JP6371808B2 - Immunochromatography detection kit - Google Patents

Description

  The present invention relates to a detection kit comprising a conjugate reagent and an immunochromatographic test strip. In particular, the test strip is a test strip in which a third antibody and a saccharide against a detection target are retained so as to be eluted separately from the first antibody contained in the conjugate reagent and the second antibody for immunochromatographic detection. To a detection kit. The present invention also relates to a detection method using the detection kit.

A method for detecting an antigen as a substance to be detected in a sample using an immunochromatographic test strip is as follows. First, the conjugate in which the first antibody is immobilized on the label is brought into contact with the sample. By these contacts, a complex of an antigen, which is a substance to be detected in the sample, and a conjugate is formed. The immunochromatographic test strip has a capture site (detection site) to which the second antibody is immobilized. When the complex is provided to the carrier, the complex is captured in the capture site while expanding in the carrier. The presence / absence and strength of the complex labeling substance can be detected by visual observation or optical means.
Here, the presence mode of the conjugate in detection using immunochromatography includes a mode in which the conjugate portion exists as a conjugate portion on a part of the test strip, typically on the sample pad, downstream from the sample supply portion. . Or there exists a form which exists as a conjugate reagent, such as a conjugate chip apart from a test strip (patent document 1). The conjugate chip is obtained by applying a conjugate to a filter that filters a sample liquid.
In the former case where the conjugate is present as part of the test strip, when the sample is dropped into the sample supply section, the sample will be in the sample from when it reaches the conjugate section downstream of the sample supply section. The binding reaction between the antigen to be detected and the conjugate starts. Therefore, the enhancement of the color intensity at the capture site (detection site) stops when all the conjugates in the conjugate part have flowed.
On the other hand, when the liquid obtained by filtering the sample with the conjugate chip is dropped onto the sample pad of the test strip, the liquid that is a mixture of the sample and the conjugate is dispersed all over the sample pad. Therefore, the binding reaction between the antigen and the conjugate in the sample starts immediately after dropping, and the color intensity of the capture site (detection site) continues to increase until the liquid flow of the mixture reaches equilibrium.
For example, Patent Document 2 is known as a method for adjusting sensitivity in detection using immunochromatography. Patent Document 2 discloses a method of reacting an analysis target sample with a sensitivity adjusting substance in order to reduce the analysis target component. Specifically, the sensitivity-modulating substance is an antibody labeled with an indistinguishable label (for example, a white substance that cannot be distinguished from a test strip), which is different from an antibody labeled with a distinguishable substance. The analysis is performed by holding the antibody labeled upstream with the substance upstream of the immobilization part. The analysis target component partially moves on the chromatographic carrier while reacting with the sensitivity control substance, and the analysis target component that does not react with the sensitivity control substance reacts with the labeling substance and is fixed by the detection unit.
In this method, although a certain effect of reducing the analyte can be expected, the presence mode of the labeled antibody corresponds to the case where the labeled antibody exists as a part of the test strip of the above-described presence mode. The problem that the flow continues until equilibrium is reached does not arise.
Further, in Patent Document 3, in immunochromatographic detection of a specimen such as fecal occult blood, a predetermined amount of a specimen is contacted with an antibody immobilized on a sample pad in advance and then bound, and then an unbound specimen. Is disclosed by binding to and detecting antibodies bound to colored latex.
Similarly to Patent Document 2, this method corresponds to the case where the labeled antibody is present as a part of the test strip in the above-described manner, and in the first place, the enhancement of the color intensity is continued until the flow of the liquid sample reaches equilibrium. The problem of continuing does not arise.

International Publication WO2004 / 081568 Pamphlet JP-A-10-48210 JP-A-8-101197

  When the conjugate is mixed with a liquid sample and supplied to the test strip, the color intensity at the detection site is kept from increasing for a long time, and the capture at the detection site of the complex is completed within a certain time. It is an object of the present invention to provide a detection kit and a detection method using immunochromatography so that the enhancement of the activity stops.

