JP4109245B2 - Analysis apparatus and analysis method - Google Patents

Description

  The present invention relates to an analysis apparatus and an analysis method for using an immunochromatography method.

  As a qualitative or quantitative (semi-quantitative) method for trace substances contained in biological samples such as blood and urine, an immunological measurement method is widely used because of its high sensitivity. Among these methods, the so-called immunochromatography method using chromatography is easy to operate and requires a short time for the examination. Therefore, it is currently widely used for clinical tests in hospitals and laboratory tests in laboratories. ing.

  As an apparatus using this immunochromatography method, a porous membrane such as a membrane is used, and a flow-through method in which a sample or a reagent flows in a filtration format perpendicular to the membrane surface, or one end along the membrane surface is used. There is a lateral flow system that allows flow from one end to the other. These are widely used as general examinations because they are easy to operate and require a short time for analysis.

  The most common immunochromatography method for detecting an analyte (for example, antigen) is to analyze a specific binding substance (for example, an antibody) with various labels on the analyte on a chromatographic material. A complex (for example, an antibody-antigen complex) of a product and a labeled specific binding substance is formed and confirmed (detected) by various means. In this case, the labeling substance used includes radioisotope elements, enzymes, metal sols, nonmetal sols, colored latex, dyes, pigments, chemiluminescent substances, fluorescent substances, and the like.

  At present, the detection principle that is mainly prevalent in immunochromatography is the sandwich method. This sandwich method is a method in which a specific binding substance for an analysis target is immobilized on a chromatographic material, and the analysis target in the sample solution and the labeled specific binding substance are brought into contact with each other. At that time, the labeled binding substance binds between the immobilized specific binding substance and the analyte to be bound to the immobilized specific binding substance. Therefore, as the amount of the analysis target in the sample solution increases, more labeled specific binding substance binds. . Therefore, the amount of labeled specific binding substance bound is a direct measure of the amount of analyte. For example, when a colored labeling substance is used, the color becomes darker as the amount of the analyte is larger.

  However, among the components to be analyzed, C-reactive protein (CRP) that is an inflammatory marker, serum amyloid A, IgE that is an index of allergic symptoms, urinary albumin that is an index of kidney damage, etc. are usually low concentrations In the measurement of components with a wide dynamic range where the concentration range fluctuates up to a very high concentration when there is an abnormality, if the concentration of the analyte in the sample solution greatly exceeds the measured concentration range of the reagent, the analyte excess Suppresses the formation of the sandwich complex, which may result in a lower apparent measurement value, possibly suggesting an erroneous measurement value whose concentration is within the normal value range. This phenomenon is called a hook phenomenon or a prozone phenomenon, and is a big problem in a measurement system using the principle of the sandwich method.

  JP-A-59-23251 discloses a first binding substance capable of binding to an antigen present in a liquid layer, a labeled third binding substance, and a second binding substance capable of binding to the first binding substance. A measurement reagent kit used as a solid-phased antibody is described. Thereby, it is set as the solid-phased antibody corresponding to all the measurement items.

  However, JP-A-59-23251 is a method limited to the sandwich method and cannot be applied to all items measured immunochemically in a laboratory. For example, T3, T4 It is not suitable for the measurement of haptens such as thyroid hormone, cortisol, estradiol, digitoxin.

  Thus, the sandwich method is suitable for the measurement of an analyte having two different binding sites for specific binding substances such as two different antibodies. On the other hand, however, it has the disadvantage of requiring a great deal of labor in preparation and production of the two types of antibodies.

  On the other hand, a measurement method known as a competitive method of the detection principle is that a specific binding substance for an analyte is immobilized on a chromatographic material, and the analyte in the sample solution is contacted with the labeled analyte or a modified form thereof. It is something to be made. As a result, the free analyte present in the sample solution competes with the labeled analyte or its denatured substance, and a complex formation reaction between the labeled analyte or its denatured substance and the immobilized specific binding substance occurs. The analyte in the sample solution is measured by inhibiting (eg, antigen-antibody reaction). For this reason, in the competitive method, the amount of labeling substance bound to the immobilized substance is an indirect measure of the amount of substance to be measured. The competitive method is based on the measurement principle, and if the concentration of the antigen to be measured contained in the sample solution greatly exceeds the upper limit of the measurement, the complex formation inhibition is not attenuated, so whether the high antigen concentration is low or not. It has a feature that such erroneous measurement values are not given.

  The immunoassay method using the competitive method is the same as the sandwich method in that the qualitative or quantitative (semi-quantitative) of the analyte is determined by the presence or absence of coloring with a colored label. However, it differs from the sandwich method in that it is determined that the analyte is below detection sensitivity when colored as a result, and has a concentration above detection sensitivity when not colored. That is, in the competitive method, when a colored labeling substance is used, the greater the amount of the analyte, the lighter the coloring. Therefore, at the present time when the sandwich method is mainly used in the field of medical care, particularly clinical examination, etc., it may be confusing to adopt the determination by the immunoassay method using the competitive method.

  As an immunoassay method using a competitive method, there are disclosed a flow-through type and a strip type (lateral flow type) in JP-A Nos. 2001-281250 and 7-325085.

  Japanese Patent Application Laid-Open No. 2002-189027 discloses an antigen detection method by a competition method using a combination of labeled antibodies having a plurality of different binding affinities. In this method, when the concentration of the antigen present in the sample is low, only the antibody with high affinity binds to the antigen, and as the antigen concentration increases, the antigen also binds to the binding of the antibody with low affinity. As a result, the detectable antigen concentration range (measurement range) is expanded compared to the conventional competitive method.

  Japanese Patent No. 3424831 discloses a method for measuring the concentration of an analyte that combines a competitive or non-competitive method of the prior art. This method combines the competitive method, which achieves the highest sensitivity when detecting low-concentration samples, with the sandwich method, which is suitable for detecting high-concentration samples. Can be measured over a wide range of concentrations.

Japanese Patent Application Laid-Open No. 8-54393 describes a competition method in which the measurement result based on the signal derived from the activity of the labeling enzyme is directly proportional to the amount of the analyte. In contrast to the conventional competitive method, the amount of labeled analyte is inversely proportional to the amount of analyte, whereas this method measures the amount of labeled analyte that was unreacted in the competitive reaction. Although it is a method, the amount of the labeling substance is an indirect method in which the result is obtained as a measure of the amount of the measuring substance. However, the result of measuring the amount of labeled analyte that could not be reacted is not straightforward when it is intended to measure the amount of analyte. In addition, it is necessary to use a plurality of devices for measurement, which is inconvenient.
JP 2002-189085 A Japanese Patent No. 3424831 JP-A-8-54393

  As described above, although the prozone phenomenon is a problem, the sandwich method is still the mainstream as an immunoassay method because the amount of the analyte is proportional to the intensity of coloring at the time of determination. Accordingly, an object of the present invention is to provide an analysis apparatus capable of performing the same determination as that of the sandwich method while taking advantage of the competitive method that the sample solution does not need to be diluted and the problem of the prozone phenomenon does not occur. And to provide an analysis method.

In the analyzer according to the first aspect of the present invention, the reagent zone and the judgment zone are arranged along the wet development direction of the sample solution, and the reagent A and the reagent B each having different visible labels are separated in the reagent zone. The competing reactant can be generated by the competitive reaction between the analyte in the sample liquid and the reagent B with respect to the reagent A accompanying the wet development of the sample liquid from the reagent area to the judgment area. The reagent C that captures the competitive reactant is immobilized in the determination area, and the analyte in the sample liquid is analyzed based on the degree of overlap of the visible signal from the competitive reactant captured by the reagent C. It is characterized in that the density can be detected.
Reagent A: Labeled a-labeled first specific binding substance in which a first specific binding substance that specifically binds to the analyte in the sample solution is labeled with a visible label a Reagent B; Specific binding to the first specific binding substance Analyte to be analyzed or its denatured product is labeled b-analyzed object or denatured product thereof labeled with a visible label b whose visual signal is different from that of visible label a Reagent C; Specificity of labeled a-labeled first specific binding substance Second specific binding substance capable of specifically binding to the first specific binding substance without impairing

In the analyzer according to the second aspect of the present invention, the reagent zone and the judgment zone are arranged along the wet development direction of the sample solution, and the reagent B and the reagent D respectively having different visible labels are separated in the reagent zone. The elution flow is maintained, and a competitive reactant can be generated by a competitive reaction between the analyte in the sample liquid and the reagent B with respect to the reagent D accompanying the wet development of the sample liquid from the reagent area to the determination area. The reagent E that captures the competitive reaction product is immobilized in the determination area, and the analyte in the sample liquid is analyzed based on the degree of overlap of the visible signal from the competitive reaction product captured by the reagent E. It is characterized in that the density can be detected.
Reagent B: Analyte that specifically binds to the first specific binding substance or a denatured product thereof Labeled b-analyte or a denatured product thereof labeled with a visible label b that has a different visible signal from the visible label a Reagent D: Labeled a in which a first specific binding substance that specifically binds to the analyte in the sample solution and a third specific binding substance that does not bind to the first specific binding substance and the analyte are labeled a First specific binding substance / third specific binding substance Reagent E; Can bind specifically to the third specific binding substance without impairing the specificity of the first specific binding substance that specifically binds to the analyte in the sample solution Fourth specific binding substance

In the analyzer according to the third aspect of the present invention, the reagent zone and the judgment zone are arranged along the wet development direction of the sample solution, and the reagent B having a visible label is held in the reagent zone so as to be able to elute and flow. Reagent A having a visual label different from that of the reagent B is directly or indirectly fixed to the determination area, and the reagent B and the analyte in the sample liquid accompanying the wet development of the sample liquid from the reagent area to the determination area It is possible to detect the concentration of the analyte in the sample solution based on the degree of overlap of the visible signal due to competitive capture by the reagent A.
Reagent A: Labeled a-labeled first specific binding substance in which a first specific binding substance that specifically binds to the analyte in the sample solution is labeled with a visible label a Reagent B; Specific binding to the first specific binding substance Analyzed analyte or denatured product thereof is labeled b-analyzed analyte or denatured product thereof that is labeled with a visible label b that has a different visible signal from the visible label a

  The invention according to claim 4 is the analyzer according to claim 3, wherein the reagent C is fixed to the determination area, and the reagent A is fixed to the determination area by being coupled to the reagent C. It is characterized by being.

