of the present invention can also block a region on the surface of a colloidal gold or the like to which a specific binding substance is not bound, with a blocking agent.

The conjugate may be present as a conjugate pad, that is, it may be present in a state where it is impregnated with a dedicated pad (conjugate pad) other than the sample pad, the third pad, and the porous membrane (( Type A), it may be present as a conjugate portion in a part of the sample pad (Type B). Furthermore, apart from the test strip, it may be present as a separate conjugating reagent 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, the third pad, and the porous membrane are arranged in this order from upstream to downstream in the flow direction of the sample, and they are arranged so as to at least partially overlap the upper and lower layers. A test piece of 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 composite is then passed through a third pad, which is placed in contact with the lower surface of the conjugate pad, and unfolded into a porous membrane.

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

Next, a test piece in which the type 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 supply section and the conjugate section 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 conjugate section is a section that contains a conjugate, and the sample supply section is on the upstream side of the conjugate section.

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 a separate conjugation reagent. For example, a filter chip in which a conjugate is incorporated in the filter can be mentioned. By subjecting the sample to filter filtration using such a filter chip, the conjugate retained in the filter and the detection target are bound to form a complex (aggregate). An object to be detected can be detected by supplying this to the same test piece as that of type A except that it does not have a conjugate pad.

(Detection reagent)
In the present invention, the “detection reagent” is specifically a solution containing at least the conjugate.
The detection reagent keeps the conjugate stable and facilitates a specific binding substance such as an antibody immobilized on the conjugate when mixed with the sample, to specifically react with the detection target. Alternatively, for the purpose of rapidly and effectively dissolving and fluidizing the conjugate, for example, one or more kinds of stabilizers, solubilizing agents and the like may be included. Examples of the stabilizer, solubilizing agent and the like include bovine serum albumin (BSA), sucrose, casein, amino acids and the like.

Further, the detection reagent may also contain a known sensitizer or chelating agent such as EDTA or EGTA for the purpose of improving the detection sensitivity.
In this specification, the term “detection” or “measurement” needs to be interpreted in the broadest sense including proof of the existence of a detection target and/or quantification, and is not limited to any meaning. must not.

(Sample diluent)
In the present invention, a diluting solution 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 on the contrary, significantly accelerates the reaction to cause the label to be over-aggregated, resulting in poor expansion in the capillary phenomenon, or the target substance to be detected. A diluent having any composition may be used as long as the signal of the reaction between the target substance 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 the below-described conjugate pad with a detection reagent that specifically reacts with an object to be detected and drying it. The conjugate pad has a function of forming a complex between the detection reagent and the detection target when the sample passes through the conjugate pad. The conjugate pad may be arranged so as to contact the porous membrane on which the specific binding substance is immobilized 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.

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

(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. It can be placed between the sample pad and the porous membrane for the purpose of allowing the porous membrane to spread smoothly. 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 this 3rd Pad, among the aggregates generated by the reaction between the measurement target and the specific binding substance, the specific binding substance moves to the membrane on which the specific binding substance is immobilized, so that it cannot be spread smoothly. It is also possible to have the additional effect of previously removing the agglomerated aggregates.

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, 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 desirable to use a blood cell separation membrane in order to more reliably separate and remove blood cells that could not be removed by the sample pad alone.

(Hemagglutination agent)
In the present invention, when whole blood is used as a sample, it is desirable to use a red blood cell aggregating agent or a red blood cell binding component in addition to the use of 3rd Pad. The hemagglutination agent and the erythrocyte binding component to be used in combination are not particularly limited, but as a known example, a polycationic hemagglutination agent can be used in addition to lectins, polyclonal antibodies, and monoclonal antibodies. Examples of known polycationic red blood cell aggregating agents include polybrene, polylysine, polyacrylamine, and polyalanine. Among them, polybrene is preferable. Polybrene is a kind of a cationic polymer which has a chemical name of hexadimethrin bromide and is provided with CAS number 28728-55-4.

The usage mode of the hemagglutination agent or erythrocyte-binding component should be added to the diluent for diluting the sample, or added directly to the sample, and should also be included in the sample supply section (sample pad) of the immunochromatographic test strip. You can By 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 the surface thereof, and an opaque base material is disposed on the back surface thereof. ..