In order to solve the above problems, the present inventors need a certain amount of complex within a certain time for detection of antigen, and what should be done to suppress detection of other complexes? We conducted an intensive study. As a result, apart from the antibody at the conjugate and the detection site, free antibody against the antigen to be detected is present on the sample pad, thereby masking the antigen and suppressing the formation of unnecessary complexes. Therefore, it was thought that capture of unnecessary complex at the detection site could be suppressed. In addition, if free antibody alone is present on the sample pad, it may elute immediately and mask the complex originally required for detection. By imparting the properties and masking the complex with a slight delay, it succeeded in selectively suppressing the supplementation of the delayed complex and completed the present invention.
That is, the present invention has the following configuration.
<1>
Detection kit using immunochromatography including:
(1) A conjugate reagent in which a first antibody that immunologically reacts with a substance to be detected is immobilized on a label (2) an insoluble carrier that detects the substance to be detected in the sample by developing the sample An immunochromatographic test strip comprising:
The insoluble carrier is, in order from the upstream, a supporting part on which a saccharide and a third antibody can be eluted,
The test strip <2>, comprising: a sample supply section for supplying a sample; and a detection section on which a second antibody that immunologically reacts with a substance to be detected is immobilized.
The detection kit according to <1>, wherein the saccharide is any one or more selected from the group consisting of monosaccharides and disaccharides.
<3>
<1> or <2> The detection kit according to <1> or <2>, wherein the saccharide and the third antibody supporting part are formed in a line shape on the insoluble carrier so as to be orthogonal to the sample development direction.
<4>
The insoluble carrier includes at least a sample pad and a membrane pad separate from the sample pad,
The sample pad has a saccharide or third antibody support and a sample supply,
The membrane pad is the detection kit according to any one of <1> to <3>, which includes a detection unit.
<5>
The detection kit according to any one of <1> to <4>, wherein the label of (1) is colored latex particles or metal colloid particles.
<6>
The detection kit according to any one of <1> to <5>, wherein the target substance is hemoglobin, and the first to third antibodies are anti-hemoglobin antibodies.
<7>
The detection kit according to any one of <1> to <6>, wherein the saccharide and the third antibody-supporting portion are disposed upstream of the sample supply portion.
<8>
The detection kit according to any one of <1> to <7>, wherein the saccharide and third antibody-supporting portion is formed at an upstream end portion of the sample pad.
<9>
A detection method using immunochromatography, comprising the following steps.
(A) A step of mixing a conjugate reagent in which a first antibody that immunologically reacts to a substance to be detected is immobilized on a label, a specimen, and a specimen diluent, and obtaining a sample comprising these mixtures ( B) Step of dropping the sample obtained in (A) into the sample supply section of the following immunochromatographic test strip and developing it in an insoluble carrier Immunochromatographic test strip;
A carrier part comprising the insoluble carrier, in which a saccharide and a third antibody are eluted in order from upstream;
A sample supply section for supplying a sample; and a detection section on which a second antibody that immunologically reacts with the detection target substance is immobilized; and a conjugate with the detection target substance in the test strip (C) sample Step <10> of detecting the gate complex in the detection unit
The detection method according to <9>, wherein the saccharide is any one or more selected from the group consisting of monosaccharides and disaccharides.
<11>
<9> or <10> The detection method according to <9> or <10>, wherein the saccharide and the third antibody-supporting part are formed in a line shape on the insoluble carrier so as to be orthogonal to the sample development direction.
<12>
The insoluble carrier includes at least a sample pad and a membrane pad separate from the sample pad,
The sample pad has a saccharide or third antibody support and a sample supply,
The detection method according to any one of <9> to <11>, wherein the membrane pad has a detection unit.
<13>
The detection method according to any one of <9> to <12>, wherein the label of (1) is colored latex particles or metal colloid particles.
<14>
The detection method according to any one of <9> to <13>, wherein the substance to be detected is hemoglobin, and the first to third antibodies are anti-hemoglobin antibodies.
<15>
The detection method according to any one of <9> to <14>, wherein the saccharide and third antibody-supporting portion is disposed upstream of the sample supply portion.
<16>
The detection method according to any one of <9> to <15>, wherein the saccharide and third antibody-supporting portion is formed at an upstream end portion of the sample pad.
<17>
The detection method according to any one of <1> to <16>, wherein the specimen is feces.