In the analyzer according to claim 5, the reagent zone and the judgment zone are arranged along the wet development direction of the sample liquid, and the reagent B having the visible label is held in the reagent zone so as to be able to be eluted and flowed. The reagent E is immobilized in the area and the reagent D is immobilized in the reagent E. Between the reagent B and the analyte in the sample liquid accompanying the wet development of the sample liquid from the reagent area to the determination area. The concentration of the analyte in the sample liquid can be detected based on the degree of overlap of the visible signal by competitive capture of the reagent D.
Reagent B: Analyte that specifically binds to the first specific binding substance or a denatured product thereof Labeled b-analyte or a denatured product thereof labeled with a visible label b that has a different visible signal from the visible label a Reagent D: Labeled a in which a first specific binding substance that specifically binds to the analyte in the sample solution and a third specific binding substance that does not bind to the first specific binding substance and the analyte are labeled a First specific binding substance / third specific binding substance Reagent E; Can bind specifically to the third specific binding substance without impairing the specificity of the first specific binding substance that specifically binds to the analyte in the sample solution Fourth specific binding substance

  In the analysis apparatus according to the sixth aspect of the present invention, the reagent area and the determination area are arranged along the wet development direction of the sample liquid, specifically bind to the analyte in the sample liquid, and labeled with the visible label a. The labeled a-labeled first specific binding substance is retained in the reagent zone so as to be able to elute and flow, and the analyte or denatured substance capable of specifically binding to the labeled a-labeled first specific binding substance is immobilized in the determination zone. And the label a-modified first specificities between the analyte in the sample liquid and the analyte in the determination area or its denatured substance accompanying the wet development of the sample liquid from the reagent area to the determination area. Competitive capture for the binding substance and a visual signal independent of the competitive capture are simultaneously generated in the determination area, and analysis in the sample liquid is performed based on the degree of overlap between the independent visual signal and the visible label a. Possible to detect the concentration of the object It is characterized in that is.

The invention described in claim 7 is the analyzer according to claim 6, wherein the reagent area and the determination area are arranged along the wet development direction of the sample liquid, and the reagent A and the reagent F respectively have different visible labels. Is held in the reagent zone so as to be capable of elution flow, and the analyte or modified product thereof and the reagent G capable of binding to the reagent F are immobilized in the judgment zone, and the sample liquid from the reagent zone to the judgment zone is immobilized. The independent visual signal can be generated by the combination of the reagent F and the reagent G accompanying the wet development.
Reagent A: Labeled a-modified first specific binding substance in which the first specific binding substance that specifically binds to the analyte in the sample solution is labeled with visible label a Reagent F; Fifth specific binding substance with visible label c Labeled c-labeled fifth specific binding substance Reagent G; Sixth binding substance capable of binding to the fifth binding substance

  The invention according to claim 8 is the analyzer according to claim 6, wherein the independent visual signal is generated in the determination area before the addition of the sample liquid.

  The invention according to claim 9 is the analyzer according to claim 6 or 8, wherein the independent visual signal is generated by the reagent G immobilized in the determination area and the reagent F bound to the reagent G. It is characterized by that.

  The invention according to claim 10 is the analyzer according to claim 6 or 8, wherein the independent visual signal is a pH indicator, a dye, a colored particle or a colored vesicle immobilized on a determination area. It is characterized by.

  Invention of Claim 11 is an analyzer of any one of Claims 1-10, Comprising: The said sample liquid is urine, blood, hemolyzed blood, plasma, serum, saliva, sweat, tears, etc. It is characterized by being an extract of analytes from body fluids, water such as ponds, rivers and industrial water, and other foods and foods.

  The invention according to claim 12 is the analyzer according to any one of claims 1 to 10, wherein the analyte is a hormone, a biochemical messenger, a steroid, a drug, a drug metabolite, a polypeptide, It is selected from the group consisting of proteins, vitamins, alkaloids, monosaccharides, disaccharides or polysaccharides, and haptens.

  The invention according to claim 13 is the analyzer according to any one of claims 1 to 10, wherein the analyte is selected from the group consisting of thyroid hormone, cortisol, estrogen, progesterone or testosterone. It is characterized by being.

  The invention according to claim 14 is the analyzer according to any one of claims 1 to 10, wherein the visible label is selected from the group consisting of a granular label, a dye label, and an enzyme label. It is characterized by.

  The invention according to claim 15 is the analyzer according to claim 14, wherein the particulate label is made of a metal colloid such as gold colloid or selenium colloid, a colored lipid vesicle (ribosome) or vesicle, or colored latex. It is selected from the group consisting of.

  The invention according to claim 16 is the analyzer according to any one of claims 1 to 5, wherein the first specific binding substance and the second specific binding substance are any one of an antigen, an antibody and a receptor. It is characterized by that.

  The invention according to claim 17 is the analyzer according to any one of claims 6 and 7, wherein the fifth specific binding substance and the sixth specific binding substance that can bind to each other are an antigen, an antibody, or a combination thereof. It consists of the combination of these.

  The invention according to claim 18 is the analyzer according to any one of claims 6 and 7, wherein the first specific binding substance is a mouse-derived antibody and can bind to each other. And the sixth specific binding substance is a combination of a goat-derived antibody and an anti-goat rabbit-derived antibody.

  The analysis method of the invention according to claim 19 is a reagent A comprising a labeled a-modified first specific binding substance in which a first specific binding substance that specifically binds to an analyte in a sample solution is labeled with a visible label a, And an analyte to be specifically bound to the labeled a-labeled first specific binding substance or a denatured product thereof, or a labeled b-labeled analyte to be labeled with a visible label b having a visible signal different from the visible label a or a denatured form thereof. A reagent zone that holds the reagent B consisting of a body in a separated state so that it can be eluted, and a second specific capable of specifically binding to the first specific binding substance without impairing the specificity of the labeled a-labeled first specific binding substance The determination area on which the reagent C made of the binding substance is immobilized is arranged so that the sample liquid can be wet-developed, and the reagent solution labeled from the reagent area is added to the label a by the first specific binding substance and the label b. Chemical analysis object The denatured body elutes and infiltrates and expands into the determination area. During this period, the analyte and labeled b-analyzed analyte or its analyte are compared with the labeled a-labeled first specific binding substance due to the presence of the analyte in the sample solution. A change in the visible signal caused by the degree of overlap of the visible label a and the visible label b in the captured competitive reaction is captured by the second specific binding substance in the determination zone. In this method, the concentration of the analyte in the sample liquid is detected.

  The analysis method of the invention according to claim 20 is the first specific binding substance that specifically binds to the analyte in the sample solution, and the third specific binding substance that does not bind to the first specific binding substance or the analyte. A reagent D comprising a labeled a-labeled first specific binding substance / third specific binding substance labeled with a visible label a, and an analyte or a modified form thereof that specifically binds to the first specific binding substance are the visible label a A reagent zone that holds a reagent B consisting of a movable labeled b-analyzed analyte or a denatured form thereof, which is labeled with a visible label b having a different visible signal, in a separated state so that it can be eluted and flowed, and the first specific binding A determination area on which the reagent E made of the fourth specific binding substance that can specifically bind to the third specific binding substance without impairing the specificity of the substance is arranged so that the sample liquid can be wet-developed, Add from reagent area With the sample solution, the labeled a-labeled first specific binding substance / third specific binding substance and the labeled b-labeled analyte or its denatured substance in the reagent zone are eluted and infiltrated into the judgment zone. Because of the presence of the analyte, the analyte and the labeled b-analyte or its denatured substance undergo a competitive reaction with the labeled a-labeled first specific binding substance / third specific binding substance, and the competitive reactant is It is captured by the fourth specific binding substance in the determination zone via the third specific binding substance, and the change in the visible signal caused by the overlapping degree of the visible label a and the visible label b in the captured competitive reaction product causes a change in the sample liquid. It is characterized by detecting the concentration of the analysis object.

