JP6464308B1 - Test specimen for immunochromatography - Google Patents

Abstract

An object of the present invention is to provide a test piece capable of avoiding a so-called white spot phenomenon in an immunochromatographic test piece and performing detection with higher visibility. Another object of the present invention is to provide an immunochromatographic test piece that can prevent variations in the detection value of the test line. An immunochromatographic test piece including at least a porous membrane and having a sample supply part, a development part, and a detection part on which a specific binding substance is immobilized, the detection part being immobilized on the surface of the porous membrane. The object is solved by the test piece, wherein an opaque base material is disposed on the back surface of the porous membrane. [Selection] Figure 1

Description

  The present invention relates to an immunochromatographic test piece and an immunochromatographic detection method using the test piece.

  With the spread of point-of-care (POCT) treatment near patients, lateral flow immunochromatography detection using a test piece such as a nitrocellulose membrane has become widespread as a simple immunoassay using antigen-antibody reaction. doing.

  A test strip for immunochromatography (hereinafter sometimes simply referred to as an immunochromatographic test strip) is generally a membrane made of a porous body having a sample supply section, a development section, and a detection section, and is a labeled antibody against a detection target. However, after contact with the specimen (sample), 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, and the detection antibody is immobilized on a part of the development part. Thus, the detection unit is structured.

  Here, when the specimen is dropped onto the sample supply unit, if the specimen includes a detection target, the detection target specifically binds to the labeled antibody to form a complex, and the complex The developing part is developed in the downstream direction, and the detection part binds to the immobilized antibody for detection. 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.

  As a method for detecting the complex of the substance to be detected and the conjugate in the detection part of the immunochromatographic test strip having the above-described configuration, there is a method for calculating the absorbance (reflection absorbance) by measuring the intensity of the reflected light derived from the label. is there. The reflected absorbance is calculated, for example, by measuring the reflected light intensity of the detection line of the substance to be detected in the detection unit and the two portions in the vicinity thereof, and using the ratio. When using this method, the complex is detected. The reflected light intensity on the upstream side or the vicinity of the downstream side of the detection unit to be detected increases non-specifically, and the measurement waveform may be disturbed. In this case, if the substance to be detected is at a low concentration, the baseline cannot be drawn ignoring the disturbance in the measurement waveform, so the specific signal (peak) cannot be detected accurately, and the measurement itself is not possible. It may be possible.

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

  In a normal immunochromatography detection method using a white membrane, the disturbance of the measured waveform is a phenomenon in which the color of the relevant part of the membrane is relatively white compared to the surrounding color (so-called white color). Since this phenomenon is also observed visually as a missing phenomenon (hereinafter, sometimes simply referred to as white spot), it also hinders the visual determination of the coloration derived from the marker, causing a lack of accuracy in the determination result.

The reason why such white spots occur is not clear, but a difference between production lots of test pieces, a difference in storage state of test pieces, a difference in production conditions such as pretreatment conditions of test pieces, and the like are expected.
The following documents are known as methods for eliminating such white spots.
Patent Document 1 discloses an assay using a sample diluent added with methanol in order to prevent white spots that are often observed when immunochromatographic test strips are stored for a long period of time.

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

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

  An object of the present invention is to provide a test piece that can be detected with higher visibility by avoiding the occurrence of a so-called white spot phenomenon in an immunochromatographic test piece by an entirely different approach from the above prior art. Another object of the present invention is to provide an immunochromatographic test piece that can prevent variations in the detection value of the test line.

In order to solve the above-mentioned problems, the present inventors tried to change various elements of an immunochromatographic test piece. Surprisingly, in an immunochromatographic test piece including a porous membrane, a specific binding substance such as an antibody was used. It was found that by providing an opaque base material on the back surface of the porous membrane with the surface immobilized on the surface, white spots in the vicinity of the test line can be avoided and variation in detection values can be prevented, and the present invention has been completed. . That is, the present invention has the following configuration.
(1) An immunochromatographic test piece including at least a porous membrane and having a sample supply unit, a developing unit, and a detection unit, wherein the detection unit is fixed on the surface of the porous membrane, The said test piece characterized by the above-mentioned.
(2) The test piece according to (1), wherein the opaque base material is a member for lining the porous membrane.
(3) The immunochromatographic test piece according to (1) or (2), wherein the opaque base material is a white base material.
(4) The immunochromatographic test strip according to any one of (1) to (3), including a sample pad that bears the sample supply unit, and a conjugate pad that includes a foreign binding substance labeled with gold colloid or colored latex. .
(5) The immunochromatographic test piece 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 is disposed under the porous membrane.
(7) The immunochromatographic test piece according to any one of (1) to (6), which is a test piece 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, wherein 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 disposed on the back side of the porous membrane.
(10) The immunochromatographic detection method according to (9), wherein the opaque base material is a member for lining the porous membrane.
(11) The immunochromatographic detection method according to (9) or (10), wherein the opaque substrate is a white substrate.
(12) The test piece includes a sample pad serving as the sample supply unit, and a conjugate pad including a specific binding substance labeled with gold colloid or colored latex. The immunochromatographic detection method as 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 disposed under the porous membrane.
(15) The immunochromatographic detection method according to any one of (9) to (14), comprising 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), including a step of determining a coloration derived from the labeling substance by visual observation.
(17) A method for reducing variations in measured values in an immunochromatographic detection method,
Said method characterized in that the following test piece is used;
An immunochromatographic test piece including at least a porous membrane, having a sample supply part, a development part, and a detection part, wherein the detection part is immobilized on the porous membrane surface,
The test piece by which the opaque base material is arrange | positioned at the back side of the said porous membrane.
(18) In the immunochromatography detection method using a test piece, the method for reducing white spots on the test piece, wherein the following test piece is used;
An immunochromatographic test piece including at least a porous membrane, having a sample supply part, a development part, and a detection part, wherein the detection part is immobilized on the porous membrane surface,
The test piece by which the opaque base material is arrange | positioned at the back side of the said porous membrane.