In the method for detecting the antigen, which is a substance to be detected in the specimen, provided to the sample supply part of the immunochromatography test strip in a state in which the specimen has been previously contacted with the conjugate reagent, separately from the conjugate and the antibody for detection, The presence of saccharide and free antibody in the test strip can suppress the enhancement of the color intensity at the detection site within a certain period of time, enabling accurate detection in a short time.
Furthermore, by arranging a site for retaining free antibodies and saccharides on the upstream side in the flow direction from the sample supply unit, the conjugate or conjugate-antigen complex that flows into the upstream from the sample supply unit. The body can be effectively masked, and the detection intensity at the detection site can be controlled more effectively.

(Example 1) which is a figure which shows the time-dependent change of the color development intensity | strength in the test line at the time of holding | maintaining the antibody at a fixed density | concentration and changing the density | concentration of saccharides. (Example 2) which is a figure which shows the time-dependent change of the color development intensity | strength in the test line at the time of setting sugars as a fixed density | concentration and hold | maintaining by changing the density | concentration of an antibody. It is a figure which shows a time-dependent change of the coloring intensity in the test line at the time of hold | maintaining only saccharides (comparative example). It is a block diagram of the immunochromatographic test strip of the present invention.

(sample)
In the present invention, biological samples such as blood, urine, sputum, saliva, nasal discharge, other body fluids, and feces are used as samples. The biological sample may be used as it is as a sample, or may be diluted as appropriate with a diluent or filtered to obtain a sample.

(Substance to be detected)
The substance to be detected of the present invention may be any substance as long as it is contained in a biological sample stool as a sample and can be detected using an antigen-antibody reaction, and examples thereof include viruses, parasites, and proteins.
Examples of the virus to be detected include influenza virus, and examples of the protein to be detected include human hemoglobin in fecal occult blood, hepatitis B virus antibody, hepatitis C virus antibody, human immunodeficiency virus antibody, and the like.

(kit)
The detection kit using the immunochromatography of the present invention includes the following (1) a conjugate reagent in which a first antibody that immunologically reacts with a substance to be detected is immobilized on a label, and (2) a sample A test strip for immunochromatography comprising an insoluble carrier for detecting a substance to be detected in a sample by developing
The insoluble carrier is, in order from the upstream, a supporting part on which a saccharide and a third antibody can be eluted,
A test supply strip including a sample supply section for supplying a sample, and a detection section on which a second antibody that immunologically reacts with a substance to be detected is immobilized;
As long as it contains.
The detection kit may further include a reagent necessary for detection, a sample diluent, a test tube, a swab for collecting stool, an instruction manual, a housing for storing a test strip, and the like.

(Test strip for immunochromatography)
In the test strip of the present invention, the sample supply unit for supplying the sample is provided as a sample pad separately from the insoluble membrane having the detection unit, or on the same membrane as the insoluble membrane, upstream of the detection unit. In some cases as a sample supply section on the side. Of these, it is desirable to exist as a sample pad.
In the test strip of the present invention, there is no conjugate pad, and the conjugate is present as a conjugate reagent separately from the test strip. The conjugate reagent is added in advance to a liquid such as a specimen diluent such as a conjugate chip in which a conjugate is impregnated in a filter, a lyophilized reagent, a frozen reagent, or a dried reagent. Examples of the liquid reagent are as follows. The conjugate chip may be a filter as it is, or may be incorporated in a housing. By using such a conjugate chip and filtering and passing the specimen diluent, the conjugate in the chip and the substance to be detected can be combined to form a complex. In the case of a lyophilized reagent form, a frozen reagent form, or a dried reagent form, it can be used by adding to a substance to be detected or a sample diluent immediately before use.

(Sample pad)
When the immunochromatographic test strip of the present invention includes a sample pad, the sample pad may be a pad having a site for receiving a sample, absorbs a liquid sample in a state of being molded into the pad, Includes any substance and form that can be passed through. 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 contain a commonly used blocking reagent for the purpose of preventing / suppressing nonspecific reaction (adsorption) in the antibody-immobilized membrane described later.

