JP4437211B2 - Measuring method using immunochromatography and sample analysis tool used therefor - Google Patents

Description

【００４４】
【数１】
Ａ’（ＣＲＰ）＝Ａ（ＣＲＰ）×［Ａ（ウサギ）／Ａ（ウサギ０）］
…（１）
Ａ’（ＣＲＰ） ：補正した吸光度
Ａ（ＣＲＰ） ：λ＝６６０ｎｍの吸光度
Ａ（ウサギ） ：λ＝５００ｎｍの吸光度
Ａ（ウサギ０） ：赤ラテックスの吸光度の理論値
（Ａｂｓ．＝４０００）
【００４５】
【表１】

【００４６】
【表２】

【００４７】
【表３】

【００４８】
【表４】

【００４９】
【表５】

【００５０】
表１から表５に示すように、各試料において、補正前の吸光度［Ａ（ＣＲＰ）］のばらつき（ＣＶ）は大きかったが、補正を行うことにより、補正後吸光度［Ａ’（ＣＲＰ）］のばらつき（ＣＶ）は小さくなった。この結果から、本発明の測定方法によれば、測定精度が向上し、免疫クロマトグラフィーにおける定量分析が可能になるといえる。
【００５１】
【発明の効果】
以上のように、本発明の免疫クロマトグラフィーを用いた測定方法および検体分析用具は、定量分析が可能であり、また検出や定性分析の精度の問題も解決できる。
【図面の簡単な説明】
【図１】本発明の検体分析用具の一実施例の構成を示す斜視図である。
【図２】（Ａ）、（Ｂ）および（Ｃ）は、前記実施例の測定部における抗原抗体反応の状態を説明する模式図である。
【図３】（Ａ）および（Ｂ）は、本発明の検体分析用具のその他の実施例の測定部における抗原抗体反応の状態を説明する模式図である。
【図４】（Ａ）は、本発明の検体分析用具のさらにその他の実施例の上面図であり、（Ｂ）は、その断面図である。
【符号の説明】
１ 検体分析用具
２ 展開する検体の先端
４ 被測定物
５ 検体
１１ 展開層
１２ 血球分離部
１３ 試薬部
１４ 測定部
１５ 液吸収材
１６ 支持体
１７ 検体添加部
１８ 検出ライン
３１ 固定化された第二の抗体
３２ 第一の標識化抗体
３３、３５ 標識
３４ 第三の標識化抗体
３６ 固定化された第四の抗体
ａ、ｂ 矢印[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a measurement method capable of quantitative analysis using immunochromatography and a sample analysis tool used therefor.
[0002]
[Prior art]
Immunochromatography is a measurement method using an immune reaction (antigen-antibody reaction) and is used for detection, qualitative analysis or semi-quantitative analysis of various substances. In immunochromatography, after contacting a specimen with a first labeled antibody against the analyte, the specimen is developed in the development layer by capillary action, and the analyte is immobilized on the development layer. The complex of the analyte and the first labeled substance is captured by the second antibody, and the analyte is measured by measuring the first labeled antibody in the captured complex. It is a method. As the label, usually metal colloid or colored latex particles are used. In some cases, an enzyme is used as a label, and a substrate that changes to a substance detectable by an enzyme reaction is allowed to act on the enzyme.
[0003]
On the other hand, specimen analysis tools (also referred to as test pieces) in which a reagent film or the like is arranged on a plate-like support are widely used in many hospitals and laboratories because they can easily process a large number of specimens. Specimen analysis tools that apply graphy have been developed and are commercially available. For example, an analytical tool using an antibody against human ciliary gonadotropin (hCG) excreted in urine during pregnancy is commercially available for pregnancy diagnosis. In addition, the sample analysis tool to which immunochromatography is applied includes a reagent part containing a labeled antibody against the object to be measured, a developing layer using a nitrocellulose filter, etc., and a measuring part formed in the middle of the developing layer ( In general, the immobilized antibody is disposed on a plate-like support. Specific examples of such a sample analysis tool include, for example, US Pat. No. 4,446,232, Japanese Patent Application Laid-Open No. 55-15100, Japanese Patent Application Laid-Open No. 58-76763, Japanese Patent Application Laid-Open No. 4-64062, and Japanese Patent Application Laid-Open No. There are specimen analysis tools described in JP-A-281231, JP-A-8-94618, JP-A-8-240591, JP-A-8-285850, and the like.
[0004]
Although the sample analysis tool using such immunochromatography can detect and qualitatively analyze the object to be measured, quantitative analysis is difficult and semi-quantitative analysis is limited. In addition, there are cases where there is a problem in accuracy in the sample analysis tool to which immunochromatography is applied in detection and qualitative analysis.
[0005]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a measurement method using immunochromatography that can perform quantitative analysis of an object to be measured and can perform detection and qualitative analysis with high accuracy, and a sample analysis tool used therefor.
[0006]
[Means for Solving the Problems]
In order to achieve the above object, the measurement method using immunochromatography according to the present invention comprises contacting a specimen with a first labeling substance that specifically binds to the analyte, and then placing the specimen in a development layer. A complex of the object to be measured and the first labeling substance is formed by a second substance that is developed by capillary action and binds to the object to be measured fixed to the measurement part formed in the middle of the development layer. In the method of measuring an object to be measured by capturing and measuring the first labeling substance in the captured complex, a third labeling substance is developed together with a specimen, and the first labeling is performed. In this method, the label of the substance and the label of the third labeling substance can be distinguished from each other, and the measurement of the third labeling substance is also performed in the measurement unit.