  In the analysis method of the invention according to claim 21, the first specific binding substance that specifically binds to the analyte in the sample solution is capable of eluting and flowing the labeled a-labeled first specific binding substance labeled with the visible label a. A reagent area to be held and a determination area in which an analyte or a denatured body thereof capable of specifically binding to the labeled a-labeled first specific binding substance is immobilized so that the sample liquid can infiltrate and expand, The reagent solution labeled a-labeled first specific binding substance in the reagent zone is eluted by the sample solution added from the zone side, infiltrate and expand into the judgment zone, and is fixed to the analyte and judgment zone by the presence of the analyte in the sample solution. When the converted analyte or denatured substance thereof is competitively captured for the labeled a-labeled first specific binding substance, a visible signal independent of competitive capture is simultaneously generated in the determination area, Visible label a and the independent And detecting the concentration of the analyte by a change in the visual signal produced by the degree of overlap Miteki signal.

  According to the present invention, the concentration of the analyte is detected based on the overlapping degree of the visible label of the reagent that has been eluted with the addition of the sample solution and flowed to the determination area or the overlapping degree of the visible signal independent of the visible label. Therefore, it is possible to make the same determination as that of the sandwich method, taking advantage of the characteristic of the competitive method that the problem of the prozone phenomenon does not occur.

  In the specific binding assay, the present invention provides a label that emits a different visual signal, and one of the labels is labeled with a first specific binding substance that specifically binds to an analyte or a denatured form thereof. Using a substance and a labeled b-analyzed analyte or modified product thereof in which the other visible label is labeled on the analyte or a modified product thereof, each labeled product is separated and arranged in the reagent area, and is wetted by the sample solution. The labeled b-analyzed analyte or a modified product thereof (hereinafter referred to as labeled b-analyzed analyte) competes with the analyte in the sample liquid. One feature is that it reacts with the labeled a-labeled first specific binding substance, and the resulting competitive reaction product is captured in the determination zone. Further, depending on the degree of the presence of the visible label a and the visible label b in the competitive reaction product captured in the determination area, a visual signal is obtained in the determination area regardless of the concentration range of the analyte in the sample solution. It is also a feature of the present invention that it depends on the concentration of the analyte.

  The present invention will be specifically described by taking an immunoassay as an example. In the immunoassay, the labeled a-labeled first antibody and the labeled b-labeled antigen respectively labeled with labels having different visual signals are separated from each other without mixing and placed in the reagent zone. When the sample solution is added to this reagent zone, the labeled a-labeled first antibody and the labeled b-labeled antigen become mobile, and an antigen-antibody reaction occurs competitively due to the presence of the antigen in the sample solution, and two complexes (labeled a First antibody-antigen, or labeled a first antibody-labeled b-antigen).

  In the case of the lateral flow method, these complexes are captured by a capture antibody that specifically binds to the first antibody immobilized in the determination zone downstream of the development. Here, the capture antibody has a specificity that does not interfere with the competitive reaction. At that time, when the antigen concentration in the sample solution is low, a labeled a-labeled first antibody-labeled b-labeled antigen complex is formed predominantly and is captured by the capture antibody. On the other hand, when the antigen concentration in the sample solution is high, the labeled a-labeled first antibody-antigen complex is formed predominantly and is captured by the capture antibody.

  Here, since each of the label a and the label b has different visual signals, as a result, when the antigen concentration in the sample solution is low, the visible signal in the determination area is the visible signal where the labels a and b overlap. When the antigen concentration is high, a visible signal in the determination area appears as a visible signal of the label a.

  Specifically, for example, when the label a has a blue visible signal and the label b has a yellow visible signal, and the antigen concentration in the sample solution is low, the visible signal in the determination area is labeled. The blue signal a and the yellow color of the sign b overlap each other, and a green visible signal appears in the determination area. On the other hand, when the antigen concentration is high, the visible signal in the judgment area becomes a visible signal of only the blue color of the label a and appears in the judgment area. As a result, the color tone of the determination area changes from green to blue in correlation with the higher antigen concentration in the sample solution. In this way, the concentration of the analyte in the sample solution can be easily semi-quantified or quantified by changing the color tone.

  As described above, the present invention solves the problem of the conventional competitive method in which the signal itself decreases as the antigen concentration increases, and the negative correlation decreases in color (the antigen concentration increases when the color is light). One feature is that it can be easily and easily discriminated by the degree of mixing of signals (the degree of mixing of two colors).

  Several modifications can be made to the present invention. Below, it demonstrates based on the example of the above-mentioned immunoassay.

  In the above example, the capture antibody immobilized on the determination area specifically binds to the first antibody, but one modified example does not bind to the first antibody and specifically binds to each other. Using two specific binding substances. That is, one of these two specific binding substances is immobilized on the determination zone, and the other is bound to the label a together with the first antibody to form the labeled a-labeled first antibody / specific binding substance. As a result, the first antibody, which is an essential part of the reaction, the antigen in the sample solution, and the labeled b-labeled antigen are not involved in the three-way competitive reaction. A dedicated capturing means for capturing the formed complex is provided. As a result, the same result as the above-mentioned invention is obtained.

  In any of the above methods, when a tester adds a sample solution from the reagent zone side, a competitive reaction and a capture reaction of the complex occur until the judgment zone. A signal appears, but one of the two different visible signals is allowed to exist in the judgment area before the tester adds the sample liquid, that is, in an unused state. There is also a method in which the reaction is started by carrying out and the other visible signal overlaps the already existing visible signal.

  For example, one of them is a labeled a-labeled first antibody and a labeled b-labeled antigen, and in the case of using a capture antibody in the determination area, the labeled a-labeled first antibody and the capture antibody already bound are fixed in the determination area. In which the visible marker a is present in the same area. This can be further simplified by directly immobilizing the labeled a-labeled first antibody in the determination zone without using the capture antibody.

  Still further, in the example using two specific binding substances that do not bind to the first antibody but specifically bind to each other, the labeled a-labeled first antibody / specific binding substance and the specific binding substance in the determination area are used. There is a method in which a pre-bonded one is fixed in a determination area and a visible marker a is present in the same area.

  As described above, in any of the methods in which the visible label a is present from the beginning, the labeled b-labeled antigen is placed in the reagent zone, and the sample solution is added to determine the determination area together with the labeled b-labeled antigen due to the presence of the antigen as the analysis target Infiltrate and develop a competitive reaction in the judgment area. By these, the same result as that in which the visible signal appears in the determination area without the visible signal is obtained.

  In the above invention, the visible label is bound to both the first specific binding substance involved in the competition reaction and the analyte for the competition reaction or a modified product thereof, and the competition reaction is performed by the presence of the analyte in the sample solution. As a result, a labeled a-labeled first antibody-antigen or a labeled a-labeled first antibody-labeled b-labeled antigen complex is formed in the determination area.

  Another feature of the present invention is to use a combination of two specific binding substances that do not affect the competitive reaction and can specifically bind to each other. Specifically, in the specific binding assay, a visible label a and a visible label c, which are labels that emit different visual signals, are prepared, and one of the visible labels a and the analyte in the sample solution is specifically identified. A labeled a-labeled first specific binding substance labeled to the first specific binding substance to be bound, and the other visible label c that does not bind to the analyte and the labeled a-labeled first specific binding substance The labeled c-labeled fifth specific binding substance labeled with the binding substance is used.

  These labeled a-labeled first specific binding substance and labeled c-labeled fifth specific binding substance are arranged in the reagent zone, and when the sample solution is wetted by the addition of the sample solution, the labeled a-labeled first specific binding substance is labeled. The specific binding substance and the labeled c-labeled fifth specific binding substance become mobile and are wet-developed toward the determination area. On the other hand, an analyte or a denatured product that specifically binds to the first specific binding substance and a sixth specific binding substance that specifically binds only to the fifth specific binding substance are immobilized in the determination area. ing.

  Here, when the sample solution is added to the reagent zone side, the analyte in the sample solution and the analyte immobilized in the judgment zone or a denatured form thereof (hereinafter, referred to as “movable label a-modified first specific binding substance”). As a result, a labeled a-labeled first specific binding substance-immobilized analyte is formed in the determination area according to the concentration of the analyte in the sample solution. Specifically, the amount of the visible label a appears in the determination area as the concentration of the analyte in the sample solution increases.

  On the other hand, the addition of the sample solution simultaneously infiltrates and moves the mobile labeled c-labeled fifth specific binding substance to the determination area, and becomes “independent” regardless of the antigen concentration in the sample liquid and is immobilized in the determination area. It binds to the sixth specific binding substance thus formed, and a labeled c-fifth specific binding substance-immobilized sixth specific binding substance is formed. This is because the complex is formed regardless of the analyte concentration in the sample solution, and a certain amount of visible signal c appears in the determination area. As a result, the visible marker a overlaps a certain amount of the visible marker c. At this time, the degree of presence of the visible label a with respect to the visible label c coupled to the determination area depends on the concentration of the analysis object, and the determination area can be obtained regardless of the concentration range of the analysis object in the sample liquid. A visible signal is obtained.