The opaque substrate of the present invention arranged on the back surface of the porous membrane is a member arranged for lining the membrane because the membrane is porous, and an impermeable film or the like is used. Such an opaque substrate of the present invention is sometimes called a so-called "backing member". The base material can be backed by sticking it to the back surface of the porous membrane, or can be formed by previously laminating the porous membrane on the upper surface of the impermeable film.
The opaque substrate of the present invention may be placed on the back side of the porous membrane and may be in contact with the adhesive layer, or if the adhesiveness is ensured, the opaque substrate may be in contact without the adhesive layer. Good. When a backing sheet to be described later is provided, it is placed below the opaque base material (lining member) of the present invention.
According to the present invention, the base material on the back surface of the porous membrane is conventionally transparent, but by using an opaque base material, the color of the relevant part of the membrane is whiter than the surrounding color and the color is missing. It was possible to avoid the so-called "blank phenomenon" (hereinafter sometimes simply referred to as "blank").
The thickness of the opaque substrate of the present invention (not including the adhesive layer) may be any function as a backing member for the porous membrane, preferably 30 μm or more and 200 μm or less, and more preferably 60 μm or more and 140 μm or less. It is preferably 80 μm or more and 120 μm or less.

Opaque means that it is not transparent and that the other side cannot be seen through. It is not required to be completely opaque, but it is more preferably completely opaque.
As the opaque base material, a base material having a color similar to that of the porous membrane is preferable, and when the porous membrane is white, the base material of the present invention is also preferably a cream color or gray close to white, and among them, white is the most preferable. preferable.
The opaque substrate may be any water-impermeable and opaque substrate, and any material may be used. Examples thereof include polyethylene, polyethylene terephthalate, polyester, nylons and the like.

Further, by providing the opaque base material of the present invention on the back surface of the porous membrane, it was possible to suppress variations in measured values on the test line. The reason for this is not clear, but the fact that the hues of the front and back sides of the membrane match makes it possible to reduce the appearance non-uniformity of the surface component (nitrocellulose) and the optical means of the backing sheet placed below the membrane. It is estimated that simultaneous reproducibility is improved by avoiding the influence of reflected light.

As the porous membrane (hereinafter sometimes simply referred to as a membrane), an arbitrary material can be used. Examples 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 papers and nitrocellulose membranes sold by Merck Millipore, Toyo Roshi Kaisha, GE Healthcare, and the like. Further, by appropriately selecting the pore size and the structure of this porous membrane, it is possible to control the rate at which the immune complex of the labeled specific binding substance and the detection target flows through the membrane. By controlling the rate of flow through the membrane, it is possible to adjust the amount of the labeled specific binding substance that binds to the specific binding substance immobilized on the membrane. It is desirable to optimize the immunochromatographic test piece in consideration of the combination with other constituent materials.

(Immobilization of specific binding substance on porous membrane)
Immobilization of a specific binding substance such as an antibody against the detection target of the present invention on the 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 adjusted to a predetermined concentration, and the solution is applied to the porous membrane in a specific symbol such as a dot or a + symbol. 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 to form a “control line”. Is common. 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 line”.

In the case of the lateral flow type, by using 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 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 amount of the specific binding substance immobilized 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 in the case of the lateral flow type. It can be optimized by adjusting the discharge speed from the nozzle of the above device. Particularly, in the case of the lateral flow type, 0.5 to 2 μL/cm is preferable.
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 vertically to a 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 binding specific binding substance to the object, in the case of the lateral flow type, from the conjugate pad, the detection reagent is developed by capillary action, each of It may be arranged so as to sequentially pass through the lines coated with the specific binding substance. 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 located downstream thereof. At this time, it is desirable that the distance between the lines be sufficiently large so that the signal of the labeled 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 labeled 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 phosphate buffer solution, Tris buffer solution, Good's buffer solution and other commonly used buffer solutions. 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 may further contain salts such as NaCl, stabilizers such as sucrose and preservatives, 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 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 porous membrane on which the specific binding substance is immobilized as described above may be referred to as “specific binding substance immobilized membrane”.

(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 on the most downstream side of the test piece, 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.