[0007]
In the measurement method using immunochromatography, quantitative analysis is difficult, and in some cases, the accuracy of detection and qualitative analysis deteriorates because the development of labeled substances (conjugates) and specimens in the development layer is uneven. Due to being. For this reason, in the measurement part (antibody etc. fixed) formed in the middle of the development layer, the concentration of the complex of the measurement object and the labeled antibody etc. to this becomes non-uniform. As a result, the signal intensity (absorbance, etc.) from the label becomes non-uniform and quantitative analysis becomes difficult, which causes a decrease in the accuracy of detection and qualitative analysis. Therefore, in the measurement method of the present invention, the third labeling substance (standard substance) is developed together with the first labeling substance, and the measurement of the third labeling substance is also performed in the measurement unit. In this way, even if the development of the labeling substance or the sample is not uniform, the measurement value of the third labeling substance can be taken as a reference, thereby enabling quantitative analysis. For example, the measurement object can be quantified from the ratio of the measurement value of the first labeling substance to the measurement value of the third labeling substance. Further, according to the measurement method of the present invention, since the reference is clear, there is no possibility of a decrease in accuracy of detection or qualitative analysis.
[0008]
In the measurement method of the present invention, it is preferable that the third labeling substance binds to the second substance immobilized on the measurement unit. Thereby, since the third labeled substance can be captured in the measurement unit, the reference becomes clearer.
[0009]
In the measurement method of the present invention, it is preferable that a fourth substance is immobilized on the measurement unit, and the third labeled substance is bound to the immobilized fourth substance. In this way, by immobilizing the fourth substance that captures only the third labeled substance, the third labeled substance can be captured more efficiently in the measurement unit, so that the standard is further increased. It becomes possible to clarify. The third labeled substance may be bound to the immobilized second substance and the immobilized fourth substance, or may be bound only to the immobilized fourth substance. May be.
[0010]
In the measurement method of the present invention, the first labeled substance is a first labeled antibody, the immobilized second substance is a second antibody immobilized, and the third label The activating substance is preferably a third labeled antibody. In addition, when the immobilized fourth substance is included, it is preferable that this is the immobilized fourth antibody.
[0011]
In this case, the first antibody and the second antibody may be the same antibody, but may be different antibodies (for example, derived from a mouse and a rat). The fourth antibody is preferably an antibody against the third antibody.
[0012]
In the measurement method of the present invention, it is preferable that the label of the first labeling substance and the label of the third labeling substance are colored particles having different colors. This is because both the labels can be easily identified by an optical method if the colored particles have different colors.
[0013]
Next, the sample analysis tool of the present invention is a sample analysis tool used in the measurement method of the present invention, and has a reagent part, a development layer, and a measurement part formed in the middle of the development layer, A second substance that binds to the object to be measured is immobilized on the measurement part, and the reagent part has a third labeling substance and a first labeling substance that specifically binds to the object to be measured. . The third labeling substance is preferably a substance that binds to the immobilized second substance.
[0014]
In the sample analysis tool of the present invention, a fourth substance may be further immobilized on the measurement unit, and the third labeled substance may be a substance that binds to the immobilized fourth substance. . In this case, the third labeled substance may be a substance that binds to both the immobilized second substance and the immobilized fourth substance, or the immobilized fourth substance. It may be a substance that binds only to the substance.
[0015]