  According to the present invention, white spots in the vicinity of the test line can be avoided, so that an immunochromatographic test piece having excellent visibility at the time of visual detection and having little variation in measured values by optical means can be provided.

It is a graph which shows the dispersion | variation in the measured value at the time of performing immunochromatography measurement with the immunochromatography test piece using the porous membrane which made the backing member the transparent base material (comparative example) and the white base material (this invention). (Test Example 2, the substance to be detected is on the medium concentration side) It is a graph which shows the dispersion | variation in the measured value at the time of performing immunochromatography measurement with the immunochromatography test piece using the porous membrane which made the backing member the transparent base material (comparative example) and the white base material (this invention). (Test Example 3, low concentration of detected substance) It is a graph which shows the dispersion | variation in the measured value at the time of performing immunochromatography measurement with the immunochromatography test piece using the porous membrane which made the backing member the transparent base material (comparative example) and the white base material (this invention). (Test Example 4, the substance to be detected is on the high concentration side) It is a perspective view which shows the immunochromatographic test piece of this invention. A is the whole immunochromatographic test piece, B shows a porous membrane.

(Immunochromatographic specimen)
The immunochromatographic test strip of the present invention includes a porous membrane having at least a “sample supply unit”, a “development unit”, and a “detection unit”, and a labeled specific binding substance that binds to a detection target is present. In order to be able to reach the detection part after passing through the development part after contact with the specimen, it is retained so that it can be eluted at the development start part of the development part, and a specific binding substance for detection is immobilized on a part of the development part. And constitutes a detector.

  As an example that embodies these, a sample pad that serves as a sample supply unit, a conjugate pad that retains a labeled specific binding substance that binds to a detection target substance so that it can be eluted, and serves as a part of a development unit, and for detection And a test piece including a porous membrane in which a specific binding substance is immobilized on a part thereof and serves as a development part and a detection part. That is, a typical test piece for immunochromatography of the present invention has the following configuration.

(1) Sample pad to which analyte is supplied (2) Conjugate pad arranged downstream of the sample pad and in which the conjugate sensitized with the first specific binding substance is retained on the surface of the gold colloid so as to be eluted (3) A porous membrane that is arranged downstream of the conjugate pad and has a detection portion on the surface on which a second specific binding substance that binds to the conjugate-conjugate complex is immobilized. The porous membrane has an opaque substrate disposed on the back side.

Here, the sample pad, the conjugate pad, and the porous membrane may each constitute a separate carrier, or the two may constitute one carrier, and the sample pad, the conjugate may be formed from upstream to downstream. Any embodiment is included as long as it is composed of a pad and a porous membrane.
In addition to the above-described configuration, the immunochromatographic test piece also includes an immunochromatographic test piece further provided with one or more of an absorption pad and a 3rd pad.
The test piece is usually arranged on a solid support (hereinafter sometimes referred to as a backing sheet) such as a plastic adhesive sheet.

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

The detection method using the immunochromatographic test strip of the present invention is performed as follows.
When the specimen is dropped onto the sample supply unit, when the detection target is contained in the specimen, the detection target specifically binds to the labeled specific binding substance to form a complex. The complex develops the development part in the downstream direction and binds to the specific binding substance immobilized on the detection part. 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. By detecting this, the detection target is qualitatively or Quantitative analysis is possible. In addition to the determination of coloration by visual observation, the detection includes a method of detecting an optical parameter derived from the label by an optical means. When using optical means, it can be automated as well as manual, and continuous measurement is also possible.