  The present invention will be described specifically by taking an immunoassay as an example. In the immunoassay, a labeled a mouse-derived antibody and a labeled c goat-derived antibody labeled with a visible label a and a visible label c, each having a different visual signal, are arranged in the reagent zone, An anti-goat IgG antibody that reacts with a goat-derived antibody, and an antigen or a denatured antigen are immobilized. Therefore, when the sample solution is added to the reagent zone side, the labeled a mouse-derived antibody and the labeled c goat-derived antibody become mobile and infiltrate and develop into the judgment zone. The labeled a mouse-derived antibody undergoes a competitive reaction with the antigen in the sample solution and the antigen immobilized in the determination area or the denatured antigen. As a result, a labeled a mouse-derived antibody-immobilized antigen (or denatured antigen) is formed in the determination area according to the antigen concentration in the sample solution.

  On the other hand, the labeled c-goat-derived antibody that becomes mobile simultaneously undergoes an antigen-antibody reaction with the anti-goat IgG antibody immobilized in the determination area, regardless of the presence or absence of the antigen in the sample solution. c-modified goat-derived antibody-immobilized anti-goat IgG antibody) is formed in the determination area. At this time, when the antigen concentration in the sample solution is low, the labeled a mouse-derived antibody-immobilized antigen is predominantly formed in the determination area. On the other hand, when the antigen concentration in the sample solution is high, the labeled a mouse-derived antibody-immobilized antigen is hardly formed. As a result, the amount of visible label a in the determination area decreases as the antigen concentration increases.

  At this time, a labeled c-goat-derived antibody-immobilized anti-goat IgG antibody is simultaneously formed regardless of the antigen concentration in the sample solution. Here, since each visible label a and visible label c have different visual signals, as a result, when the antigen concentration in the sample solution is low, the visible signals in the judgment area are the visual labels a and c. When the antigen concentration is high, the visible signal in the determination area appears as a visible signal of the visible marker c. More specifically, when the visible marker a is a yellow visible signal and the visible marker c is a blue visible signal, in the determination area, the antigen concentration becomes high due to a mixture of yellow and blue on the side where the antigen concentration is low. As it turns blue. As a result, it becomes easy to know the antigen concentration by color discrimination.

  In the above example, the visible label c is generated in the determination area “independently” using a combination of a goat-derived antibody and an anti-goat IgG antibody, but this can also be modified in some ways.

  One of the modifications is, for example, a method in which an antibody-antigen complex and an antibody specific for the antigen are used in combination, and the same result can be obtained. Even without using such a combination method, a substance that emits a visible signal similar to the visible marker b when the determination area is wetted by the addition of the sample solution is immobilized in advance in the determination area. The same result can be obtained by. An example of this is a pH indicator that changes from colorless to colored when the sample liquid infiltrates the determination area. These are those that are not involved in the three-part competitive reaction of the first antibody, which is an essential part of the reaction, the antigen in the sample solution, and the immobilized denatured antigen. Means for simultaneously indicating the coloration of the pH indicator by the sample solution is provided. As a result, the same result as the above-mentioned invention is obtained.

  In the above example, when the tester adds a sample solution from the reagent zone side, the complex reaction until the determination zone and capture of the complex that emits a visual signal independent of the reaction, or pH As a further modification, a visible signal appears in a determination area where there is no visible signal due to a colored substance such as an indicator. However, as a further modification, one of the two different visible signals is tested. Before the person adds the sample solution, that is, in the unused state from the beginning, the reaction is started by carrying out the test, and the other visible signal overlaps the existing visible signal. There is also a method.

  For example, in one of the examples described above, for example, a method of immobilizing a labeled c-labeled goat-derived antibody-anti-goat IgG antibody complex in the determination area in advance, or the labeled c-labeled goat-derived antibody in the determination area. There are a method of immobilizing in advance and a method of immobilizing the visible label c directly in the same manner. By these, the same result as that in which the visible signal appears in the determination area without the visible signal is obtained.

  Examples of the analysis target in the present invention include proteins, glycoproteins, antibodies, enzymes, saccharides, cells, bacteria and viruses, drugs, chemical substances, and the like.

  Among the above analytes are hormones, biochemical messengers, steroids, drugs, drug metabolites, polypeptides, vitamins, alkaloids, etc., especially haptens such as thyroid hormone, cortisol, estrogen, progesterone or testosterone It is done.

  The specific binding substance in the present invention is a substance that specifically binds to a substance having a specific structure, and is an antigen, antibody, nucleic acid sequence fragment, effector molecule, receptor molecule, enzyme and its inhibitor, avidin, biotin, sugar Examples include chain compounds and lectins.

  The signal in the present invention is a color visually confirmed in the visible range. In addition, the bond in the present invention refers to an ionic bond, a covalent bond, a coordinate bond, etc. in physical adsorption and chemical bond.

  Examples of the particulate labeling substance in the present invention include metal colloids such as gold colloids and selenium colloids, colored lipid vesicles (ribosomes), vesicles, and the like, each having a signal. In addition, there are white metal colloids, silica particles, white latex, lipid vesicles (ribosomes) and vesicles that do not have signals themselves, and these bind signal substances (various dyes, pigments, dyes, fluorescent dyes, etc.). Or it has a signal by enclosing.

  The analyzer used in the immunoassay method includes the following structure when exemplified by the lateral flow method. That is, a “sample solution addition zone” for adding a certain amount of sample solution, a “reagent zone”, a “development zone” and a “determination zone” where the reagent is eluted by the sample solution, and a “sample zone” for absorbing the sample solution "Absorption zone". The sample liquid addition area, the reagent area, the development area, the determination area, and the absorption area are sequentially arranged so that the sample liquid added to the sample liquid addition area infiltrates the determination area through the development area.

  A specific binding substance that specifically binds to the labeled specific binding substance is immobilized in the judgment area, and the labeled specific binding substance and the labeled analysis target are in the reagent area in a state in which they can be eluted and flowed by the sample solution. Or a modified product thereof is kept in a separated state.

  When the sample solution is added to the sample solution addition zone, the sample solution begins to infiltrate, and the sample solution is mixed with the labeled analyte or its denatured substance and the labeled specific binding substance carried in the reagent zone. Depending on the concentration of the analyte in the sample solution, a “labeled specific binding substance-analyte” complex or a “labeled specific binding substance-labeled analyte or its modified substance” complex is formed. The formed complex flows together with the sample solution in the development area, reaches the determination area, where it is captured by the immobilized specific binding substance. As a result, the "labeled specific binding substance-analyte-immobilized specific binding substance" complex or the "labeled specific binding substance-labeled analyte or its modified substance-immobilized specific binding substance" complex is judged. Formed in the area. Therefore, the captured “labeled specific binding substance-analyte object—immobilized specific binding substance” complex and “labeled specific binding substance-labeled analyte or modified product thereof—immobilized specific binding substance” complex. The concentration of the analyte in the sample solution can be measured by measuring the amount of the label in the sample.

  Another apparatus of the present invention includes the following structure. That is, a “sample solution addition zone”, “reagent zone”, “development zone”, “determination zone” and “absorption zone” are provided in the same manner as described above, and the sample solution added to the sample solution addition zone passes through the development zone. A sample solution addition area, a reagent area, a development area, a determination area, and an absorption area are sequentially arranged so as to infiltrate the determination area.

  The reagent zone contains a labeled specific binding substance A and a labeled specific binding substance B, and the determination zone binds to the analyte to be bound to the specific binding substance A or a modified form thereof and the specific binding substance B. The specific binding substance C to be immobilized is immobilized. Here, the labeled specific binding substance B and the specific binding substance C do not participate in the competitive reaction system of the labeled specific binding substance A and the analyte or its denatured substance, and emit signals independently. In this case, unlike the analyzer described above, it is not necessary to separate the two types of labeling reagents in the reagent zone.

  When the sample solution is added to the sample solution addition zone, the sample solution begins to infiltrate, and on the one hand, the sample solution is mixed with the labeled specific binding substance A and the labeled specific binding substance B carried on the reagent zone. The competing reactant is obtained in the determination area by flowing through the development area together with the sample liquid due to the presence of the analyte in the sample liquid. On the other hand, a complex “labeled specific binding substance B-immobilized specific binding substance C” is formed regardless of the presence or absence of the analyte in the sample solution, and is independent of the competitive reaction. Send a signal. As a result, the degree of overlap between the visible signal from the competing reactant and the independent visible signal changes according to the concentration of the analyte in the sample solution, and the concentration of the analyte can be measured.

  The material used in the development area and the judgment area is a hydrophilic material, and any material that easily absorbs an aqueous solution such as a sample solution may be used as the hydrophilic material. Any material can be used as long as it can secure and adjust the time required for the body to react sufficiently with the labeled specific binding substance or the immobilized specific binding substance. Specific examples include porous membranes such as nitrocellulose, nylon, and cellulose acetate, crosslinked dextran, cellulose filter paper, and glass fiber paper. Among them, nitrocellulose adsorbs proteins and is easy to immobilize. Cellulose filter paper or the like can be easily fixed by activation with a crosslinking agent or the like in advance.