By using the sample analysis tool of the present invention, it is possible to quantitatively analyze the object to be measured, and to perform detection and qualitative analysis with high accuracy.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 shows an example of the sample analysis tool of the present invention. As shown in the figure, in the sample analysis tool 1, a reagent part 13 is formed on one end (left end part in the figure) of the development layer 11, and a blood cell separation part 12 is laminated on the reagent part 13. A liquid absorbing material 15 is disposed on the other end (right end portion in the figure) of the spread layer 11. A measuring unit 14 is formed in the middle of the development layer 11.
[0017]
The size of the sample analysis tool 1 is generally 5 to 200 mm in total length, 1 to 50 mm in width, 0.5 to 30 mm in thickness, and 0.1 to 50 mm in width of the measurement unit.
[0018]
Although not shown, the spread layer 11 is usually formed on a support. As said support body, what was formed from polystyrene, polyester, cellulose acetate etc. can be used, for example, As the shape, any of film shape, sheet shape, and plate shape may be sufficient. In addition, when the measurement in the measurement unit is performed by irradiating light from the back side (the side opposite to the development layer arrangement side) of the support, the support is preferably transparent, specifically, transparent polyethylene terephthalate. (PET) is preferred. In addition, a porous membrane is usually used for the spreading layer, and for example, a cellulose membrane, a cellulose derivative membrane such as cellulose acetate or nitrocellulose, a glass filter, a filter paper, and the like can be used. The development layer can be formed on the support by a conventional method. For example, the porous film may be fixed on the support using a double-sided tape or an adhesive.
[0019]
The blood cell separation unit 12 is an arbitrary component provided when, for example, the analysis target is a plasma component in whole blood and it is necessary to remove the blood cells. Therefore, in the case of a sample such as urine that does not require separation of blood cells, it is not necessary to provide the blood cell separation unit 12. A glass filter or the like is usually used for the blood cell separation unit.
[0020]
In the reagent part 13, the first labeled antibody and the third labeled antibody are usually arranged as reagents. As shown in FIG. 2A, the second antibody 31 immobilized is disposed in the measurement unit 14. In FIG. 2, the same parts as those in FIG. The antibody is not particularly limited, and may be any of immunoglobulin (Ig) G, IgA, IgM, IgE, and IgD. In addition, these antibodies may be either polyclonal or monoclonal. The antibody that binds to the analyte can be prepared by an ordinary method using animals such as mice, rats, and goats. An antibody that binds to the immobilized second antibody can also be prepared by a conventional method.
[0021]
The second antibody 31 can be immobilized on the measurement unit 14 of the development layer 11 by a conventional method.
[0022]
The label of the antibody is not particularly limited, and gold colloid, colored latex particles, radioactive label, fluorescent dye label, enzyme label and the like can be used. However, it is necessary that the label of the first labeled antibody and the label of the third labeled antibody can be distinguished from each other. Accordingly, it is preferable that the labels have different colors from each other (absorption spectra do not overlap) or are optically distinguishable. Examples of such a combination of labeling substances include gold colloid and colored latex particles (for example, blue latex particles), but are not particularly limited as long as the two labeling substances can be optically distinguished. The labeling of the antibody can be performed by a conventional method. For example, when an antibody and the gold colloid are mixed in a buffer solution, the antibody is physically adsorbed on the gold colloid or the like.
[0023]
In the present invention, the reagent part 13 may be formed separately from the development layer 11 as shown in FIG. 1, or may be formed in the development layer. When the reagent part is formed separately from the spreading layer, a filter paper or the like may be impregnated with the reagent and disposed on one end of the spreading layer as shown. Further, when the reagent part is formed in the development layer, the predetermined part of the development layer may be impregnated with the reagent. Moreover, the reagent part may be one or plural.
[0024]
In addition to the antibody, the reagent to be arranged in the reagent part is a hemolytic reagent such as a surfactant, a pH adjusting agent such as a buffering agent, or a blocking agent such as casein, depending on the type of specimen or the type of analyte. Etc. may be arranged.
[0025]
The liquid absorbing material 15 disposed on the other end of the development layer 11 is for assisting the capillary phenomenon in the development layer and holding the specimen that has flowed through the measurement unit 14. As the liquid absorbent material, filter paper or the like can be used.
[0026]