(Sample pad)
In the present invention, a “sample pad” is a part that bears a sample supply unit that receives a sample, absorbs a liquid sample in a state of being molded in the pad, and allows the liquid and the component of the detection object to pass through. Any substance and form are included.
The sample pad of the present invention can be pretreated to improve water permeability.
In addition, when measuring whole blood, a hemagglutinating agent can be included. In this case, it should just be contained in at least one part of a sample pad, and can also be contained in the whole.

  Specific examples of materials suitable for the sample pad include, but are not limited to, glass fiber (glass fiber), acrylic fiber, hydrophilic polyethylene material, dry paper, paper pulp, and fabric. Preferably, a fiberglass pad is used. The sample pad can also have the function of a conjugate pad described later. In addition, the sample pad may contain a blocking reagent that is usually used as necessary without departing from the object of the present invention and without affecting the reaction system.

(Conjugate)
In the present invention, the conjugate refers to a label in which a specific binding substance or a control substance that binds to a detection target is immobilized.
The label used in the present invention can be sensitized (immobilized) with a specific binding substance such as an antibody to form a conjugate, and is contacted with a sample to detect an object (such as an antigen) in the sample. As long as it can play a role as a label in the method to be performed, gold colloid particles, platinum colloid particles, color latex particles, magnetic particles, and the like can be mentioned. And colloidal gold is even more desirable. These particle sizes may be adjusted so as to obtain the desired detection sensitivity of the measurement object according to each type. For example, the particle size of the gold colloid particles is preferably 20 to 100 nm, more preferably. It is 30-100 nm, and 60 nm is particularly preferable.
The conjugate of the present invention can also block a region where a specific binding substance is not bound on the surface of a colloidal gold or the like with a blocking agent.

  Conjugates may exist as a conjugate pad, that is, in a state where the conjugate pad is impregnated in a dedicated pad (conjugate pad) other than a sample pad, a third pad, or a porous membrane ( Type A) may be present as a conjugate part in a part of the sample pad (Type B). Furthermore, it may exist as a separate conjugate reagent so as to be mixed with the specimen separately from the test piece (type C).

Hereinafter, a typical example of a test piece of 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 the upstream to the downstream in the sample flow direction, and are arranged so that at least a part of the upper and lower layers overlap each other. A test piece of such an arrangement example is shown in FIG.

  When a sample containing an object to be detected is supplied to the sample pad of such a test piece, the object to be detected flows through the sample pad to the conjugate pad on the downstream side. In the conjugate pad, the detection target and the conjugate come into contact with each other and pass through the pad while forming a complex. The composite is then passed through a third pad placed in contact with the bottom surface of the conjugate pad and deployed 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 absorbance, reflected light, fluorescence, magnetism and the like derived from the labeling substance.

Next, a test piece of which type of 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 part and the conjugate part are formed in a part of the sample pad.
The sample supply part is a part for supplying a sample containing a detection target, the conjugate part is a part containing a conjugate, and the sample supply part is on the upstream side of the conjugate part.

Next, a specimen having a type C conjugate will be described.
The difference from the type A test strip is that the conjugate pad is not present as a test strip and the conjugate is present as a separate conjugate reagent. For example, a filter chip in which a conjugate is incorporated in a filter can be mentioned. By using such a filter chip, the sample is filtered and the conjugate held in the filter and the detection target are combined 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, the “detection reagent” is specifically a solution containing at least a conjugate.
The detection reagent keeps the conjugate in a stable state and promotes a specific binding substance such as an antibody immobilized on the conjugate to react specifically with the detection target when mixed with the sample. 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 and solubilizing agent include bovine serum albumin (BSA), sucrose, casein, amino acids and the like.

The detection reagent may also contain EDTA or EGTA, which are known sensitizers or chelating agents, as necessary for the purpose of improving detection sensitivity.
In this specification, the term “detection” or “measurement” must be interpreted in the broadest sense including proof and / or quantification of the existence of the detection target, and is limitedly interpreted in any sense. must not.

(Sample diluent)
In the present invention, a diluent may be used when the sample needs to be diluted depending on the concentration of the detection target in the sample. The diluted solution significantly inhibits the reaction between the detection target and the specific binding substance, or conversely accelerates the reaction significantly, causing the label to overaggregate, resulting in poor development in the capillary phenomenon, or the detection target. Diluents of any composition may be used as long as signal detection of the reaction between the detection target and the specific binding substance according to the concentration of the substance is possible.

(Conjugate pad)
In the present invention, the “conjugate pad” is obtained by impregnating a detection reagent that specifically reacts with a detection target with a material suitable for a conjugate pad described later 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 be in contact with a porous membrane on which a specific binding substance is immobilized. Alternatively, the specimen that has been placed in contact with the sample pad and that has passed through the sample pad by capillary flow is received, and then the specimen is transferred to the 3rd Pad that is in contact with the surface different from the contact surface with the sample pad by capillary flow. You may arrange as follows.

  Suitable materials for the conjugate pad include, but are not limited to, paper, cellulose blend, nitrocellulose, polyester, acrylonitrile copolymer, glass fiber or non-woven fiber such as rayon. Preferably, a fiberglass pad is used.