(Backing sheet)
In the present invention, the backing sheet is a solid phase for supporting the test piece, and a sheet made of a plastic and having an adhesive attached thereto is usually used. Examples of the material for the backing sheet include, but are not limited to, polystyrene, polyester, polypropylene, vinyl chloride and the like. The color of the backing sheet is not particularly limited, but it is preferably the same color as the membrane, and when the membrane is white, the backing sheet is also preferably white.
The thickness of the backing sheet of the present invention (not including the pressure-sensitive adhesive layer) may be sufficient as long as it can function as a support member for a test piece, preferably 10 μm or more and 600 μm or less, more preferably 150 μm or more and 350 μm or less, and 200 μm Most preferably, it is larger than 320 μm.

(Top film)
In the present invention, the top film refers to a sheet-like member that covers the uppermost surface of the test piece, and enhances the mechanical strength of the porous membrane on which the specific binding substance is immobilized, or during the assay. Used to prevent evaporation (drying) of water. As the material of the top film, any material can be used as long as it can achieve the above purpose, and examples thereof include polyester. The top film can be coated with an adhesive material or laminated. In the present invention, it has been found that the whiteout phenomenon, which is enhanced by coating the test piece with the top film, can be reduced by making the backing member of the membrane opaque. Therefore, the effect of the present invention becomes more remarkable in the test piece provided with the top film.
The thickness of the top film of the present invention (not including the pressure-sensitive adhesive layer) may be such that it can exert a function of protecting the membrane and preventing evaporation of water 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 even more preferable.

(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 mounted in a simple container (housing), and such a stored and mounted state is called a "device". 5 and 6 show an example of the device of the present invention in a top view and a sectional view.

(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 supported on the porous membrane, or the state of being supported 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 the sample of the present invention also includes a sample that is appropriately diluted with a diluent, extracted and/or filtered. The blood sample includes red blood cells, plasma, serum and the like in addition to whole blood.
The blood sample also includes a sample collected by a blood collection tube to which an anticoagulant such as EDTA or heparin is added at the time of collecting blood.

(Detection target)
The detection target of the present invention is a substance present in biological samples such as blood (whole blood), erythrocytes, serum, plasma, urine, saliva or sputum, for example, CRP (C-reactive protein), IgA, IgG. , Inflammation-related markers such as IgM, fibrin degradation products (for example, 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), infectious disease-related markers such as HBV (hepatitis B virus), HCV (hepatitis C virus), chlamydia trachomatis, gonococcus, allergen specific Examples thereof include IgE (immunoglobulin E), hormones, drugs and the like. Among these, D-dimer, CRP, BNP, H-FABP, cTnI and the like, which are highly demanded to use whole blood as a sample, are more preferable.

(Specific binding compound)
In the present invention, as the specific binding substance for the substance to be measured carried on the insoluble carrier particles such as gold colloid or the porous membrane, proteins, peptides, amino acids, lipids, sugars, DNA, RNA, receptors, haptens There are no particular restrictions on the origin of the molecular weight, such as high and low molecular weight and natural or synthetic, and examples thereof include an antibody or an antigen that can be used in an immunological assay utilizing an immune reaction.

(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 it 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 prepared by cell fusion with cells (myeloma cells).

As the antibody of the present invention, it is possible to use not only the whole antibody molecule but also 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 gene recombination technology etc. Antibodies (chimeric specific binding substance, humanized antibody, fully humanized antibody, etc.) may be used. Examples of functional fragments of antibodies include F(ab′) ₂ , Fab′ and single chain antibodies (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, the antibody for immobilizing the label (first antibody) and the antibody for immobilizing the 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 the second antibody is used 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)
A detection kit using the immunochromatographic test strip of the present invention is an immunochromatographic test strip having at least a porous membrane, a sample supply part, a developing part, and a detection part to which an antibody is immobilized, and the detection part is a porous membrane. The test piece is fixed on the surface of the porous membrane, and an opaque substrate is arranged on the back surface of the porous membrane.
The detection kit also includes other reagents necessary for detection (for example, a detection reagent containing a conjugate), a sample diluent, a test tube, a swab for collecting feces, an instruction manual, a housing for storing test pieces, and the like. But it's okay.
Hereinafter, specific examples of the present invention will be described, but they are merely examples and the present invention is not limited thereto.

[Test Example 1] Confirmation test of whiteout phenomenon avoidance effect Comparison of the whiteout phenomenon avoidance effect in the case of using the antibody-immobilized membrane lined with the white base material of the present invention compared with the case in which the conventional transparent base material is lined did.