Next, based on FIG. 1 and FIG. 2, the measurement using the sample analysis tool will be described with an example in which whole blood is used as a sample and a specific component in plasma is a measurement target. In this sample analysis tool, a first labeled antibody against a specific component (measurement object) in plasma and a third labeled antibody against an immobilized second antibody are arranged in the reagent part 13, A second antibody against a specific component in the plasma is immobilized on the measurement unit 14. The label of the antibody is colored latex particles having different colors.
[0027]
First, as shown by an arrow a in FIG. 1, whole blood 5 is dropped on the blood cell separator 12. Then, only the plasma component in the whole blood moves to the reagent part 13 and contacts with the reagent here. By this contact, a specific component in plasma and the first labeled antibody form a complex. Next, as shown by the arrow b, the plasma component develops in the development layer 11. At this time, as indicated by the wavy line 2, the development of the composite or the like becomes uneven. In this development, the third labeled antibody is similarly developed. And the said composite_body | complex which reached | attained the measurement part 14 is capture | acquired by the 2nd antibody 31 fixed as shown in FIG.2 (B). In the figure, 4 indicates a specific component in plasma, 32 indicates a first labeled antibody, and 33 indicates colored latex particles. In addition, the third labeled antibody developed together with the complex is also captured by the immobilized antibody 31 when it reaches the measurement unit 14 as shown in FIG. In addition, the third labeled antibody may be captured by the second antibody 31 capturing the complex. In the figure, 34 indicates the third labeled antibody, and 35 indicates colored latex particles. FIG. 2 schematically shows the antigen-antibody reaction, and the size of the label is different from the actual one. Then, the measurement unit 14 is irradiated with light to measure the absorbance of each of the two colored latex particles. From these absorbance measurements, the amounts of the first labeled antibody and the third labeled antibody are determined, and the amount of the first labeled antibody is determined based on the amount of the third labeled antibody. Quantify the amount of a specific component (substance to be measured) in plasma.
[0028]
In the present invention, the sample to be measured is preferably liquid, but is not limited thereto. This is because even a solid material can be developed if dissolved or dispersed in a liquid such as a buffer solution.
[0029]
Next, in the sample analysis tool of the present invention, in the measurement unit 14 shown in FIG. 1, in addition to the immobilized second antibody, the immobilized fourth antibody that binds to the third labeled antibody. An example in which the antibody is arranged is shown in FIG. 3, the same parts as those in FIGS. 1 and 2 are denoted by the same reference numerals. FIG. 3 schematically shows the antigen-antibody reaction, and the size of the label is different from the actual one.
[0030]
As shown in FIG. 3 (A), the immobilized second antibody 31 and the immobilized fourth antibody 36 are arranged in the measurement part of the development layer 11. Then, when the third labeled antibody developed together with the complex reaches the measuring unit 14, it is captured by the immobilized fourth antibody 36 as shown in FIG. Other than this, the measurement is performed in the same manner as in the above example. The fourth antibody 36 is not particularly limited as long as it binds to the third labeled antibody, and can be prepared in the same manner as the other antibodies described above, and can be immobilized by a conventional method. it can.
[0031]
【Example】
Example 1
As shown below, the sample analysis tool of the present invention was prepared, and the human C-reactive protein (human CRP) antigen was measured using the sample analysis tool.
[0032]
(1) Preparation of immobilized antibody membrane Anti-human CRP antibody (mouse-derived: manufactured by OY Mediax, the same hereinafter) as the second antibody, and anti-rabbit IgG antibody (goat-derived: Nordic Immunological Laboratories) as the fourth antibody Were added to physiological saline to a concentration of 1 mg / ml and mixed to obtain a solid-phased antibody solution. This solid-phased antibody solution was directly applied on a nitrocellulose membrane (High Flow Membrane: manufactured by Millipore) using a microdroplet coating machine Biodot 2000 (manufactured by Biodot) at a coating amount of 1 μl / cm. After coating on the wire, it was dried with dry air at 40 ° C. for 30 minutes to produce a solid-phased antibody membrane.
[0033]
(2) Preparation of conjugate pad A blue latex solution (particle size 500 nmφ: manufactured by Bangs) and a red latex solution (particle size 500 nmφ: manufactured by Bangs) having different maximum absorption peaks were prepared. These were added to different 0.1M borate buffers (pH 8.5), and the two kinds of mixed solutions were each centrifuged (15,000 rpm, 10 minutes).