  If necessary, the conjugate pad 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 a sample component labeled with a label. High antigenic proteins such as KLH (Scattle hemocyanin) may be included. These control reagents are components (substances) that are unlikely to exist in the sample, and can be appropriately selected.

(3rd Pad)
In the present invention, 3rd Pad is a reaction component between a specimen and a detection reagent, in which a component unnecessary for detection of a detection target is removed, and a specific binding substance is immobilized as a component necessary for the reaction. The porous membrane can be disposed between the sample pad and the porous membrane for the purpose of enabling smooth development. For example, it is desirable to remove blood cells, insoluble blood cell fragments and the like as components unnecessary for detection. Also, the 3rd Pad has an aggregate that is generated by the reaction between the object to be measured and the specific binding substance, and moves to the membrane on which the specific binding substance is immobilized, so that it cannot be expanded smoothly. It is also possible to have an additional effect of previously removing the aggregates.

  3rd Pad includes any substance and form that allows the liquid, the component to be detected, and the detection reagent to pass through. Specific examples include, but are not limited to, glass fiber (glass fiber), acrylic fiber, hydrophilic polyethylene material, dry paper, paper pulp, and woven fabric. 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 specimen, 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 using only the sample pad.

(Hemagglutinating agent)
In the present invention, when whole blood is used as a specimen, it is desirable to use a hemagglutinating agent and an erythrocyte binding component in addition to the use of the 3rd Pad. The hemagglutinating agent and erythrocyte binding component to be used in combination are not particularly limited, but as a known example, a polycationic hemagglutinating agent can be used in addition to a lectin, a polyclonal antibody, and a monoclonal antibody. Examples of known polycationic hemagglutinating agents include polybrene, polylysine, polyacrylamine, polyalanine and the like, among which polybrene is preferred. Polybrene, whose chemical name is called hexadimethrin bromide, is a kind of cationic polymer to which CAS number 28728-55-4 is assigned.

  The usage mode of the hemagglutinating agent and the red blood cell binding component should be added to the dilution solution for diluting the sample, or added directly to the sample, and included in the sample supply part (sample pad) of the immunochromatographic test strip. Can do. According to such a use mode, red blood cells in whole blood are aggregated.

(Porous membrane)
The porous membrane used in the immunochromatographic test piece of the present invention is characterized in that a specific binding substance for detection is immobilized on the surface, and an opaque base material is disposed on the back surface. .

The opaque base material of the present invention disposed on the back surface of the porous membrane is a member disposed for lining the membrane because the membrane is porous, and an impermeable film or the like is used. Such an opaque base material of the present invention is sometimes called a so-called “lining member”. The base material can be backed by bonding to the back surface of the porous membrane, or can be formed by previously laminating the porous membrane on the upper surface of the water-impermeable film.
The opaque base material of the present invention may be disposed on the back side of the porous membrane, may be in contact with the adhesive layer, or may be in contact without the adhesive layer as long as the adhesiveness is ensured. Also good. In addition, when providing the backing sheet mentioned later, it arrange | positions under the opaque base material (backing member) of this invention.
In the present invention, the base material on the back surface of the porous membrane is conventionally transparent, but by making the base material opaque, the color of the corresponding part of the membrane is whiter than the surrounding color, and the color is lost. It was possible to avoid the so-called “white spot phenomenon” (hereinafter, simply referred to as white spot).

Opaque means that it is not transparent and the other side cannot be seen through, and does not require complete opacity, but is more preferably completely opaque.
As the opaque base material, a base material having the same color as the porous membrane is preferable, and when the porous membrane is white, the base material of the present invention is preferably a cream or gray color close to white, among which white is the most preferable.
The opaque base material may be any base material that is impermeable and opaque, and any material can be used. Examples thereof include polyethylene, polyethylene terephthalate, polyester, and nylons.

  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 in the test line. The reason for this is not clear, but it is due to the appearance of the surface component (nitrocellulose) being reduced in uniformity and the optical means of the backing sheet placed on the lower layer of the membrane because the colors of the membrane surface and the back surface match. It is estimated that simultaneous reproducibility is improved by avoiding the influence of reflected light.

  As a porous membrane (hereinafter sometimes simply referred to as a membrane), any material can be used. Examples thereof include, but are not limited to, porous membranes such as polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, and ceramics. Specific examples include glass fiber filter paper and nitrocellulose membrane sold by Merck Millipore, Toyo Roshi, GE Healthcare and the like. In addition, by appropriately selecting the pore size and structure of the porous membrane, it is possible to control the flow rate of the immune complex of the labeled specific binding substance and the detection target through the membrane. Since the amount of the labeled specific binding substance that binds to the specific binding substance immobilized on the membrane can be adjusted by controlling the flow rate in the membrane, the pore size and structure of the membrane can be adjusted according to the present invention. It is desirable to optimize the immunochromatographic test piece in consideration of the combination with other constituent materials.

