JP2018048818A - Immunochromatography detection kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunochromatography-utilizing detection kit that can, when a conjugate is to be supplied to a test strip in a state of being mixed with a liquid sample, restrain continuous rise of the intensity of color development in the position of detection, cause capture of the complex to be completed in a certain length of time and stop the increase of the intensity of color development, and a detection method for achieving such an effect.SOLUTION: The problem is solved by causing, in an immunochromatography test strip, specific highly viscous material different from the conjugate and from an antibody in the position of detection to be sustained on a sample pad.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a detection kit comprising a conjugate reagent and an immunochromatographic test strip. In particular, the present invention relates to a detection kit in which the test strip is a test strip in which a polymer viscous substance is held on the downstream side of a sample supply section so as to be eluted. 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 exists as a conjugate portion downstream from the sample supply portion of the test strip (typically on the sample pad). 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 it exists as a conjugate chip | tip, if the liquid which filtered the sample with the said chip | tip is dripped at the sample pad of a test strip, the liquid which is a mixture of a sample and a conjugate will be disperse | distributed at once to the whole 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.
In this way, when using a conjugate chip or the like to supply the conjugate to the test strip in a state where the conjugate is mixed with a liquid sample, this is a method that prevents the color intensity at the detection site from continuing to rise for a long time. Did not exist until.

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

  In the present invention, when a conjugate is mixed with a liquid sample and supplied to a test strip using a conjugate chip or the like, the color intensity at the detection site is prevented from continuing to rise for a long time, and is combined at a fixed time. It is an object of the present invention to provide a detection kit and a detection method using immunochromatography that completes the capture at the detection site of the body and stops the enhancement of the color intensity.

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 further complex We conducted an intensive study.
As a result, the flow of the conjugate (complex) to which the antigen is bound is suppressed by allowing a specific high molecular viscosity substance to exist downstream of the sample supply unit, and the capture of unnecessary complex at the detection site is suppressed. The present inventors have found that this can be done and have completed the present invention.
That is, the present invention has the following configuration.
<1> A detection kit using immunochromatography including 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 (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 a sample supply unit for supplying a sample in order from upstream,
A polymer viscous material carrying part on which a polymer viscous material is carried,
And a detection part on which a second antibody that immunologically reacts with the substance to be detected is immobilized,
The detection kit according to <1>, wherein the test strip <2> the viscous polymer substance has a molecular weight of 6000 or more and a viscosity of 4 to 1000 mPa · S.
<3> The detection kit according to <1> or <2>, wherein the polymer viscous substance-carrying part is 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 sample supply part and a polymer viscous substance carrying part,
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 <4>, wherein the polymer viscous substance-carrying part is disposed on the downstream side of the sample supply part of the sample pad.
<6> The detection kit according to any one of <1> to <5>, wherein the label of (1) is a colored latex or a gold colloid.
<7> The detection kit according to any one of <1> to <6>, wherein the substance to be detected is hemoglobin, and the first and second antibodies are anti-hemoglobin antibodies.
<8> A detection method using immunochromatography, which includes 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 sample supply unit comprising the insoluble carrier and supplying a sample in order from upstream;
A detection target in the sample of the test strip (C), comprising: a support part on which a high molecular viscosity substance is supported; and a detection part on which a second antibody that immunologically reacts with the target substance is immobilized. <9> The detection method according to <8>, wherein the detection unit detects a complex of the substance and the conjugate, and <9> the high molecular viscosity substance has a molecular weight of 6000 or more and a viscosity of 4 to 1000 mPa · S.
<10> The detection method according to <8> or <9>, wherein the supporting part of the polymer viscous material is formed in a line shape on the insoluble carrier so as to be orthogonal to the sample development direction.
<11> The insoluble carrier includes at least a sample pad and a membrane pad separate from the sample pad,
The sample pad has a sample supply part and a polymer viscous substance carrying part,
The detection method according to any one of <8> to <10>, wherein the membrane pad has a detection unit.
<12> The detection method according to <11>, wherein the polymer viscous substance supporting unit is disposed on the downstream side of the sample supply unit of the sample pad.
<13> The detection method according to any one of <8> to <12>, wherein the label of (1) is a colored latex or a gold colloid.
<14> The detection method according to any one of <8> to <13>, wherein the substance to be detected is hemoglobin, and the first and second antibodies are anti-hemoglobin antibodies.
<15> The detection method according to any one of <8> to <14>, wherein the specimen is feces.

  In the method for detecting the antigen, which is a substance to be detected in the specimen, by providing the specimen in contact with the conjugate reagent in advance to the sample supply section of the immunochromatography test strip, a high level is provided downstream of the sample supply section of the test strip. Presence of molecular viscous substances suppresses the inflow of excess antigen to the detection site and suppresses the increase in color intensity at the detection site within a certain time, enabling accurate detection in a short time. It was.