[0034]
Then, 1 ml of an anti-human CRP antibody solution (concentration 1 mg / ml) obtained by mixing the anti-human CRP antibody (derived from mouse) in physiological saline in advance is added to the blue latex precipitate obtained by the centrifugation and stirred. And left at room temperature for 1 hour. This solution was centrifuged (15,000 rpm, 10 minutes), and the resulting precipitate was added to phosphate buffered saline (pH 7.4) containing 1% by weight bovine serum albumin (BSA) and 5% by weight saccharose. A latex-labeled antibody solution A, which is a first labeled antibody, was prepared by dispersing in 1.5 ml.
[0035]
On the other hand, 1 ml of a rabbit-derived IgG solution (concentration 1 mg / ml) in which a rabbit-derived IgG (manufactured by Nordic Immunological Laboratories) is mixed with physiological saline is added to the red latex precipitate obtained by the above centrifugation and stirred. And left at room temperature for 1 hour. To this mixed solution was further added 1 ml of a 4 wt% blocking agent (Block Ace: Snow Brand) solution, stirred, and allowed to stand at room temperature for 1 hour. The precipitate obtained by centrifuging this solution (15,000 rpm, 10 minutes) was dispersed in 1.5 ml of phosphate buffered saline (pH 7.4) containing 1% by weight of BSA. Latex labeled antibody solution B, which is a third labeled antibody, was prepared. The composition of the phosphate buffered physiological saline is shown below.
[0036]
(Phosphate buffered saline composition)
NaCl 8.0 g / liter KCl 0.2 g / liter Na ₂ HPO ₄ · 12H ₂ O 2.9 g / liter KH ₂ HPO ₄ 0.2 g / liter
These two types of latex-labeled antibody solutions A and B were mixed at a volume ratio of 80:20 to obtain a conjugate solution. Then, a glass filter (product number AP25: manufactured by Millipore) was immersed in the vat containing the conjugate solution and impregnated, and then lyophilized to prepare a conjugate pad.
[0038]
(3) Preparation of specimen analysis tool Glass filter (Product No. AP25: manufactured by Millipore), solid-phased antibody membrane, conjugate pad, filter paper (Product No. AP10: manufactured by Millipore) and PET film (Tetron film U- 22: manufactured by Teijin Ltd.) was used to prepare a sample analysis tool as shown in FIG. FIG. 4A is a top view of the sample analysis tool, and FIG. 4B is a cross-sectional view taken along the II direction of the top view. In the figure, the same parts as those in FIGS. As shown in the figure, the developing layer 11 (immobilized antibody membrane) having an immobilized antibody is disposed on the center of the support 16 (the PET film), and one end of the developing layer 11 (left side in the figure). The reagent part 13 (conjugate pad) was arranged on the support 16 so as to cover the end part. Then, the specimen addition part 17 was formed by arranging the glass filter so as to cover the entire surface of the reagent part 13 and one end (left end part in the figure) of the support 16. Further, the liquid absorbent 15 (the filter paper) is formed so as to partially overlap the other end (right end in the figure) of the spreading layer 11 and to cover the other end (right end in the figure) of the support 16. ) Was placed.
[0039]
The sample analysis tool has a total length of 59 mm, a width of 7 mm, a maximum thickness of 550 μm, and a minimum thickness of 370 μm. The support 16 has a length of 59 mm, a width of 7 mm, and a thickness of 250 μm. The specimen adding portion 17 has a length of 18 mm, a width of 7 mm, and a thickness of 300 μm. The reagent part 13 has a length of 5 mm, a width of 7 mm, and a thickness of 100 μm. The spreading layer 11 has a length of 25 mm, a width of 7 mm, and a thickness of 120 μm. The liquid absorbent 15 has a length of 18 mm, a width of 7 mm, and a thickness of 200 μm.
[0040]
Using the thus prepared sample analysis tool, human CRP antigen was detected as follows.
[0041]
(Preparation of sample solution)
A 20 M Tris-HCl buffer (pH 7.0) containing 0.1 wt% Tween-20 (manufactured by Nacalai Tesque) and 1 wt% BSA was prepared in advance. Then, five samples were prepared by adding human CRP antigen to this buffer at five concentrations (50 μg / liter, 10 μg / liter, 15 μg / liter, 25 μg / liter, 50 μg / liter). Eight specimens were prepared.
[0042]
(Measuring method)
The sample solution is spotted on the sample addition portion of the sample analysis tool, and after 10 minutes, a densitometer (Flying Spot Scanner CS-9000: manufactured by Shimadzu Corporation) is used on the detection line 18 shown in FIG. The absorbance at two wavelengths was measured under the following conditions, and the obtained absorbance curve was integrated to obtain a measured value. Then, this measured value was substituted into the following formula (1), and the corrected absorbance was calculated. These results are shown in Tables 1 to 5 below.
[0043]
(Measurement condition)