(Immobilization of specific binding substance to 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 generally performed by a well-known method. For example, in the case of the flow-through type, the specific binding substance described above is adjusted to a predetermined concentration, and the liquid is applied to the porous membrane in a certain amount, such as a specific symbol such as a dot or +. 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 be a “control line”. It is common. In addition, the specific binding substance that binds to the control reagent can be immobilized at a position different from the specific binding substance that binds to the detection target to form a “control line”.

In the case of the lateral flow type, a device having a mechanism that can move the specific binding substance to a predetermined concentration and move the liquid in a horizontal direction while discharging the liquid from the nozzle at a constant speed is used. It is performed by applying the porous membrane in a line shape. At this time, the concentration of the specific binding substance is preferably 0.1 to 5 mg / mL, and more preferably 0.5 to 3 mg / mL. In addition, the amount of specific binding substance immobilized on the porous membrane can be optimized by adjusting the amount applied to 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 apparatus. In particular, in the case of the lateral flow type, 0.5 to 2 μL / cm is preferable.
In the present invention, the term “flow-through membrane assay” refers to a method in which a sample solution or the like is developed so as to pass perpendicularly to the porous membrane, and the term “lateral flow membrane assay” This refers to a method in which the sample liquid or the like is developed so as to move in a parallel direction with respect to the porous membrane.

  In the present invention, the position of application of the binding-specific binding substance to the target object on the porous membrane is the lateral flow type, and the detection reagent is developed from the conjugate pad by capillary action in the case of the lateral flow type. What is necessary is just to arrange | position so that a specific binding substance may pass through the coated line in order. It is preferable to arrange so that the line to which the specific binding substance that binds to the detection target is applied is upstream, and the line to which the control specific binding substance is applied is located downstream thereof. At this time, it is desirable that the distance between the lines is sufficiently long so that the signal of the label can be detected. Even 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 label can be detected.

  The specific binding substance solution to be applied to the porous membrane can usually be prepared using a predetermined buffer solution. Examples of the buffer solution include a buffer solution usually used such as a phosphate buffer solution, a Tris buffer solution, and a Good buffer solution. The pH of the buffer is preferably in the range of 6.0 to 9.5, and may be set as appropriate according to the nature of the specific binding substance used. For example, a pH 8.0 buffer can be used for the anti-cTnI antibody described below. The buffer may further contain salts such as NaCl, stabilizers such as sucrose, preservatives, preservatives such as procrine, and the like. In addition to salts such as NaCl that are added to adjust the ionic strength, salts such as sodium hydroxide that are added in the step of adjusting the pH of the buffer solution are also included. After the specific binding substance is immobilized on the porous membrane, blocking can also be carried out by coating a portion other than the specific binding substance immobilization site with a normally used blocking agent in the form of a solution or vapor. In the present specification, a porous membrane on which a specific binding substance is immobilized as described above may be referred to as a “specific binding substance-immobilized membrane”.

(Absorption pad)
In the present invention, the absorption pad is a part having liquid absorbency 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 piece, and in the flow through type, for example, it may be provided at the lower part of the specific binding substance-immobilized membrane. As the absorbent pad, for example, filter paper can be used, but is not limited thereto.

(Backing sheet)
In the present invention, the backing sheet is a solid phase for supporting a test piece, and a sheet in which an adhesive material is attached to a plastic sheet is usually used. Examples of the material for the backing sheet include, but are not limited to, polystyrene, polyester, polypropylene, and vinyl chloride. The color of the backing sheet is not particularly limited, but is preferably the same color as the membrane. When the membrane is white, the backing sheet is also preferably white.

(Top film)
In the present invention, the top film refers to a sheet-like member that covers the uppermost surface of a test piece, and increases the mechanical strength of a porous membrane on which a specific binding substance is immobilized, Used to prevent moisture evaporation (drying). As the material of the top film, any material can be used as long as the object can be achieved. Examples thereof include polyester, and the top film can be coated on the test piece with an adhesive material or can be laminated. In the present invention, it has been found that the white spot phenomenon strengthened by covering the test piece with the top film can be weakened by making the membrane backing member opaque. Therefore, the effect of the present invention becomes more remarkable in the test piece provided with the top film.

(Detection device)
The immunochromatographic test strip of the present invention is suitable in consideration of the size of the test strip, the addition method / position of the specimen, the specific binding substance immobilization position on the specific binding substance immobilization membrane, the signal detection method, etc. It can be stored and mounted in a simple container (housing), and the state stored and mounted in this way is called a “device”.