(Sugars and antibodies)
The saccharide supported in the support portion of the test strip of the present invention may be any saccharide that can control the elution rate of free antibody, and among these, one or more selected from monosaccharides and disaccharides are preferable. Specifically, examples of monosaccharides include glucose, galactose, fructose, and mannose. Examples of disaccharides include sucrose, lactose, maltose, trehalose, and fructose.
The third antibody carried in the carrying part of the strike strip of the present invention may be an antibody against the detection target, as long as it is an antibody that binds to the detection target or a complex of the detection target and the conjugate. Therefore, it may be the same antibody as the first antibody contained in the conjugate or the second antibody immobilized on the detection unit, or may be a different antibody. Moreover, a monoclonal antibody or a polyclonal antibody may be sufficient. The third antibody is not immobilized on the test strip, unlike the antibody bound to the solid phase, such as a conjugate, or the antibody immobilized on the membrane, such as a detection antibody. It is desirable that the antibody is a free antibody that is retained so that it can be eluted.

  In the present invention, when the test strip has a sample pad, the saccharide and the antibody are preferably carried on the upstream side of the sample supply unit in the sample pad. In addition, when the sample supply unit and the detection unit are provided on the same insoluble membrane without the sample pad, it is desirable that the insoluble membrane is supported on the upstream side of the sample supply unit. Further, it is even more desirable that it is carried on the most upstream part, that is, on the upstream end of the insoluble membrane on the upstream side of the sample supply part.

As a method for allowing saccharides and antibodies to be supported on a sample pad or an insoluble membrane so as to be eluted, a method of applying a solution containing saccharides and a solution containing antibodies to a sample pad or the like, or immersing and drying the sample pad in these solutions Is mentioned.
The saccharide and the antibody may be dried by applying different solutions, respectively, or may be applied and dried by mixing a solution in which the saccharide and the antibody are mixed.

Prepare a liquid containing the above saccharide or antibody at a predetermined concentration, and use a device that has a mechanism that can move the liquid in a horizontal direction while discharging the liquid from the nozzle at a constant speed. The sample can be applied to a sample pad or an insoluble membrane carrier and dried so that it can be eluted. The concentration of the saccharide solution is preferably 0.1 to 10%, more preferably 0.5 to 8%, and even more preferably 1 to 5%. The concentration of the antibody solution is preferably 0.05 to 5 mg / mL, more preferably 0.1 to 1 mg / mL, and still more preferably 0.1 to 0.7 mg / mL. In addition, the amount of sugar or antibody supported on the sample pad or insoluble membrane carrier can be optimized by adjusting the discharge rate from the nozzle of the above apparatus in the case of the lateral flow type, and preferably 1 to 20 μL / cm. More preferred is 5 to 10 μL / cm.
Moreover, it is desirable that the support portion is formed in a line shape so as to be orthogonal to the flow direction, and the width of the line is preferably 1 to 10 mm, more preferably 2 to 8 mm, and even more preferably 3 to 5 mm.

When a sample containing a conjugate reagent, a substance to be detected, and a specimen diluent is supplied to the sample pad of such a test strip, the pad is placed while the substance to be detected and the conjugate contact to form a complex. pass. Thereafter, the complex is developed into an insoluble membrane on which the detection antibody is immobilized.
Since the second antibody that immunologically reacts with the substance to be detected is immobilized on a part of the insoluble membrane, the complex is bound and immobilized on the second antibody. become. The immobilized complex is detected by a means for detecting absorbance, reflected light, fluorescence, magnetism, etc. derived from the conjugate labeling substance.
Here, the operation of the present invention will be described. Since the sample supplied to the sample supply unit diffuses in all directions on the membrane, there are those that develop downstream and those that wrap around upstream. Of these, those that wrap around upstream develop later and cause the color development intensity in the detection unit to be increased for a long time. The action of the saccharide of the present invention and the third antibody probably masks the substance to be detected and the complex of the substance to be detected and the conjugate by reacting with the sample that circulates upstream. It seems that the binding of can be suppressed.