  A hydrophilic material is also used in the sample solution addition zone, the reagent zone, and the absorption zone, and examples of the hydrophilic material include the same materials as described above. However, the analyte in the sample solution and the reagent that can be used in the present invention. A material that adsorbs a kind is not preferable. However, such adsorption and the like can be prevented by pretreatment with polyvinyl alcohol or other masking agents.

  1 and 2 show a test piece of a lateral flow type apparatus used in the present invention.

  As shown in FIG. 1, the test piece 11 is being fixed on the base_sheet | mounting_paper 1 with which each member has an adhesive surface on one side. The reagent paper 5 is disposed at the lower end (upstream end) of the determination paper 3 with a slight overlap so that the liquid infiltrates and communicates in a capillary manner over the whole. Reagent paper 6 is provided. Further, a sample solution addition member 7 is disposed so as to partially cover the reagent paper 6.

  On the other hand, the absorbent body 2 is disposed at the top (downstream end) of the determination paper 3 with a slight overlap so that the liquid can infiltrate and communicate in a capillary manner. The absorber 2 acts as an absorption pump for the sample liquid flowing from the sample liquid addition member 7 through the reagent paper 6, the reagent paper 5, and then the judgment paper 3 by capillary action. The reagent paper 5 contains a labeled a-labeled first specific binding substance, and the reagent paper 6 contains a labeled b-labeled analyte or a modified product thereof. These reagents can freely move through the determination paper 3 to the absorber 2 by adding a sample solution. A second specific binding substance 10 that specifically binds to the first specific binding substance is immobilized in the determination line section 4 in the determination paper 3.

  FIG. 2 shows the result of using the test piece 11 for analysis. 3, 4, and 5 schematically show the results in the determination line unit 4.

  In FIG. 1, one end of the sample solution addition member 7 of the test piece 11 is brought into contact with a sample solution (not shown) that may contain an analysis object. The sample solution absorbed by the sample solution addition member 7 instantaneously infiltrates the reagent paper 6 and then the reagent paper 5, and accompanying this, the labeled b-analyzed analyte or its denatured product 8 and the labeled a-formed first The specific binding substance 9 is dissolved or dispersed in the sample solution and moves toward the determination paper 3 together with the sample solution.

  When the analyte is present in the sample liquid, the analyte and the labeled b-analyzed analyte or its denatured substance 8 are applied to the labeled a-labeled first specific binding substance 9 while moving toward the determination paper 3. Compete with each other and a competitive reactant is formed. When the competitive reaction product reaches the determination line unit 4, it is captured by the second specific binding substance 10 immobilized thereon, and the determination line unit 4 is colored in a line according to the degree of the presence of the label a and the label b. Is confirmed.

  Here, a labeled a-labeled first specific binding substance-analyte or a labeled a-labeled first specific binding substance-labeled b-analyzed object or a denatured form thereof is formed as the competitive reaction product. For example, when blue latex particles are used as the label a and red latex particles are used as the label b, when the concentration of the analyte in the sample solution is low, the determination line unit 4 indicates “labeled a first specific binding substance (blue)”. A purple line derived from the complex of “labeled b-analyzed analyte or modified product thereof (red)” appears as shown in FIG.

  On the contrary, when the concentration of the analyte in the sample solution is high, a blue line derived from the complex “labeled a first specific binding substance (blue) -analyte” appears in the determination line section 4. FIG. 5 schematically shows.

  When the analyte in the sample solution is present in a moderate amount, the determination line unit 4 displays “labeled a-labeled first specific binding substance (blue) —labeled b-labeled analyte or a modified product thereof (red) ) "And a complex of" labeled a-labeled first specific binding substance (blue) -analyte ", a blue to purple line is formed in the mixed color, as schematically shown in FIG. become.

  6 to 8 are schematic diagrams when the third specific binding substance 25 and the fourth specific binding substance 26 are used in place of the second specific binding substance 10. FIGS. 9 to 11 are schematic diagrams when the labeled a-labeled first specific binding substance 9 is immobilized in advance in the determination area 4.

  FIG. 12 and FIG. 13 are test pieces of another typical lateral flow system used for the analysis according to the present invention, and show the operation principle thereof.

  As shown in FIG. 12, the test piece 23 is fixed on a mount 13 in which each member has an adhesive surface on one side. A reagent paper 17 is disposed at the lower end of the judgment paper 15 with a slight overlap so that the liquid is infiltrated and communicated in a capillary manner, and a sample liquid addition member 18 is provided so as to partially cover the reagent paper 17. It is arranged.

  On the other hand, the absorber 14 is disposed on the top of the judgment paper 15 with a slight overlap so that the liquid is infiltrated in a capillary manner. The absorber 14 acts as an absorbent for the sample liquid flowing from the sample liquid adding member 18 through the reagent paper 17 and then the determination paper 15 by capillary action. The reagent paper 17 contains a labeled a-labeled first specific binding substance and a labeled c-labeled fifth antibody-antigen complex.

  These reagents can freely move to the absorber 14 through the determination paper 15 by adding a sample solution. In the determination line section 16 in the determination paper 15, an analyte to be specifically bound to the labeled a-labeled first specific binding substance or a modified form thereof is immobilized, and a labeled c-labeled fifth antibody-antigen is immobilized. A sixth antibody that binds only to the antigen of the complex is also immobilized.

  FIG. 13 shows a case where the test piece 23 is used for analysis. 14 to 16 schematically show the results.

  In FIG. 12, one end of the sample solution adding member 18 of the test piece 23 is brought into contact with a sample solution (not shown) that may contain an analysis object. The sample solution absorbed in the sample solution addition member 18 instantaneously infiltrates into the reagent paper 17 and is mixed and supported thereon, and the labeled a-labeled first specific binding substance 19 and the labeled c-labeled fifth antibody-antigen complex 20 are supported. Is dissolved or dispersed in the sample solution and moves toward the determination paper 15 together with the sample solution.

  When the analyte is present in the sample solution, it can be bound to the labeled a first specific binding substance 19 while moving toward the determination paper 15. When the determination line unit 16 is reached, the combined complex (labeled a-specific first binding substance-analyte) does not bind to the analyte immobilized on the determination line unit 16 or its denatured product 21 and is labeled. A colored line derived from a is not confirmed.

  Conversely, when there is no analysis target in the sample solution, the labeled a-labeled first specific binding substance binds to the immobilized analysis target in the determination line unit 16 or a denatured product thereof. A colored line derived from the label a is confirmed. On the other hand, the labeled c-labeled fifth antibody-antigen complex 20 that has moved at the same time is combined with the sixth antibody 22 immobilized on the determination line portion 16 to form a complex (labeled c-labeled fifth antibody-antigen-immobilized group 6 antibody complex), and as a result, a colored line derived from the label c is confirmed regardless of the presence or absence of the analyte in the sample solution.

  For example, when red latex particles are used for the label a and blue latex particles are used for the label c, when there is no analyte in the sample, the determination line unit 16 displays “labeled first specific binding substance (red)”. And a purple line derived from the “labeled c-labeled fifth antibody (blue) -antigen” complex appears as shown in FIG. On the other hand, when the concentration of the analyte in the sample solution is high, only the blue line derived from the “labeled c-labeled fifth antibody (blue) -antigen” complex appears in the determination line section 16, schematically. As shown in FIG. When the analyte in the sample solution is present in an intermediate amount, “labeled a-labeled first specific binding substance (red)” and “labeled c-labeled first antibody (blue) -antigen are displayed in the determination line section 16. "The composites are mixed, and the mixed color changes from blue to purple lines, as shown schematically in FIG.

  17 to 19 are schematic diagrams in the case where the pH indicator 28 and the like are immobilized independently in advance.

  FIG. 20 illustrates a summary corresponding to claims 1 to 5, where “No 1” is in claim 1, “No 2” is in claim 2, and “No 3” is in claims 3 and 4. , “No 4” corresponds to claim 3, and “No 5” corresponds to claim 5.

  FIG. 21 illustrates a summary corresponding to claims 6 to 10, wherein “No 6” is claimed in claims 6 and 7, “No 7” is claimed in claims 8 and 9, and “No 8” is claimed. “No 9” corresponds to claims 6 and 10.

  Hereinafter, the present invention will be specifically described based on examples.

[Example 1]
In Example 1, semi-quantitative measurement of estrogen is performed, and the following steps are included.

(1-a) Production of Estrone-17-O (carboxymethyl) oxime Synthesized according to the method described in the publication “Biological Science 38 (5) 376-378 (by Takeshi Tanaka)”.

(1-b) Production of Estrone-BSA Conjugate Synthesized according to the method described in the publication “Biological Science 38 (5) 376-378 (by Kenji Tanaka)”.

(1-c) Production of red latex labeled estrone-BSA 0.1 ml of red latex suspension (solid content weight 10%) having a particle size of about 0.1 μm was placed in a microtube and 25 mM MES (pH 6.1). Add 0.9 ml of buffer and dilute. Centrifugation is performed at 6000 rpm for 30 minutes using a microcentrifuge, and the supernatant is discarded by decantation, leaving the settled particles in a tube. Add 1.0 ml of 25 mM MES (pH 6.1) buffer again to disperse the particles. This washing operation is repeated three more times. The finally obtained settled particles are dispersed by adding 1.0 ml of 25 mM MES (pH 6.1) buffer to obtain a latex particle suspension having about 1% weight solids.