It is the graph which showed the color development intensity in a detection line at the time of carrying PEG 2K on a sample pad. Same as for PEG 4K. Same as for PEG 6K. Same as for PEG 20K. Same as for pullulan. Same as for Dextran 40K. Same as for Dextran 200K. Same as for PVP K-25. Same as for PVP K-90. Same as for sucrose and glycerol. 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 as a sample and can be detected using an antigen-antibody reaction, and examples thereof include viruses, bacteria, 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 is:
(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) the substance to be detected is detected by developing the sample. An immunochromatographic test strip with an insoluble carrier,
The insoluble carrier is a sample supply unit for supplying a sample in order from upstream,
A polymer viscous material carrying part on which a polymer viscous material is carried,
And a detection part on which a second antibody that immunologically reacts with the substance to be detected is immobilized,
Including the test strip.
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 portion (sample supply unit) for receiving a sample, absorbs a liquid sample in a state of being molded into the pad, And any substance and form that the detection object can pass 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.

(Polymer viscous material)
The polymer viscous substance to be carried on the test strip of the present invention may be any substance capable of aggregating the antigen-conjugate complex in the sample and controlling the flow to the detection part, and has a viscosity of 4 to 1000 mPa. -S is preferable. This is because if it is less than 4 mPa · S, the ability to aggregate the complex is poor, and if it is greater than 1000 mPa · S, the complex is rapidly aggregated, and the detection section cannot obtain the required sensitivity. The molecular weight is preferably 6000 or more. More preferably, the viscosity is 4 to 1000 mPa · S and the molecular weight is 6000 or more. The viscosity is more preferably 5 to 500 mPa · S, and still more preferably 10 to 200 mPa · S. The molecular weight is more preferably 20,000 or more, and even more preferably 20,000 to 400,000. Specifically, any one or more selected from polyethylene glycol (PEG) having a molecular weight of 6000 or more, pullulan, dextran, polyvinylpyrrolidone (PVP), or the like is preferable.

  In the present invention, when the test strip has a sample pad, the high molecular viscosity substance needs to be carried on the downstream side of the sample supply portion of the sample pad. Further, when the sample supply section and the detection section are provided on the same insoluble membrane without the sample pad, it is desirable that the insoluble membrane is carried on the downstream side of the sample supply section. Further, it is more desirable that the sample is also carried on the downstream side of the sample supply unit and downstream of the sample supply unit and upstream of the overlapping portion with the membrane.

  As a method for supporting a polymer viscous substance on a sample pad or an insoluble membrane in such a manner that it can be eluted, there are a method in which a solution containing a polymer viscous substance is applied to a sample pad or the like, or a sample pad is immersed in these solutions and dried. Can be mentioned.

Prepare a liquid containing the above-mentioned polymer viscous substance at a predetermined concentration, and use a device that has a mechanism that can move the liquid in the 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. It is preferable to adjust the concentration of the polymer viscous substance solution so as to obtain the above-mentioned preferable viscosity. In addition, the loading amount of the polymer viscous substance on the sample pad or the insoluble membrane carrier can be optimized by adjusting the discharge speed from the nozzle of the above apparatus in the case of the lateral flow type, and is 1 to 20 μL / cm. Preferably, 5 to 10 μL / cm is more preferable.
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 viscous polymer of the present invention is probably due to the reaction between the sample and the conjugate that circulates upstream, and these complexes aggregate and do not flow further downstream. The downstream supply is stopped. In other words, since the complex is captured at the polymer viscous material carrying site, only the portion that has passed without being captured is bound by the detection unit. Therefore, it seems that the coupling | bonding in a detection part 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 (registered trademark), 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 and second antibodies that immunologically react with the substance to be detected used in the present invention are antibodies that can bind to the substance to be detected, and are immobilized on a label and a detection unit described later.
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.

[Embodiment 1] Effect confirmation test of the present invention The control effect in the detection reaction of hemoglobin was confirmed by including a polymer viscous substance in the downstream of the sample supply portion 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 sintered filter was impregnated with conjugate (colored latex-labeled anti-hemoglobin antibody) and GSA (Goat Serum Albumin) -sensitized latex as a control reagent and dried to prepare a conjugate chip.

2. Production of test device (1) Production of sample pad of the present invention 10 mM phosphate buffered saline solution (pH 7.2) containing the following high molecular viscosity substances at concentrations of 1%, 5% and 10% (hereinafter referred to as PBS) Solution) is applied to the surface of the sample pad downstream of the sample supply part ((g) in FIG. 11) and dried, and the sample pad of the present invention having a polymer viscous substance supporting part is dried. Produced. A sample pad in which only a PBS solution containing no polymer viscous substance was applied and dried was also prepared. In addition, a sample pad was prepared by replacing the solution containing the polymer viscous substance with sucrose (33%) and glycerol (30%). Table 1 shows the results of measuring the viscosity (mPa · s) of each polymer viscous substance and each concentration of the polymer substance using a viscometer.
<Polymer viscous material>
PEG 2K (Wako Pure Chemical Industries)
PEG 4K (Wako Pure Chemical Industries)
PEG 6K (Wako Pure Chemical Industries)
PEG 20K (Wako Pure Chemical Industries)
Pullulan (produced by Hayashibara Biochemical Research Institute)
Dextran 40K (Wako Pure Chemical Industries)
Dextran 200K (Wako Pure Chemical Industries)
PVP K-25 (Wako Pure Chemical Industries)
PVP K-90 (Wako Pure Chemical Industries)