1. Preparation of immunochromatographic 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 (60 nm)
To 5000 mL of purified water heated to 93° C., 10 mL of a 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, the mixture was reacted for 10 minutes while 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 Anti-cTnI diluted with 39.3 μg/mL of 2 mM Tris-hydrochloric acid buffer (pH 7.0) to 200 mL of 60 nm gold colloid solution (pH 8.0) at 1 OD/mL. Monoclonal antibody (10 mL) was added, and the mixture was stirred at room temperature for 10 minutes. To the mixed solution of the gold colloid and the antibody, 10 mL of purified water containing 0.5% (w/v) Neo Protein Saver (Toyobo Co., No. NPS-301) was added, and the mixture was stirred at room temperature for 5 minutes. .. Then, it was centrifuged at 11900×g at 10° C. for 45 minutes. After removing the supernatant, 10 mL of 0.2% (w/v) Neo Protein Saver aqueous solution was added to the obtained precipitate to suspend the conjugate, and an anti-cTnI antibody conjugate was obtained.

(Iii) Preparation of gold colloid solution (40 nm)
To 5000 mL of purified water heated to 73° C., 10 mL of a 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, the mixture was reacted for 10 minutes while stirring, and then the reaction solution was boiled. Then, it was cooled in ice water to prepare a gold colloid solution having an average particle diameter of 40 nm.
This gold colloid solution having an average particle diameter of 40 nm was adjusted to 1 OD/mL with purified water at the absorbance at the maximum absorption wavelength of the gold colloid.

(Iv) Preparation of Gold Colloid Labeled KLH (KLH Conjugate) for Control Line With respect to 200 mL of 40 nm gold colloid solution (pH 6.1) at 1 OD/mL, 375 μg/mL with 2 mM phosphate buffer solution (pH 6.1) 2.67 mL of KLH (manufactured by Sigma), which had been dissolved, was added thereto, and the mixture was stirred at room temperature for 10 minutes. 20 mL of a 10% bovine serum albumin (BSA) aqueous solution was added to the mixed solution of the gold colloid and KLH, and the mixture was stirred at room temperature for 5 minutes. Then, the mixture was centrifuged at 10° C. for 45 minutes, the supernatant was removed, and 10.7 mL of Conjugate Dilution Buffer (Scripts) was added to the obtained precipitate to suspend the conjugate, thereby obtaining a KLH conjugate. It was

2) Preparation of conjugate pad Anti-cTnI antibody conjugate 3 OD, KLH conjugate 0.75 OD, 0.5% Lipidure BL-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 were mixed to prepare a conjugate solution, and a fixed volume of glass fiber pad (Merck Millipore) was used to prepare 1 volume of the pad volume. .2 times the volume was soaked. 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) The anti-cTnI monoclonal antibody was used as a test line in 10 mM PB (containing 0.09% NaN ₃ ) containing 2.5% sucrose (pH 8.0). It was diluted to 3 mg/mL.
Further, 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% NaN ₃ ) (pH 7.2) containing 2.5% sucrose.
The anti-cTnI monoclonal antibody was placed at a position inside one end of the short side of a 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. Then, the test line was formed by applying it in a line shape. 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 adjusted to prepare a sample pad pretreatment solution.
A glass fiber pad (Lydall) was appropriately cut into a required size, a sample pad pretreatment solution was soaked in a volume 1.5 times the volume of the pad, and dried in a dry oven at 70° C. for 45 minutes. Used as a sample pad.

5) Preparation of immunochromatographic test piece The antibody-immobilized porous membrane (b-1) lined with each of the above-mentioned base materials (b-2) was attached to the backing sheet (a), and the anti-cTnI antibody (c ), and then the application part is arranged in the order of the anti-KLH antibody (d), and the 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. The absorbent pad (h) was placed and mounted.
Finally, the surfaces of the absorbent pad and the antibody-immobilized membrane were covered with the top film (i) so that a part of the antibody-immobilized membrane was exposed. In this way, an immunochromatographic test piece was produced by cutting into a structure in which the respective constituent elements were superposed. FIG. 4 shows a schematic configuration diagram of the immunochromatographic test piece.
Further, the test piece may be stored and mounted in a dedicated 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 part, (o) is a detection window part, (L), (m) shows a housing, (j) shows an immunochromatographic test piece.