(Third pad)
The third pad is desirably arranged as necessary depending on the properties of the sample, and any third pad may be used as long as it can permeate the complex of the substance to be detected and the conjugate in the sample. Specifically, glass fiber (glass fiber), acrylic fiber, hydrophilic polyethylene material, dry paper, paper pulp, woven fabric and the like are included, and in particular, the porous member is made of polysulfone or cellulose acetate. desirable.
The pore size of the porous third pad of the present invention is preferably 1 to 100 μm in average pore size, more preferably 10 to 100 μm, still more preferably 20 to 80 μm, and most preferably 25 to 70 μm. This is because if it is less than 1 μm, clogging is caused and the flow of the sample itself becomes poor, and if it is more than 100 μm, it is considered that the nonspecific reaction substance cannot be captured.

(Insoluble membrane)
The insoluble membrane used in the present invention has at least one detection unit on which a first antibody that immunologically reacts with a substance to be detected is immobilized. Immobilization of an antibody that reacts immunologically with a substance to be detected on an insoluble membrane carrier can be performed by a conventionally known method. In the case of a lateral flow type immunochromatographic reagent, a device having a mechanism capable of preparing a liquid containing the above-mentioned antibody at a predetermined concentration and moving it horizontally while discharging the liquid from the nozzle at a constant speed, etc. Can be immobilized by applying the liquid to an insoluble membrane carrier in a line and drying. The antibody concentration in the solution is preferably 0.1 to 5 mg / mL, more preferably 0.5 to 2 mg / mL. Further, the amount of the antibody immobilized on the insoluble membrane carrier can be optimized by adjusting the ejection speed from the nozzle of the above apparatus in the case of the lateral flow type, and is preferably 0.5 to 2 μL / cm. .
The measurement method using the lateral flow type immunochromatography reagent is a measurement method in which the sample is developed so as to move in a parallel direction with respect to the insoluble membrane carrier by capillary action.
A solution containing the above antibody at a predetermined concentration can be prepared by adding the antibody to a buffer solution. Examples of the buffer include normal buffers such as phosphate buffer, Tris buffer, and Good's buffer. The pH of the buffer is preferably in the range of 6.0 to 9.5, more preferably 6.5 to 8.5, and even more preferably 7.0 to 8.0. The buffer may further contain salts such as sodium chloride, stabilizers such as sucrose, preservatives, preservatives such as procrine, and the like. The salts include those added for the purpose of adjusting the pH of the buffer solution, such as sodium hydroxide, in addition to those included for adjusting the ionic strength, such as sodium chloride.
After immobilizing the antibody on the insoluble membrane, blocking can also be carried out by coating a part other than the part where the antibody is immobilized by using a commonly used blocking agent in solution or vapor.
It should be noted that a control capture reagent conventionally used in immunochromatographic reagents may be immobilized on the insoluble membrane. The control capture reagent is a reagent for ensuring the reliability of the assay, and captures the control reagent contained in the conjugate reagent. For example, when KLH labeled with a conjugate reagent is included as a control reagent, an anti-KLH antibody or the like corresponds to the control capture reagent. The position at which the control capture reagent is immobilized can be appropriately selected to suit the design of the assay system.

  As the membrane constituting the insoluble membrane used in the present invention, a known membrane conventionally used as an insoluble membrane carrier for an immunochromatographic reagent can be used. For example, there are membranes composed of fibers made of polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, ceramics, and the like. Specific examples include glass fiber filter paper and cellulose filter paper commercially available from Sartorius, Millipore, Toyo Roshi, Whatman and the like. Of these, Sartorius and UniSart CN140 are preferable. In addition, by appropriately selecting the pore size and structure of the insoluble membrane carrier, it is possible to control the speed at which the complex of the conjugate and the substance to be detected in the sample flows through the insoluble membrane carrier.