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

(Sample)
In the detection method of the present invention, the “specimen” including the detection target refers to blood, urine, sputum, saliva, nasal discharge, nasal wiping liquid, pharyngeal wiping liquid, other body fluids, biological samples such as feces, and the like. A biological sample may be used as a specimen as it is, and a specimen diluted as appropriate with a diluent, extracted and / or filtered is also included in the specimen of the present invention. Blood samples include whole blood, red blood cells, plasma, serum and the like.
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 blood collection.

(Detection target)
The detection target of the present invention is a substance present in a biological sample such as blood (whole blood), red blood cells, serum, plasma, urine, saliva or sputum, such as 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 (cardiac fatty acid binding protein), cardiac troponin I (cTnI), metabolism-related markers such as adiponectin, CEA (carcinoembryonic antigen), AFP (α-) Fetoprotein), CA19-9, CA125, PSA (prostate specific antigen) Tumor markers, HBV of (B hepatitis virus), HCV (C hepatitis virus), Chlamydia trachomatis, infections associated markers, such as Neisseria gonorrhoeae, allergen-specific IgE (immunoglobulin E), hormones, such as a drug is exemplified. Among these, D dimer, CRP, BNP, H-FABP, cTnI, and the like, which are highly desired to use whole blood as a specimen, are more preferable.

(Specific binding compound)
In the present invention, specific binding substances for insoluble carrier particles such as colloidal gold and a measurement target substance supported on a porous membrane include proteins, peptides, amino acids, lipids, carbohydrates, DNA, RNA, receptors, haptens. There are no particular restrictions on the molecular weight, natural or synthetic origin, and examples include antibodies or antigens that can be used in immunological assays utilizing immune reactions.

(Antibodies used in the present invention)
The antibody to the detection target used in the present invention is not limited in any way as long as it is an antibody that reacts specifically with the detection target, and even a polyclonal antibody is a monoclonal antibody. May be. More preferably, it is a monoclonal antibody. In general, a hybridoma producing the antibody is a myeloma of the same kind as the spleen cell 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)). Cells (myeloma cells) can be produced by cell fusion.

As the antibody of the present invention, a functional fragment of an antibody having antigen-antibody reaction activity can be used in addition to the whole antibody molecule. In addition to those obtained through the immunization process for general animals (mouse, goat, sheep, etc.), the amino acid sequence of the animal species is different from the animal immunized with the immunogen (substance to be measured) by genetic recombination technology. It may be an antibody (such as a chimera-specific binding substance, a humanized antibody, or a fully humanized antibody). Examples of the functional fragment of the antibody include F (ab ′) ₂ , Fab ′, and single chain antibody (scFv), which are fragments having antigen-antibody reaction activity. Functional fragments of these antibodies can be produced by treating the antibody obtained as described above with a proteolytic enzyme (for example, pepsin or papain).

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

(kit)
The detection kit using the immunochromatographic test strip of the present invention is an immunochromatographic test strip including at least a porous membrane, and having a sample supply section, a developing section, and a detection section on which an antibody is immobilized, wherein the detection section is a porous membrane. What is necessary is just to include the said test piece characterized by being fixed to the surface of this and the opaque base material being arrange | positioned at the back surface of the said porous membrane.
This detection kit includes other reagents necessary for detection (for example, detection reagents including conjugates), specimen dilutions, test tubes, swabs for stool collection, instruction manuals, housings for specimen storage, etc. But you can.

Hereinafter, specific examples of the present invention will be described, but they are merely illustrative and the present invention is not limited thereto.

[Test Example 1] Confirmation test of whitening phenomenon avoidance effect Comparison of whitening phenomenon avoidance effect when using an antibody-immobilized membrane lined with a white base material of the present invention compared with a case where a white background is 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 5% (w / v) tetrachloroauric (III) acid aqueous solution was added and reacted for 10 minutes with stirring, and then the reaction solution was boiled. Thereafter, the mixture was cooled in ice water to prepare a colloidal gold 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 at an absorbance at the maximum absorption wavelength of the gold colloid.

(Ii) Preparation of anti- cTnI antibody conjugate Anti- cTnI diluted 69.3 μg / mL with 2 mM Tris-HCl buffer (pH 7.0) with respect to 200 mL of the above 1 OD / mL 60 nm colloidal gold solution (pH 8.0) 10 mL of monoclonal antibody was added and stirred at room temperature for 10 minutes. 10 mL of purified water containing 0.5% (w / v) Neo Protein Saver (Toyobo, 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. . Then, it centrifuged at 11900xg for 45 minutes at 10 degreeC. After removing the supernatant, 10 mL of 0.2% (w / v) Neo Protein Saver aqueous solution was added to the resulting precipitate to suspend the conjugate to 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) aqueous triammonium citrate solution was added and mixed with stirring. Subsequently, 10 mL of 5% (w / v) tetrachloroauric (III) acid aqueous solution was added and reacted for 10 minutes with stirring, and then the reaction solution was boiled. Then, it cooled in ice water and prepared the gold colloid solution whose average particle diameter is 40 nm. This gold colloid solution having an average particle diameter of 40 nm was adjusted to 1 OD / mL with purified water at an absorbance at the maximum absorption wavelength of the gold colloid.