(2) Production of Test Strip FIG. 11 shows a schematic configuration diagram of the immunochromatographic test strip of the present invention.
The antibody-immobilized membrane (b) is attached to the plastic backing sheet (a), and the sample pad (e) of the present invention having the high-viscosity substance supporting portion (d) prepared in (1) above is mounted thereon, An absorbent pad (f) was arranged 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 supply portion of the sample pad from the sample addition window portion of the test device. The color intensity of the line was measured and digitized at each time point of 2.5, 5, 7.5, 10, 15, 20, and 30 minutes after the dropping. 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 and discussion The results are shown in FIGS.
3 and 8, PEG 6K and PVP K-25 showed a slight hemoglobin detection reaction inhibitory effect at a concentration of 10% (viscosity: 5.44, 4.36).
In particular, from FIG. 4, FIG. 5, FIG. 7, and FIG. 9, PEG 20K is 5% (viscosity 4.14), 10% (viscosity 11.4), pullulan is 5% (viscosity 14.0), 10% ( Viscosity 86.8), Dextran 200K 5% (viscosity 4.34), 10% (viscosity 13.0), PVP K-90 5% (viscosity 30.3), 10% (viscosity 160.0) Thus, the detection reaction suppressing effect of hemoglobin was observed.
On the other hand, FIG. 1, FIG. 2, FIG. 6 show that PEG 2K, PEG 4K, and dextran 40K have any concentration (viscosity: 1.0 to 2.14, 1.03 to 2.68, 1. 11 to 3.85), the detection reaction inhibitory effect of hemoglobin was not recognized.
Further, from FIG. 10, the sucrose and glycerol concentrations of 30% or higher (viscosity of 3.27 and 2.08, respectively) did not show the effect of suppressing the detection reaction of hemoglobin.
From this, it was found that the viscosity of the polymer viscous substance is required to be 4.0 or more in order to obtain the detection reaction suppressing effect.

  In the method of detecting the antigen, which is a substance to be detected in the specimen, by providing the specimen in contact with the conjugate reagent in advance to the sample supply section of the immunochromatography test strip, the downstream side of the sample supply section of the test strip. In the presence of a specific high molecular viscosity substance, the enhancement of the color intensity at the detection site can be suppressed within a certain time, and the reaction can be controlled.

(A) Backing sheet (b) Antibody-immobilized membrane (c1) Test line (c2) Control line (d) Polymer viscous material carrier (e) Sample pad (f) Absorption pad (g) Sample supply unit

Claims (15)

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 a sample supply unit for supplying a sample in order from upstream,
A polymer viscous material carrying part on which a polymer viscous material is carried,
And a detection part on which a second antibody that immunologically reacts with the substance to be detected is immobilized,
Including the test strip The detection kit according to claim 1, wherein the high molecular viscosity substance has a molecular weight of 6000 or more and a viscosity of 4 to 1000 mPa · S. The detection kit according to claim 1 or 2, wherein the polymer viscous substance supporting part is 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 sample supply part and a polymer viscous substance carrying part,
The detection kit according to claim 1, wherein the membrane pad has a detection unit. The detection kit according to claim 4, wherein the polymer viscous substance carrying portion is disposed on the downstream side of the sample supply portion of the sample pad. The detection kit according to any one of claims 1 to 5, wherein the label of (1) is colored latex or gold colloid. The detection kit according to any one of claims 1 to 6, wherein the substance to be detected is hemoglobin, and the first and second antibodies are anti-hemoglobin antibodies. 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 sample supply unit comprising the insoluble carrier and supplying a sample in order from upstream;
A detection target in the sample of the test strip (C), comprising: a support part on which a high molecular viscosity substance is supported; and a detection part on which a second antibody that immunologically reacts with the target substance is immobilized. A step of detecting a complex of a substance and a conjugate in a detection unit The detection method according to claim 8, wherein the high molecular viscosity substance has a molecular weight of 6000 or more and a viscosity of 4 to 1000 mPa · S. The detection method according to claim 8 or 9, wherein the polymer viscous substance-supporting portion is formed in a line shape on the insoluble carrier so as to be perpendicular to the sample development direction. The insoluble carrier includes at least a sample pad and a membrane pad separate from the sample pad,
The sample pad has a sample supply part and a polymer viscous substance carrying part,
The detection method according to claim 8, wherein the membrane pad has a detection unit. The detection method according to claim 11, wherein the polymer viscous substance carrying portion is disposed on the downstream side of the sample supply portion of the sample pad. The detection method according to claim 8, wherein the label of (1) is a colored latex or a gold colloid. The detection method according to claim 8, wherein the substance to be detected is hemoglobin, and the first and second antibodies are anti-hemoglobin antibodies. The detection method according to claim 8, wherein the specimen is feces.