  The immunochromatographic test strip is preferably disposed on a solid support such as a plastic adhesive sheet. The solid support is composed of a material that does not interfere with the capillary flow of the sample and conjugate. Further, the immunochromatographic test strip may be fixed on a solid support with an adhesive or the like. In this case, the adhesive component and the like are also composed of a substance that does not interfere with the capillary flow of the sample and the conjugate. The immunochromatographic test strip is stored in an appropriate container (housing) taking into account the size of the immunochromatographic test strip, the method and position of sample addition, the position where the insoluble membrane detector is formed, and the signal detection method. -It can be mounted and used, and the state stored and mounted in this way is called "device".

(Antibodies that react immunologically with the substance to be detected)
The first to third antibodies that immunologically react with the target substance used in the present invention are antibodies that can bind to the target substance. The first and second antibodies are immobilized on a label and detection unit described later. The third antibody is releasably retained on the insoluble membrane having the saccharide and upstream of the sample pad or the detection part.
These first to third antibodies may be the same, but the first and second antibodies are preferably different. The antibody may be a polyclonal antibody or a monoclonal antibody. Moreover, a functional antibody fragment, Fab, Fab prime, etc. may be sufficient.

(Marker)
As the label used in the present invention, a known label conventionally used for a test strip for immunochromatography can be used. For example, colloidal metal particles such as gold colloid particles and platinum colloid particles, colored latex particles, magnetic particles, and fluorescent particles are preferable, and gold colloid particles and colored latex particles are particularly preferable.

(Conjugate reagent)
The conjugate used in the present invention is obtained by immobilizing an antibody that immunologically reacts with a substance to be detected on the label as described above. Examples of a method for immobilizing an antibody that immunologically reacts with a substance to be detected to a label include physical adsorption and chemical bonding, and is generally immobilized by physical adsorption. The conjugate in the present invention exists as a separate conjugate reagent so as to be mixed with the specimen separately from the test strip. The conjugate reagent may be a conjugate as it is, or may contain other components and components other than the conjugate as necessary. For example, the form of the conjugate chip | tip with which the conjugate impregnated in the filter is mentioned. By using the conjugate chip and filtering the specimen diluent, the conjugate and the substance to be detected can be combined to form a complex.
It is desirable that the conjugate chip is incorporated in a cap having an opening and a nozzle that are fitted with the specimen diluent container. Such a conjugate chip is sometimes called a conjugate cap. The filtration method using the conjugate cap is as follows. The conjugate liquid is passed through the conjugate chip in the conjugate cap by fitting the conjugate cap into the opening of the specimen diluent container, inverting the specimen diluent container, and pressing the container. As a result, the diluent containing the conjugate is discharged from the nozzle. This is provided to the sample supply of the immunochromatographic test strip.

The immunochromatographic test strip of the present invention may further contain other reagents and structures depending on the measurement conditions and the type of sample.
Examples of other reagents include blocking agents that prevent non-specific reactions.
Other configurations include, for example, an absorption pad that controls the development of the sample by absorbing the sample that has moved and passed through the insoluble membrane.
The production of the immunochromatographic test strip of the present invention can be carried out by appropriately modifying or altering the method described in the examples.

(Other)
In this specification, the meaning of upstream or downstream is used to mean the upstream side or the downstream side in the direction of sample flow. That is, when the test pad of the present invention is laminated so that the sample pad, the third pad, and the insoluble membrane partially overlap from above, the sample pad is the most upstream and the insoluble membrane is the downstream. In addition, an absorbent pad (also referred to as an end pad) may be laminated on the downstream side of the insoluble membrane so as to overlap. In this case, the absorbent pad is the most downstream.
In addition, the specimen diluent in this specification is also referred to as a specimen extract. In addition, the sample diluent has an action of developing the sample, and may be expressed as a developing solution.

[Example 1] Effect confirmation test of the present invention (1)
The control effect in the detection reaction of hemoglobin was confirmed by including each concentration of sugar and anti-hemoglobin antibody in the upstream end of the sample pad so as to be eluted. Hereinafter, a mouse monoclonal antibody was used as the colored latex-labeled anti-hemoglobin antibody, and a goat polyclonal antibody was used as the anti-hemoglobin antibody of the sample pad and test strip.
1. Preparation of Conjugate Chip A conjugate filter was prepared by impregnating a sintered filter with GSA (Goat Serum Albumin) -sensitized latex as a conjugate (colored latex-labeled anti-hemoglobin antibody) and a control reagent.