(Iv) Preparation of gold colloid-labeled KLH (KLH conjugate) for control line 375 μg / mL with 2 mM phosphate buffer (pH 6.1) with respect to 200 mL of 40 nm gold colloid solution (pH 6.1) of the above 1 OD / mL 2.67 mL of KLH (manufactured by Sigma) dissolved so as to be added was added and stirred at room temperature for 10 minutes. 20 mL of 10% bovine serum albumin (BSA) aqueous solution was added to the mixture of the gold colloid and KLH, and the mixture was stirred at room temperature for 5 minutes. Thereafter, the mixture is centrifuged at 10 ° C. for 45 minutes, and the supernatant is removed. Then, 10.7 mL of Conjugate Dilution Buffer (Scripps) is added to the resulting 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 Conjugate Dilution Buffer are mixed to make a conjugate solution, and a fixed volume of glass fiber pad (Merck Millipore) is added to 1 of the pad volume. .. Doubled in volume. The conjugate pad was dried by heating in a dry oven at 70 ° C. for 45 minutes.

3) Preparation of anti-cTnI monoclonal antibody-immobilized membrane (antibody-immobilized membrane) Anti-cTnI monoclonal antibody for test line was prepared with 10 mM PB (containing 0.09% NaN ₃ ) (pH 8.0) containing 2.5% sucrose. Dilute to 3 mg / mL.
For control lines, a rabbit anti-KLH polyclonal antibody (Bethyl) was diluted to 1 mg / mL with 10 mM PB (containing 0.09% NaN ₃ ) (pH 7.2) containing 2.5% sucrose.
Using the immunochromatographic dispenser “XYZ3050” (BIO DOT), the above-mentioned anti-cTnI monoclonal antibody is placed at a position inside one end of the short side of the nitrocellulose membrane lined with a transparent or white substrate shown in Table 1 at 1 μL / cm. Was applied to form a test line. An anti-KLH polyclonal antibody was similarly applied at a distance of about 4 mm from the position of the test line to form a control line. The membrane 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 to prepare a sample pad pretreatment solution.
A glass fiber pad (Lydall) was appropriately cut to the required size, soaked the sample pad pretreatment solution by 1.5 times the pad volume, and dried in a dry oven at 70 ° C. for 45 minutes. A sample was used as a sample pad.

5) Preparation of immunochromatographic test piece The antibody-immobilized porous membrane (b-1) lined with each of the substrates (b-2) above is attached to the backing sheet (a), and the anti-cTnI antibody (c ), And then the coating part was arranged in the order of 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 placed and mounted, and the sample pad (g) prepared in 4) above is placed and mounted so as to overlap this conjugate pad, and the opposite end is placed on the opposite end. An absorbent pad (h) was placed and mounted. Finally, the surfaces of the absorption pad and the antibody-immobilized membrane were covered with a top film (i) so that a part of the antibody-immobilized membrane was exposed. Thus, the immunochromatographic test piece was produced by cutting each structural element into a superposed structure. In the assay, the test piece may be stored and mounted in a dedicated plastic housing (having a sample addition window and a detection window, not shown in FIG. 4) to form an immunochromatographic test detection device. . FIG. 4 shows a schematic configuration diagram of an immunochromatographic test piece.

2. Detection or measurement by immunochromatography method 120 μL of 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. The results are shown in Table 1.
<Outline evaluation criteria>
Twelve or twenty samples were visually evaluated. When even one of the samples was recognized as white, “white” was judged as “existing”, and when zero, “white” was judged as “no”.

3. Results When using a nitrocellulose membrane lined with a transparent substrate as an antibody-immobilized membrane, white spots were observed just before the test line, but they were lined with the white substrate of the present invention. When a nitrocellulose membrane was used as an antibody-immobilized membrane, no white spots were observed. Therefore, more accurate evaluation becomes possible by improving the visibility in the visual evaluation of the immunochromatographic test.
In addition, this time, white spots are determined and evaluated by visual observation. However, in the case of optical measurement by an apparatus, the waveform is prevented from being disturbed and the baseline can be set correctly.

[Test Example 2] Confirmation test of CV reduction effect Comparison of measured values when optically measured using an antibody-immobilized membrane lined with a white base material of the present invention is compared with a case where a conventional transparent base material is lined. did.

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

2. Detection or measurement by immunochromatography method 120 μL of plasma specimen solution containing 40 or 60 pg / mL myocardial troponin I (cTnI) was added to the sample pad window of the immunochromatographic test device prepared above, and 15 minutes later, immunochromato reader rapid peer (Sekisui) The color amount (absorbance) of the test line was measured using a medical company, and the CV value (variation) thereof 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 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 lined with the transparent base material is used as the antibody-immobilized membrane. Improved measurement reproducibility.