  This suspension is mixed with 700 μg of estrone-BSA obtained in (1-b) above, and the mixed suspension is stirred at room temperature for 4 hours by a rotary mixer. This suspension is centrifuged at 6000 rpm for 30 minutes in a microcentrifuge, and the supernatant is discarded by decantation. The obtained settled latex particles are dispersed in 1.0 ml of 25 mM MES (pH 6.1) buffer containing bovine serum albumin (BSA) at a weight of 0.5%, and stirred with a rotary mixer at room temperature for 1 hour. The suspension was centrifuged at 6000 rpm for 30 minutes with a microcentrifuge, the supernatant liquid was discarded by decantation, and the settled particles were washed with 1.0 ml of 25 mM MES (pH 6.1) buffer by the above washing method. Perform 3 washing operations. Finally, it is dispersed in 25 mM MES (pH 6.1) buffer containing 0.5% BSA and stored at 4 ° C. until use.

(1-d) Production of Blue Latex Labeled Anti-Estradiol Antibody 0.1 ml of blue latex suspension (solid content 10%) having a particle size of about 0.4 μm is taken in a microtube and 25 mM MES (pH 6.1) Add 0.9 ml of buffer and dilute. Centrifugation is performed at 6000 rpm for 30 minutes using a microcentrifuge, and the supernatant is discarded by decantation, leaving the settled particles in a tube. Add 1.0 ml of 25 mM MES (pH 6.1) buffer again to disperse the particles. This washing operation is repeated three more times. The finally obtained settled particles are dispersed by adding 1.0 ml of 25 mM MES (pH 6.1) buffer to obtain a latex particle suspension having about 1% weight solids. This suspension is mixed with 300 μg of the anti-estradiol antibody, and the mixed suspension is stirred with a rotary mixer at room temperature for 4 hours. This suspension is centrifuged at 6000 rpm for 30 minutes in a microcentrifuge, and the supernatant is discarded by decantation.

  The obtained settled latex particles are dispersed in 1.0 ml of 25 mM MES (pH 6.1) buffer containing bovine serum albumin at 0.5% by weight, and stirred at room temperature for 1 hour with a rotary mixer. The suspension was centrifuged at 6000 rpm for 30 minutes with a microcentrifuge, the supernatant liquid was discarded by decantation, and the settled particles were washed with 1.0 ml of 25 mM MES (pH 6.1) buffer by the above washing method. Perform 3 washing operations. Finally, it is dispersed in 25 mM MES (pH 6.1) buffer containing 0.5% BSA and stored at 4 ° C. until use.

(1-e) Preparation of reagent paper 5 and reagent paper 6 1% by weight of each of red latex labeled estrone-BSA and blue latex labeled anti-estradiol antibody obtained in (1-c) and (1-d) above Dilute with 100 mM Tris buffer containing 1% BSA and 2% by weight sucrose. At this time, the red latex labeled estrone-BSA and the blue latex labeled anti-estradiol antibody are both diluted to 10% by volume. 1 ml of the obtained solution was applied to a 10 × 200 mm reagent paper sheet (manufactured by Whatman, F075-17), dried in a hot air dryer at 80 ° C. for 5 minutes, and a bag containing a desiccant at room temperature until use. Save to.

(1-f) Preparation of determination paper 3 A 25 × 200 mm Whatman nitrocellulose membrane (PuraBind, A-RP, 8 μm pore diameter) was fixed on a desk, and an anti-mouse IgG antibody (1 mg / mg) was placed at a position 10 mm from one end. ml) The solution is applied using a dispenser so as to form a line having an application width of about 1.0 mm. After application, it is dried in a hot air dryer at 80 ° C. for 5 minutes and stored at room temperature until used in a bag containing a desiccant. Thereby, the determination paper 3 on which the determination line portion 4 in FIG. 1 is formed is obtained.

(1-g) Preparation of test piece and analytical device A sheet 1 (thickness 250 μm, length 125 mm, width 180 mm, manufactured by Lintec) on one side was fixed on a desk with the adhesive side up, and 30 mm from one end The determination paper 3 is pasted to the position in parallel with one end thereof. Subsequently, the reagent paper 5 is bonded in parallel to the one end at a position 22 mm from the one end. By the bonding, the determination paper 3 and the reagent paper 5 are continuously provided with an overlap of about 2 mm. Next, the reagent paper 6 is bonded in parallel with the one end at a position of 14 mm from the one end. By this bonding, the reagent paper 5 and the reagent paper 6 are continuously provided with an overlap of about 2 mm.

  Furthermore, one end is put together and a sample solution adding member 7 made of Benise (manufactured by Asahi Kasei Co., Ltd.) having a length of 30 mm and a width of 200 mm is bonded so as to partially cover the reagent paper 6. In addition, a glass fiber filter paper of 40 mm length and 200 mm width made of GF-F (manufactured by Whatman) is bonded to the position 32 mm from the other end of the mount 1 in parallel with the other end to make the absorber 2. Here again, the judgment paper 3 and the absorbent body 2 are continuously provided with an overlap of about 2 mm. Furthermore, the other end is put together, and a protective cover 12 made of a transparent ester film (manufactured by Lintec Co., Ltd.) having a length of 110 mm and a width of 200 mm is attached with the adhesive surface side down. The laminated sheet thus laminated is sequentially cut into strips by a rotary cutter at intervals of 6 mm in width so as to cross each member at a right angle. This completes about 28 analyzers and stores them in a desiccant bag at room temperature until used in analytical tests.

(1-h) Analytical Test Method An estradiol-17-glucuronide (manufactured by Sigma, hereinafter referred to as E ₂ 17G) solution diluted to a predetermined concentration (0 to 5000 ng / ml) with a physiological saline solution was prepared as a sample solution. . The analyzer prepared as described above was placed on a horizontal desk, and 100 μl of each concentration of E ₂ 17G solution was added from one end of the sample solution addition member 7 with a micropipette, and the color tone of color development at the judgment line unit 4 was observed. The results are shown in Table 1.

As shown in Table 1, at 0 ng / ml, the judgment line portion shows reddish purple, and as the E ₂ 17G concentration increases in the order of 100 ng / ml, 500 ng / ml, and 2500 ng / ml, the red color tone of the judgment line portion increases. As a result, an overall color change from reddish purple to blue was observed.

[Example 2]
In this example, semi-quantitative measurement of estrogen is performed.

(2-a) Production of Estrone-17-O (carboxymethyl) oxime The product produced in Example 1 was used.

(2-b) Production of Estrone-BSA Combined Product The product produced in Example 1 was used.

(2-c) Production of colloidal gold-labeled anti-estradiol antibody Colloidal gold particles used in the immunoassay are commercially available in various particle sizes and can be easily produced by those skilled in the art.

  The pH of the 40 nm colloidal gold is adjusted to 6.0 with 100 mM aqueous potassium carbonate solution, and 2 ml of anti-estradiol antibody is added to 100 ml of the colloid with stirring. The mixture is slowly stirred for an additional 5 minutes. Subsequently, 5 ml of casein is added to the mixture and similarly stirred slowly for 5 minutes. The resulting mixture is purified by centrifugation at 5500 G for 45 minutes at 4 ° C. The pelleted colloidal gold-labeled anti-estradiol antibody is resuspended in distilled water to 5 ml and stored at 4 ° C. until use.

(2-d) Production of Blue Latex Labeled Anti-β-hCG Mouse Monoclonal Antibody 0.1 ml of blue latex suspension (solid content weight 10%) having a particle size of about 0.3 μm is placed in a microtube, and 25 mM MES ( pH 6.1) Add 0.9 ml of buffer and dilute. Centrifugation is performed at 6000 rpm for 30 minutes using a microcentrifuge, and the supernatant is discarded by decantation, leaving the settled particles in a tube. Add 1.0 ml of 25 mM MES (pH 6.1) buffer again to disperse the particles. This washing operation is repeated three more times. The finally obtained settled particles are dispersed by adding 1.0 ml of 25 mM MES (pH 6.1) buffer to obtain a latex particle suspension having about 1% weight solids. This suspension is mixed with 700 μg of anti-β-hCG mouse monoclonal antibody, and the mixed suspension is stirred by a rotary mixer at room temperature for 4 hours. This suspension is centrifuged at 6000 rpm for 30 minutes in a microcentrifuge, and the supernatant is discarded by decantation.

  The obtained settled latex particles are dispersed in 1.0 ml of 25 mM MES (pH 6.1) buffer containing bovine serum albumin at 0.5% by weight, and stirred at room temperature for 1 hour with a rotary mixer. The suspension was centrifuged at 6000 rpm for 30 minutes with a microcentrifuge, the supernatant liquid was discarded by decantation, and the settled particles were washed with 1.0 ml of 25 mM MES (pH 6.1) buffer by the above washing method. Perform 3 washing operations. Finally, it is dispersed in 25 mM MES (pH 6.1) buffer containing 0.5% BSA and stored at 4 ° C. until use.