2. Preparation of test device (1) Preparation of sample pad of the present invention A solution containing sucrose 0-7% and anti-hemoglobin antibody 0-0.5 mg / mL was applied to the end surface of the sample pad at 10 μL / cm and dried. Thus, a sample pad of the present invention having a saccharide and a third antibody support was prepared. (In the figure, notation such as S1% -0.5 mg / mL indicates the concentration of sucrose and antibody contained in the sample pad. For example, S1% -0.5 mg / mL indicates sucrose 1% and antibody 0.5 mg / mL. (The same is true for Figures 2 and 3. Control shows the case without sucrose or antibody.)
(2) Production of Test Strip FIG. 4 shows a schematic configuration diagram of the immunochromatographic test strip of the present invention.
An antibody-immobilized membrane (b) is attached to a plastic backing sheet (a), and a sample pad (e) of the present invention having a saccharide and third antibody support (d) prepared in (1) above is applied thereon. The absorbent pad (f) was placed and mounted on the opposite end. In the antibody-immobilized membrane, an anti-hemoglobin antibody and a control reagent are immobilized on an insoluble membrane in a line shape perpendicular to the flow direction. Each pad is laminated so that the upper and lower pads are in contact with a part thereof. The line consisting of the anti-hemoglobin antibody is called the test line (c1), and the line consisting of the control reagent is called the control line (c2).
Thus, the structure which piled up each component was cut | disconnected by the fixed width | variety, and the test strip for immunochromatography was produced.
The test strip was accommodated in a plastic housing having a sample addition window part and a detection window part (not shown) to produce a test device.

3. Specimen Diluent Human hemoglobin was added to a specimen diluent containing 10 mM PBS so as to be 120 ng / mL to obtain a measurement sample.

4). Test Method The measurement sample was filtered with a conjugate chip, and 5 drops were dropped on the sample pad from the sample addition window of the test device. The color development intensity of the line was measured at each time point of 2, 5, 7, 10, 15, 20, and 30 minutes after the dropping, and digitized. The hemoglobin in the sample passes through the filter inside the conjugate chip, and forms a complex with the colored latex-labeled anti-hemoglobin antibody in the course of passage. The complex is detected by developing the membrane, binding to the anti-hemoglobin antibody immobilized on the test line.

5. Test results The results are shown in FIG.
When a sample pad containing a constant concentration of antibody and each concentration of sucrose was used, the effect of suppressing the detection reaction of hemoglobin was observed at any sucrose concentration. In particular, good results were confirmed at sucrose concentrations of 1 to 3%.

[Example 2] Effect confirmation test of the present invention (2)
Sugar and anti-hemoglobin antibody at various concentrations were included in the upstream end of the sample pad so as to be able to elute, and the control effect in the hemoglobin detection reaction was confirmed.

1. Test Method A solution containing 5% sucrose and 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL or 0.7 mg / mL of antibody was applied to the end surface of the test strip sample pad at 10 μL / cm. A sample pad was prepared and a test strip was manufactured in the same manner as in Example 1 except that it was dried.

2. Test results The results are shown in FIG. When a sample pad containing a certain concentration of sugar and each concentration of anti-hemoglobin antibody was used, an inhibitory effect on the hemoglobin detection reaction was observed at any antibody concentration.

[Reference Example 1]
In order to compare with the effect of the present invention, the control effect in the detection reaction of hemoglobin was confirmed by including only sugar at the upstream end of the sample pad so as to be eluted.

1. Test Method A sample pad was prepared and tested in the same manner as in Example 1 except that a solution containing sucrose 0, 5%, and 10% was applied to the end surface of the sample pad of the test strip at 10 μL / cm and dried. A strip was produced.
2. Test results The results are shown in FIG. When a sample pad containing no sugar and containing only sugar was used, no effect of suppressing the detection reaction of hemoglobin in the sample was observed.