[0044]
[Expression 1]
A ′ (CRP) = A (CRP) × [A (rabbit) / A (rabbit 0)]
... (1)
A ′ (CRP): corrected absorbance A (CRP): λ = 660 nm absorbance A (rabbit): λ = 500 nm absorbance A (rabbit 0): theoretical value of red latex absorbance (Abs. = 4000)
[0045]
[Table 1]

[0046]
[Table 2]

[0047]
[Table 3]

[0048]
[Table 4]

[0049]
[Table 5]

[0050]
As shown in Tables 1 to 5, each sample had a large variation (CV) in absorbance [A (CRP)] before correction, but by performing correction, the absorbance after correction [A ′ (CRP)] The variation (CV) of the value was reduced. From this result, it can be said that according to the measurement method of the present invention, the measurement accuracy is improved and quantitative analysis in immunochromatography becomes possible.
[0051]
【The invention's effect】
As described above, the measurement method and the sample analysis tool using the immunochromatography of the present invention can perform quantitative analysis, and can solve the problem of accuracy of detection and qualitative analysis.
[Brief description of the drawings]
FIG. 1 is a perspective view showing the configuration of an embodiment of a sample analysis tool of the present invention.
FIGS. 2A, 2B, and 2C are schematic diagrams illustrating the state of an antigen-antibody reaction in the measurement unit of the example.
FIGS. 3A and 3B are schematic diagrams illustrating the state of an antigen-antibody reaction in a measurement unit of another example of the sample analysis tool of the present invention. FIGS.
4A is a top view of still another embodiment of the sample analysis tool of the present invention, and FIG. 4B is a cross-sectional view thereof.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Specimen analysis tool 2 Specimen tip 4 to be developed 4 Measured object 5 Specimen 11 Developed layer 12 Blood cell separation part 13 Reagent part 14 Measurement part 15 Liquid absorbent 16 Support body 17 Specimen addition part 18 Detection line 31 Second immobilized Antibody 32 of first labeled antibody 33, 35 Labeled 34 Third labeled antibody 36 Immobilized fourth antibody a, b Arrow

Claims (6)

After contacting the specimen with the first labeling substance that specifically binds to the analyte, the specimen is developed in the spreading layer by capillary action,
The second substance that binds to the measurement object immobilized on the measurement unit formed in the middle of the development layer captures the complex of the measurement object and the first labeling substance,
In the method for measuring an object to be measured by measuring the first labeled substance in the captured complex,
A third labeling substance to expand together with the specimen, observed including that performed also measured the third labeling substance in the measuring section,
The label of the first labeling substance and the label of the third labeling substance are distinguishable from each other ,
The measurement of the third labeled substance is immobilized on the measurement unit by the second substance or the fourth substance that can be bound to the third labeled substance and is immobilized on the measurement unit. And measuring the third labeled substance,
A measurement method comprising quantifying an object to be measured from a ratio of a measurement value of the first labeling substance to a measurement value of the third labeling substance . The first labeled substance is the first labeled antibody, the immobilized second substance is the second immobilized antibody, and the third labeled substance is the third labeled antibody. The measuring method according to claim 1 . The first labeled substance is the first labeled antibody, the immobilized second substance is the second immobilized antibody, and the third labeled substance is the third labeled antibody. There, the measurement method of claim 1, wherein the fourth substances immobilized is fourth antibody which is immobilized. The measurement method according to any one of claims 1 to 3 , wherein the label of the first labeling substance and the label of the third labeling substance are colored particles having different colors. It is a sample analysis tool used for the measuring method as described in any one of Claims 1-4 , Comprising: It has a reagent part, an expansion | deployment layer, and the measurement part formed in the middle of the said expansion | deployment layer, The said measurement A second substance that binds to the analyte is immobilized on the part, and the reagent part includes a third labeled substance and a first labeled substance that specifically binds to the analyte Tools. It is a sample analysis tool used for the measuring method as described in any one of Claims 1-4 , Comprising: It has a reagent part, an expansion | deployment layer, and the measurement part formed in the middle of the said expansion | deployment layer, The said measurement A second substance that binds to the fourth substance and the analyte is immobilized on the part, and the reagent part is specific to the third labeled substance and the analyte that bind to the fourth substance. A sample analysis tool having a first labeling substance that binds to the sample.

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