(2-e) Production of “blue latex labeled β-hCG mouse monoclonal antibody-hCG” complex 1.5 ml of 500 IU hCG solution was placed in a microtube, and the blue latex labeled β obtained in (2-d) above was obtained. Add 100 μl of hCG mouse monoclonal antibody and suspend. The suspension is stirred with a rotary mixer for 30 minutes at room temperature. This suspension is centrifuged at 12000 rpm for 30 minutes in a microcentrifuge, and the supernatant is discarded by decantation. The obtained settled particles are washed three times with 1.0 ml of 20 mM PB (pH 7.4) buffer by the above washing method. Finally, it is dispersed in 25 mM HEPES (pH 7.0) buffer containing 0.5% BSA and stored at 4 ° C. until use.

(2-f) Preparation of reagent paper 17 1 of the colloidal gold-labeled anti-estradiol antibody and blue latex-labeled β-hCG mouse monoclonal antibody-hCG complex obtained in (2-c) and (2-e) above Dilute with 100 mM Tris buffer containing wt% BSA and 2 wt% sucrose. At this time, the gold colloid-labeled anti-estradiol antibody and the “blue latex labeled β-hCG mouse monoclonal antibody-hCG” complex are both diluted to 10% by volume. 1 ml of the obtained solution was applied to a 10 × 200 mm reagent paper sheet (manufactured by Whatman, F075-17), dried in a hot air dryer at 80 ° C. for 5 minutes, and a bag containing a desiccant at room temperature until use. Save to.

(2-g) Preparation of determination paper 15 A 25 × 200 mm Whatman nitrocellulose membrane (PuraBind, A-RP, 8 μm pore diameter) was fixed on a desk, and a β-hCG mouse monoclonal antibody solution was placed at a position 10 mm from one end. And a mixed solution of estrone-BSA solution (0.5 mg / ml) are applied using a dispenser so as to form a line having a coating width of about 1.0 mm. After application, it is dried in a hot air dryer at 80 ° C. for 5 minutes and stored at room temperature until used in a bag containing a desiccant. As a result, the determination paper 15 on which the determination line portion 16 in FIG. 12 is formed is obtained.

(2-h) Preparation of test piece and analyzer An adhesive sheet 13 (thickness 250 μm, length 125 mm, width 180 mm, manufactured by Lintec Co., Ltd.) on one side was fixed on a desk with the adhesive side up, and 30 mm from one end The determination paper 15 is pasted to the position in parallel with one end thereof. Subsequently, the reagent paper 17 is bonded in parallel with the one end at a position 22 mm from the one end. By the bonding, the determination paper 15 and the reagent paper 17 are continuously provided with an overlap of about 2 mm. Furthermore, one end is put together, and a sample solution addition member 18 made of Benrize (manufactured by Asahi Kasei Co., Ltd.) having a length of 30 mm and a width of 200 mm is pasted so as to partially cover the reagent paper 17. Further, a glass fiber filter paper of 40 mm length and 200 mm width made of GF-F (manufactured by Whatman) is bonded to a position 32 mm from the other end of the mount 13 in parallel with the other end to make the absorbent body 14. Here again, the determination paper 15 and the absorber 14 are connected with an overlap of about 2 mm. Furthermore, the other ends are put together, and a protective cover 24 made of a transparent ester film (manufactured by Lintec Co., Ltd.) having a length of 110 mm and a width of 200 mm is attached with the adhesive side facing down. The laminated sheet thus laminated is sequentially cut into strips by a rotary cutter at intervals of 6 mm in width so as to cross each member at a right angle. This completes about 28 analyzers and stores them in a desiccant bag at room temperature until used in analytical tests.

(2-i) Analytical Test Method An E ₂ 17G solution diluted to a predetermined concentration (0 to 5000 ng / ml) with a physiological saline solution as a sample solution was prepared. The analyzer produced as described above was placed on a horizontal desk, and 100 μl of each concentration of E ₂ 17G solution was added from one end of the sample solution addition member 18 with a micropipette, and the color tone of the color developed at the judgment line unit 16 was observed. The results are shown in Table 2.

As shown in Table 2, at 0 ng / ml, the judgment line portion showed reddish purple, and as the E ₂ 17G concentration increased in the order of 10 ng / ml, 50 ng / ml, 250 ng / ml, 500 ng / ml, 1000 ng / ml, As a result, a change in the color tone from purple to purple was observed as a whole.

It is a perspective view of the analyzer of one embodiment of the present invention. It is a perspective view which shows the result of having used the analyzer of FIG. 1 for the analysis. It is a schematic diagram which shows the combined state in the determination line part in the analyzer of FIG. It is a schematic diagram which shows the combined state in the determination line part in the analyzer of FIG. It is a schematic diagram which shows the combined state in the determination line part in the analyzer of FIG. It is a schematic diagram which shows the binding state in the determination line part at the time of using a 3rd specific binding substance and a 4th specific binding substance. It is a schematic diagram which shows the binding state in the determination line part at the time of using a 3rd specific binding substance and a 4th specific binding substance. It is a schematic diagram which shows the binding state in the determination line part at the time of using a 3rd specific binding substance and a 4th specific binding substance. It is a schematic diagram which shows the binding state in the determination line part at the time of fix | immobilizing the label a 1st specific binding substance to the determination area. It is a schematic diagram which shows the binding state in the determination line part at the time of fix | immobilizing the label a 1st specific binding substance to the determination area. It is a schematic diagram which shows the binding state in the determination line part at the time of fix | immobilizing the label a 1st specific binding substance to the determination area. It is a perspective view of the analyzer of another embodiment of the present invention. It is a perspective view which shows the result of having used the analyzer of FIG. 12 for the analysis. It is a schematic diagram which shows the combined state in the determination line part in the analyzer of FIG. It is a schematic diagram which shows the combined state in the determination line part in the analyzer of FIG. It is a schematic diagram which shows the combined state in the determination line part in the analyzer of FIG. It is a schematic diagram at the time of fixing the pH indicator in the determination area. It is a schematic diagram at the time of fixing the pH indicator in the determination area. It is a schematic diagram at the time of fixing the pH indicator in the determination area. It is a schematic diagram which shows the generalization of this invention. It is a schematic diagram which shows another generalization of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,13 Mount 2,14 Absorber 3,15 Judgment paper 4,16 Judgment line part 5,17 Reagent paper 6 Reagent paper 7,18 Sample liquid addition member 8 Labeled b-analysis object or its modified substance 9,19 Label a-specific first binding substance 10 second specific binding substance 11, 23 test piece 12, 24 protective cover 20 labeled c-labeled fifth antibody-antigen complex 21 analyte or denatured substance 22 sixth antibody 25 third specific binding Substance 26 Fourth specific binding substance 27 Labeled a first specific binding substance / third specific binding substance 28 pH indicator

Claims (21)