[Test Example 3] CV reduction effect confirmation test (low concentration side)
A test was conducted to determine whether or not the variation reduction effect of Test Example 2 appears even when the substance to be detected is at a low concentration.

1. Production of immunochromatographic device Immunochromatographic test in the same manner as in Test Example 1 except that two lots of a membrane (CN140) lined with a transparent substrate and a porous membrane (CN150) lined with a white substrate of the present invention were used. Pieces were produced.

2. Detection or measurement by immunochromatography The measurement was performed in the same manner as in Test Example 2 except that a plasma sample solution containing 15 pg / mL myocardial 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 base material of the present invention is used as the antibody-immobilized membrane, the membrane lined with the transparent base material is immobilized with the antibody even when the concentration of the target to be detected is low. Compared to the membrane, the CV value decreased, and the reproducibility of the measurement was improved. Similar results were obtained even when two different lots were used.

[Test Example 4] Confirmation test of CV reduction effect (high concentration side)
A test was conducted to determine whether the variation reducing effect of Test Example 2 appears even when the substance to be detected is at a high concentration.

1. Manufacture of an immunochromatographic device An immunochromatographic test piece was manufactured in the same manner as in Test Example 1 except that a membrane backed by a transparent substrate (CN140) and a porous membrane backed by a white substrate of the present invention (CN150) were used. did.

2. Detection or measurement by immunochromatography The 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 base material of the present invention is used as the antibody-immobilized membrane, the porous membrane lined with the transparent base material is used as the antibody even if the concentration of the detection target is high. Compared to the case of using an immobilized membrane, the CV value was greatly reduced, and the reproducibility of the measurement was greatly improved.

  An immunochromatographic test piece comprising at least a porous membrane of the present invention, and having a sample supply part, a development part, and a detection part on which a specific binding substance is immobilized, wherein the detection part is immobilized on the surface of the porous membrane. According to the test piece in which the opaque base material is disposed on the back surface of the porous membrane, white spots near the test line can be avoided.

In addition, an immunochromatographic test strip with little variation in measured values can be provided.
According to the present invention, the visibility in the visual determination is improved by avoiding white spots in the test piece, and also the variation in the measured value in the optical measurement is reduced. Therefore, 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) Absorbent pad (i) Top film

Claims (14)

Wherein at least the porous membrane, the sample supply unit, development unit, a immunochromatographic test strip having a detector, the detector is fixed to the surface of the porous membrane, the the back surface of the porous membrane An opaque base material for backing a porous membrane is disposed , and a backing sheet is disposed under the opaque base material . The immunochromatographic test piece according to claim 1, wherein the opaque substrate is a white substrate. The sample sample pad responsible for supplying portion, and colloidal gold or immunochromatographic test strip of claim 1 or 2 comprising a conjugate pad including a singular binding substance labeled with colored latex. The immunochromatographic test strip according to any one of claims 1 to 3 , wherein the specific binding substance is an antibody. The immunochromatographic test piece according to any one of claims 1 to 4 , which is a test piece for detection by optical means. An immunochromatographic detection kit comprising the immunochromatographic test piece according to any one of claims 1 to 5 . An immunochromatographic detection method, wherein the following test piece is used;
A porous membrane having at least a detection portion; an opaque base material for backing the porous membrane is disposed on the back side of the porous membrane; and a backing sheet is further disposed under the opaque base material Immunochromatographic test piece. The immunochromatographic detection method according to claim 7 , wherein the opaque substrate is a white substrate. The immunochromatographic detection method according to claim 7 or 8 , wherein the test piece includes a sample pad serving as the sample supply unit and a conjugate pad containing a specific binding substance labeled with gold colloid or colored latex. The immunochromatographic detection method according to any one of claims 7 to 9 , wherein the specific binding substance is an antibody. The immunochromatographic detection method according to any one of claims 7 to 10 , comprising a step of detecting an optical parameter derived from the labeling substance by optical means. By visual observation comprises determining a color derived from the labeling substance, immunochromatography detection method according to any one of claims 7-10. A method for reducing variations in measured values in an immunochromatographic detection method,
Said method characterized in that the following test piece is used;
An immunochromatographic test piece including at least a porous membrane, having a sample supply part, a development part, and a detection part, wherein the detection part is immobilized on the porous membrane surface,
The porous opaque substrate for backside backing of those porous membranes of the membrane is disposed, further, a test piece backing sheet is disposed under the opaque substrate. In the immunochromatography detection method using a test piece, the method for reducing white spots in the test piece, wherein the following test piece is used;
An immunochromatographic test piece including at least a porous membrane, having a sample supply part, a development part, and a detection part, wherein the detection part is immobilized on the porous membrane surface,
The test piece by which the opaque base material is arrange | positioned at the back side of the said porous membrane.