2. Detection or measurement by immunochromatography method 120 μL of a plasma sample solution containing cardiac troponin I (cTnI) at any concentration 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.
The results are shown in Table 1.
<Blank evaluation criteria>
Twelve or twenty samples were visually evaluated, and even if only one white spot was observed, the white spot was "present", and 0 was white spot "no".

3. Results When a nitrocellulose membrane lined with a transparent substrate was used as the antibody-immobilized membrane, the phenomenon of white spots was observed slightly before the test line, but it was lined with the white substrate of the present invention. 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 can be performed.
Further, this time, the white spots are judged and evaluated by visual observation, but in the case of optical measurement by the device, the 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 variations in measurement values when optical measurement is performed using the antibody-immobilized membrane lined with the white base material of the present invention compared with the case where the transparent base material is lined with a conventional transparent base material did.

1. Production of immunochromatographic device An immunochromatographic test was conducted 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 manufactured.

2. Detection or measurement by immunochromatography method 120 μL of a plasma sample solution containing 40 or 60 pg/mL cardiac troponin I (cTnI) is added to the sample pad window of the immunochromatographic test device prepared above, and 15 minutes later, the immunochromatographic rapid pier (Sekisui The amount of coloration (absorbance) of the test line was measured using Medical Co., Ltd., and the CV value (variation) thereof was calculated. CV stands for Coefficient of Variation and can be obtained by dividing the standard deviation by the average value. The results are shown in Figure 1.

3. Results When the porous membrane backed by the white base material of the present invention is used as the antibody-immobilized membrane, the CV value is reduced as compared with the case where the membrane backed by the transparent base material is used as the antibody-immobilized membrane, The reproducibility of measurement is improved.

[Test Example 3] CV reduction effect confirmation test (low concentration side)
It was tested whether the variation reducing effect of Test Example 2 appears even when the substance to be detected has a low concentration.

1. Production of immunochromatographic device Immunochromatographic test was carried out in the same manner as in Test Example 1 except that 2 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 manufactured.

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

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

[Test Example 4] CV reduction effect confirmation test (high concentration side)
It was tested whether or not the variation reducing effect of Test Example 2 appears even when the substance to be detected has a high concentration.

1. Production of immunochromatographic device An immunochromatographic test piece was produced 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 The CV value (variation) was calculated in the same manner as in Test Example 2 except that a plasma sample solution containing 400 pg/mL cardiac troponin I (cTnI) was used. Results are shown in FIG.

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

An immunochromatographic test strip comprising at least a porous membrane of the present invention, a sample supply section, a developing section, and a detection section having a specific binding substance immobilized thereon, wherein the detection section is immobilized on the surface of the porous membrane. Therefore, according to the test piece in which the opaque base material is arranged on the back surface of the porous membrane, white spots near the test line can be avoided.

Further, it is possible to provide an immunochromatographic test strip with little variation in measured values.
According to the present invention, not only the visibility in visual judgment is improved by avoiding blank areas of the test piece, but also the variation in the measured values in the optical measurement is reduced, so that more accurate measurement is possible in any detection method. 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) Absorption pad (i) Top film (j) Immunochromatographic test piece (k) Immunochromatographic detection device (L) Upper housing (m) Lower housing (n) Sample addition window (O) Detection window

Claims (4)

An immunochromatographic test strip including at least a porous membrane, 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 thickness is provided on the back surface of the porous membrane. An opaque substrate having a size of 30 μm or more and 200 μm or less is arranged. An immunochromatographic detection method, characterized in that the following test piece is used;
An immunochromatographic test strip comprising a porous membrane having at least a detection part, and an opaque base material having a thickness of 30 μm or more and 200 μm or less is arranged on the back side of the porous membrane. A method for reducing variations in measurement values in an immunochromatography detection method,
The above method characterized by using the following test pieces;
At least a porous membrane, a sample supply unit, a developing unit, an immunochromatographic test strip having a detection unit, the detection unit is immobilized on the surface of the porous membrane,
A test piece in which an opaque substrate having a thickness of 30 μm or more and 200 μm or less is arranged on the back side of the porous membrane. A method for reducing white spots in a test piece in an immunochromatographic detection method using the test piece, wherein the following test piece is used:
At least a porous membrane, a sample supply unit, a developing unit, an immunochromatographic test strip having a detection unit, the detection unit is immobilized on the surface of the porous membrane,
A test piece in which an opaque substrate having a thickness of 30 μm or more and 200 μm or less is arranged on the back side of the porous membrane.

Images