A reagent zone and a judgment zone are arranged along the wet development direction of the sample liquid, and reagent A and reagent B each having a different visible label are held in the reagent zone in a separated state so as to be able to be eluted and judged from the reagent zone. A competitive reaction product can be generated by a competitive reaction between the analyte in the sample solution and the reagent B with respect to the reagent A accompanying the wet development of the sample solution to the area, and the reagent C that captures this competitive reaction product. Is immobilized in the determination area, and it is possible to detect the concentration of the analyte in the sample solution based on the degree of overlap of the visible signals from the competing reactant captured by the reagent C. Characteristic analyzer.
Reagent A: Labeled a-labeled first specific binding substance in which a first specific binding substance that specifically binds to the analyte in the sample solution is labeled with a visible label a Reagent B; Specific binding to the first specific binding substance Analyte to be analyzed or its denatured product is labeled b-analyzed object or denatured product thereof labeled with a visible label b whose visual signal is different from that of visible label a Reagent C; Specificity of labeled a-labeled first specific binding substance Second specific binding substance capable of specifically binding to the first specific binding substance without impairing A reagent zone and a judgment zone are arranged along the wet development direction of the sample liquid, and reagent B and reagent D each having a different visible label are held in the reagent zone so as to be able to be eluted and flowed, and judged from the reagent zone. A competitive reaction product can be generated by a competitive reaction between the analyte in the sample solution and the reagent B with respect to the reagent D accompanying the wet development of the sample solution to the area, and the reagent E that captures the competitive reaction product Is immobilized in the determination area, and it is possible to detect the concentration of the analyte in the sample solution based on the degree of overlap of the visible signals from the competing reactant captured by the reagent E. Characteristic analyzer.
Reagent B: Analyte that specifically binds to the first specific binding substance or a denatured product thereof Labeled b-analyte or a denatured product thereof labeled with a visible label b that has a different visible signal from the visible label a Reagent D: Labeled a in which a first specific binding substance that specifically binds to the analyte in the sample solution and a third specific binding substance that does not bind to the first specific binding substance and the analyte are labeled a First specific binding substance / third specific binding substance Reagent E; Can bind specifically to the third specific binding substance without impairing the specificity of the first specific binding substance that specifically binds to the analyte in the sample solution Fourth specific binding substance A reagent zone and a judgment zone are arranged along the wet development direction of the sample liquid, the reagent B having a visible label is held in the reagent zone so as to be able to elute and flow, and the reagent A having a visible label different from the reagent B is Directly or indirectly fixed to the determination area, by competitive capture of reagent B between the reagent B and the analyte in the sample liquid accompanying the wet development of the sample liquid from the reagent area to the determination area An analyzer characterized in that the concentration of an analyte in a sample solution can be detected based on the degree of overlap of visible signals.
Reagent A: Labeled a-labeled first specific binding substance in which a first specific binding substance that specifically binds to the analyte in the sample solution is labeled with a visible label a Reagent B; Specific binding to the first specific binding substance Analyzed analyte or denatured product thereof is labeled b-analyzed analyte or denatured product thereof that is labeled with a visible label b that has a different visible signal from the visible label a   4. The analyzer according to claim 3, wherein a reagent C is fixed in the determination area, and the reagent A is fixed to the determination area by being coupled to the reagent C. A reagent zone and a judgment zone are arranged along the wet development direction of the sample liquid, the reagent B having a visible label is held in the reagent zone so as to be able to be eluted and flowed, and the reagent E is immobilized in the judgment zone. Reagent D is immobilized on reagent E, and visible by competitive capture of reagent D between reagent B and the analyte in the sample liquid accompanying wet development of the sample liquid from the reagent area to the determination area An analyzer characterized in that the concentration of an analyte in a sample solution can be detected based on the degree of overlap of signals.
Reagent B: Analyte that specifically binds to the first specific binding substance or a denatured product thereof Labeled b-analyte or a denatured product thereof labeled with a visible label b that has a different visible signal from the visible label a Reagent D: Labeled a in which a first specific binding substance that specifically binds to the analyte in the sample solution and a third specific binding substance that does not bind to the first specific binding substance and the analyte are labeled a First specific binding substance / third specific binding substance Reagent E; Can bind specifically to the third specific binding substance without impairing the specificity of the first specific binding substance that specifically binds to the analyte in the sample solution Fourth specific binding substance   A reagent zone and a judgment zone are arranged along the wet development direction of the sample liquid, and the labeled a-modified first specific binding substance that specifically binds to the analyte in the sample liquid and is labeled with the visible label a is provided. An analyte or a denatured form thereof, which is held in the reagent zone so as to be able to elute and flow and can specifically bind to the labeled a-labeled first specific binding substance, is immobilized in the judgment zone, and is transferred from the reagent zone to the judgment zone. Competitive capture of the labeled a-labeled first specific binding substance between the analyte in the sample liquid and the analyte in the determination area or its denatured substance with the wet development of the sample liquid, and this competitive A visible signal independent of the capture is simultaneously generated in the determination area, and the concentration of the analyte in the sample liquid can be detected based on the degree of overlap between the independent visible signal and the visible marker a. Analysis characterized by Location. A reagent zone and a judgment zone are arranged along the wet development direction of the sample liquid, and the reagent A and the reagent F each having a different visible label are held in the reagent zone so as to be able to elute and flow, and the analyte in the judgment zone Alternatively, the denatured product and the reagent G that can bind to the reagent F are immobilized, and the independent visual signal is generated by the binding of the reagent F and the reagent G accompanying the wet development of the sample liquid from the reagent zone to the determination zone. The analysis apparatus according to claim 6, wherein: can be generated.
Reagent A: Labeled a-modified first specific binding substance in which the first specific binding substance that specifically binds to the analyte in the sample solution is labeled with visible label a Reagent F; Fifth specific binding substance with visible label c Labeled c-labeled fifth specific binding substance Reagent G; Sixth binding substance capable of binding to the fifth binding substance   The analyzer according to claim 6, wherein the independent visual signal is generated in the determination area before the addition of the sample liquid.   9. The analyzer according to claim 6, wherein the independent visual signal is generated by the reagent G immobilized in the determination area and the reagent F bound to the reagent G.   9. The analyzer according to claim 6 or 8, wherein the independent visual signal is a pH indicator, a dye, a colored particle or a colored vesicle immobilized on a determination area.   The sample solution is an extract of an analyte from body fluids such as urine, blood, hemolyzed blood, plasma, serum, saliva, sweat and tears, water such as ponds, rivers and industrial water, and other foods and foods. The analyzer according to claim 1, wherein the analyzer is provided.   The analyte is selected from the group consisting of hormones, biochemical messengers, steroids, drugs, drug metabolites, polypeptides, proteins, vitamins, alkaloids, monosaccharides, disaccharides or polysaccharides, and haptens. The analyzer according to any one of claims 1 to 10.   The analyzer according to any one of claims 1 to 10, wherein the analysis object is selected from the group consisting of thyroid hormone, cortisol, estrogen, progesterone, or testosterone.   The analyzer according to any one of claims 1 to 10, wherein the visible label is selected from the group consisting of a granular label, a dye label, and an enzyme label.   The particulate marker is selected from the group consisting of metal colloids such as gold colloid and selenium colloid, colored lipid vesicles (ribosomes), vesicles, colored latex and the like. Analysis equipment.   The analyzer according to any one of claims 1 to 5, wherein the first specific binding substance and the second specific binding substance are any one of an antigen, an antibody, and a receptor.   The analyzer according to any one of claims 6 and 7, wherein the fifth specific binding substance and the sixth specific binding substance that can bind to each other comprise an antigen, an antibody, or a combination thereof. .   The first specific binding substance is a mouse-derived antibody, and the fifth specific binding substance and the sixth specific binding substance that can bind to each other are a combination of a goat-derived antibody and an anti-goat rabbit-derived antibody. The analyzer according to any one of 6 and 7.   Specific to the reagent A comprising the labeled a-labeled first specific binding substance labeled with the visible label a and the first specific binding substance that specifically binds to the analyte in the sample solution, and the labeled a-labeled first specific binding substance The analyte B or its denatured product that binds to the target is labeled with a visible label b that has a different visual signal from the visible label a, and the reagent B comprising the denatured analyte or labeled B is separated so that it can be eluted. Determination of immobilized reagent C composed of a reagent area to be held in a state and a second specific binding substance capable of specifically binding to the first specific binding substance without impairing the specificity of the labeled a-labeled first specific binding substance The area is arranged so that the sample solution can be wet-developed, and the sample liquid added from the reagent area side elutes the labeled a-labeled first specific binding substance and the labeled b-labeled analyte or its denatured substance in the reagent area. Judgment area Invasion develops, during which the analyte and the labeled b-labeled analyte or its denatured substance undergo a competitive reaction with the labeled a-labeled first specific binding substance due to the presence of the analyte in the sample solution, and the competitive reaction The analyte is captured by the second specific binding substance in the determination area, and the concentration of the analyte in the sample liquid is determined by the change in the visible signal caused by the overlapping degree of the visible label a and the visible label b in the captured competitive reaction product. An analysis method characterized by detecting.   A first specific binding substance that specifically binds to the analyte in the sample solution and a third specific binding substance that does not bind to the first specific binding substance or the analyte are labeled with the label a. A reagent D composed of a first specific binding substance / a third specific binding substance, and an analyte to be specifically bound to the first specific binding substance or a modified form thereof by a visible label b having a visible signal different from the visible label a A reagent zone that holds a reagent B consisting of a labeled mobile labeled b-analyzed analyte or a modified product thereof in a separated state so that it can be eluted and flowed, and the third specific without impairing the specificity of the first specific binding substance. The determination area in which the reagent E made of the fourth specific binding substance that can specifically bind to the specific binding substance is immobilized is arranged so that the sample liquid can be wet-developed, and by the sample liquid added from the reagent area side, Reagent area label The first specific binding substance / third specific binding substance and the labeled b-analyzed analyte or its denatured substance are eluted and infiltrated into the determination area, and during that time, the presence of the analyte in the sample liquid causes The analyte and the labeled b-analyzed analyte or its denatured substance undergo a competitive reaction with the 1 specific binding substance / third specific binding substance, and the competitive reaction product passes through the third specific binding substance in the determination area. It is captured by the fourth specific binding substance, and the concentration of the analyte in the sample liquid is detected by a change in the visible signal caused by the overlapping degree of the visible label a and the visible label b in the captured competitive reaction product. Analysis method.   A reagent zone in which the first specific binding substance that specifically binds to the analyte in the sample solution is labeled with the visible label a and holds the first specific binding substance labeled so that it can be eluted and flowed; (1) An analyte that can specifically bind to a specific binding substance or a determination area on which a denatured body thereof is immobilized is arranged so that the sample liquid can be infiltrated and expanded, and the reagent area is added by the sample liquid added from the reagent area side. The first specific binding substance labeled with a is eluted and infiltrated and expanded into the determination area, and the analysis object or its denatured body immobilized on the analysis object and the determination area due to the presence of the analysis object in the sample solution And a labeled signal a-labeled first specific binding substance, a visual signal independent of competitive capture is simultaneously generated in the determination area, and the visible label a in the area and the independent visible signal Live depending on the degree of overlap Analysis method characterized by sensing the concentration of the analyte by a change in the visual signal that.

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