  In the method for detecting the antigen, which is a substance to be detected in the specimen, provided to the sample supply part of the immunochromatography test strip in a state in which the specimen has been previously contacted with the conjugate reagent, separately from the conjugate and the antibody for detection, The presence of saccharide and free antibody in the test strip can suppress the enhancement of the color intensity at the detection site within a certain time, and the reaction can be controlled.

(A) backing sheet (b) antibody-immobilized membrane (c1) test line (c2) control line (d) saccharide and third antibody support (e) sample pad (f) absorption pad

Claims (17)

Detection kit using immunochromatography including:
(1) A conjugate reagent in which a first antibody that immunologically reacts with a substance to be detected is immobilized on a label (2) an insoluble carrier that detects the substance to be detected in the sample by developing the sample An immunochromatographic test strip comprising:
The insoluble carrier is, in order from the upstream, a carrier part on which a third antibody that binds to a saccharide and a substance to be detected or a complex of a substance to be detected and a conjugate is leached.
A sample supply section for supplying a sample, and a detection section on which a second antibody that immunologically reacts with a substance to be detected is immobilized,
Including the test strip The detection kit according to claim 1, wherein the saccharide is any one or more selected from the group consisting of monosaccharides and disaccharides. The detection kit according to claim 1 or 2, wherein the saccharide and the third antibody supporting part are formed in a line shape on the insoluble carrier so as to be orthogonal to the developing direction of the sample. The insoluble carrier includes at least a sample pad and a membrane pad separate from the sample pad,
The sample pad has a saccharide or third antibody support and a sample supply,
The detection kit according to claim 1, wherein the membrane pad has a detection unit. The detection kit according to any one of claims 1 to 4, wherein the label (1) is colored latex particles or metal colloid particles. The detection kit according to any one of claims 1 to 5, wherein the substance to be detected is hemoglobin, and the first to third antibodies are anti-hemoglobin antibodies. The detection kit according to any one of claims 1 to 6, wherein the saccharide and the third antibody-supporting portion are disposed upstream of the sample supply portion. The detection kit according to any one of claims 1 to 7, wherein the saccharide and the third antibody carrying part are formed at an upstream end of the sample pad. A detection method using immunochromatography, comprising the following steps.
(A) A step of mixing a conjugate reagent in which a first antibody that immunologically reacts to a substance to be detected is immobilized on a label, a specimen, and a specimen diluent, and obtaining a sample comprising these mixtures ( B) Step of dropping the sample obtained in (A) into the sample supply section of the following immunochromatographic test strip and developing it in an insoluble carrier Immunochromatographic test strip;
Wherein comprising an insoluble carrier, the carrier part of a third antibody that binds the upstream and order saccharide and the substance to be detected or the detection target substance and the complex of the conjugate is carried so as to be eluted,
A sample supply section for supplying a sample, and a detection section on which a second antibody that immunologically reacts with a substance to be detected is immobilized,
A step of detecting a complex of a substance to be detected and a conjugate in the test strip (C) sample in a detection unit; The detection method according to claim 9, wherein the saccharide is any one or more selected from the group consisting of monosaccharides and disaccharides. The detection method according to claim 9 or 10, wherein the saccharide and the third antibody supporting part are formed in a line shape on the insoluble carrier so as to be orthogonal to the developing direction of the sample. The insoluble carrier includes at least a sample pad and a membrane pad separate from the sample pad,
The sample pad has a saccharide or third antibody support and a sample supply,
The detection method according to claim 9, wherein the membrane pad has a detection unit. The detection method according to claim 9, wherein the label of (1) is colored latex particles or metal colloid particles. The detection method according to claim 9, wherein the substance to be detected is hemoglobin, and the first to third antibodies are anti-hemoglobin antibodies. The detection method according to any one of claims 9 to 14, wherein the saccharide and the third antibody-supporting portion are disposed upstream of the sample supply portion. The detection method according to any one of claims 9 to 15, wherein the saccharide and the third antibody carrying portion are formed at an upstream end portion of the sample pad. The detection method according to any one of claims 9 to 16, wherein the specimen is feces.

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