JP5118479B2 - Immunochromatographic analysis method and immunochromatographic analysis kit - Google Patents

Description

  The present invention relates to an immunochromatographic analysis method and an immunochromatographic analysis kit for developing a solution containing a sample from one end of a support (strip) on which a capture substance is immobilized, utilizing a capillary phenomenon, and capturing and detecting the labeled test substance. It is about.

  The immunochromatographic analysis method is a technique widely used as a simple test kit in pregnancy tests and influenza tests, and since it has the convenience of being detectable only by visual observation, the field of use tends to expand. For example, application to primary screening for determining allergens of allergic patients is an example.

  A typical example of the immunochromatographic analysis method is immunochromatography using a so-called sandwich method. In immunochromatography using the sandwich method, first, two types of antibodies that recognize different sites of the test substance and a strip are prepared, and one antibody (capture antibody) is immobilized in an area called a test line of the strip. On the other hand, for example, colloidal gold particles are labeled to form colloidal gold labeled antibodies. Then, after mixing the sample solution with the colloidal gold labeled antibody, the sample solution is absorbed at one end of the strip and developed. When the test substance is contained in the sample solution, the test substance reacts with the gold colloid-labeled antibody to form a test substance-gold colloid-labeled antibody complex, and this complex passes through the test line. The capture antibody captures the captured antibody-test substance-gold colloid-labeled antibody complex. As a result, red coloration of the colloidal gold labeled antibody is observed on the test line.

  However, in the immunochromatography method using the above-described colloidal gold labeled antibody or the like, detection may be difficult when the concentration of the test substance is low. If the concentration of the test substance in the sample solution is low, the amount of colloidal gold labeled antibody that accumulates in the test line also decreases, so the intensity of the signal (color development) derived from the colloidal gold labeled antibody in the region where the capture antibody is immobilized is, for example, the naked eye This is because it does not reach a recognizable level.

Under such circumstances, research aimed at increasing the detection sensitivity of the immunochromatography method has been promoted in various fields. For example, Patent Document 1 proposes a method for enhancing the color intensity on the test line. In the invention described in Patent Document 1, a labeling substance (for example, gold colloid) is immobilized in advance on the test line, thereby increasing the color development intensity and the emission intensity, thereby enabling detection of a low-concentration test substance. Yes.
WO / 2007/061098

  However, in the technique described in Patent Document 1, it is difficult to evenly fix a labeling substance such as a colloidal gold to each strip, which may lead to variations in the level of raising the bottom and cause erroneous detection. In the case of the technique described in Patent Document 1, a sufficient sensitizing effect may not always be obtained. The reason is that there is a certain distance between the labeling substance immobilized in advance and the labeling substance for labeling the test substance, so that the resonance plasmon effect may not be sufficiently exhibited.

  The present invention has been proposed in view of such conventional circumstances, and an immunochromatographic analysis method that can uniformly raise color development and the like and can surely express sensitization by the resonance plasmon effect. It is another object of the present invention to provide an immunochromatographic analysis kit.

  In order to solve the above-described problems, an immunochromatographic analysis method according to the present invention is an immunochromatographic analysis method in which a test substance in a sample is labeled with a labeling substance and captured and detected by a fixed capture substance, A first labeling reagent that contains a labeling substance and recognizes and binds to the test substance, and includes a labeling substance and an antibody that recognizes the same recognition site as the capture substance or the capture substance itself. It is characterized by sensitization with a two-labeling reagent.

  The immunochromatographic analysis kit according to the present invention includes a detection strip in which a capture substance is fixed at a predetermined position, a first labeling reagent that contains a labeling substance and recognizes and binds to the test substance in the sample, and a labeling substance And a second labeling reagent containing an antibody that recognizes the same recognition site as the capture substance or the capture substance itself.

  For example, when immunochromatography analysis is performed using a strip on which a capture antibody is immobilized as a capture substance, the capture antibody, the test substance, and the first labeling reagent are complexed in a region where the capture antibody is immobilized (determination unit). The labeling substance is accumulated in the determination unit. When the labeling substance accumulated in the determination unit exceeds a certain amount, color development or luminescence can be recognized with the naked eye or detected with an instrument, and the presence of the test substance can be known. However, when the test substance is at a very low concentration, the labeling substance is not sufficiently accumulated, resulting in weak color development or luminescence, which may be unrecognizable with the naked eye or undetectable by instrumental analysis.

  In the present invention, the second labeling reagent is accumulated in the determination unit to perform sensitization and solve this problem. The second labeling reagent is accumulated in the determination unit by binding to the same recognition site as the capture antibody or the capture antibody itself. Thereby, the intensity of color development or light emission in the determination unit is the color development or light emission intensity of the labeling substance derived from the first labeling reagent accumulated in the determination part and the coloration or light emission intensity of the labeling substance derived from the second labeling reagent. Become sum. That is, the color development intensity or light emission intensity in the determination unit is raised. Therefore, even if the concentration of the test substance is low, it can be easily identified. For example, when 100 or more labeling substances are accumulated and the color development can be detected (when the visual detection threshold is 100), 80 labeling substances are accumulated by the binding of the second labeling reagent. If it is possible, the accumulated value of 20 or more of the labeling substances by the first labeling reagent for labeling the test substance exceeds the threshold value, and the color development can be detected visually.

  Further, in the determination unit, the number of test substances to be captured (that is, the number of recognition sites recognized by the capture substance) is generally determined by the number of capture substances (capture antibodies). The second labeling reagent accumulates according to the number of recognition sites recognized by the capture substance and the number of capture substances. Therefore, if the number of immobilized capture substances is constant, the second labeling reagent is accumulated evenly, and color development or light emission is achieved. The level of raising will be even. Furthermore, since the second labeling reagent binds to a position close to the first labeling reagent, the distance between the labeling substances is also close, and the resonance plasmon effect is reliably exerted to obtain a sufficient sensitizing effect.

  According to the present invention, it is possible to uniformly raise (sensitize) color development and the like in immunochromatographic analysis, and it is possible to reliably realize sensitization by the resonance plasmon effect. Therefore, it is possible to reliably detect the test substance by a simple operation.

  Hereinafter, an immunochromatography analysis method and an immunochromatography analysis kit to which the present invention is applied will be described in detail with reference to the drawings.

  As described above, the immunochromatographic analysis method of the present invention is an immunochromatographic analysis method in which a test substance in a sample is labeled with a labeling substance and immobilized with a capture substance and detected, for example, immunochromatography analysis in general such as immunoassay by a sandwich method. It is possible to apply to.

  In immunoassay by the sandwich method, two types of antibodies that recognize different sites on the test substance (antigen) are used. One of the two types of antibodies (capture substance) is immobilized on a strip as a support. The region where the capture substance is immobilized becomes the determination unit. The other antibody (labeled antibody) is not immobilized on the strip, but is labeled with a labeling substance to form a first labeling reagent. In the sandwich method, a test substance labeled with a first labeling reagent is captured with a capture substance and detected.

  Alternatively, instead of simply forming a complex sandwiched between the labeling reagent and the capture substance and performing detection, the test substance and the specific reaction substance are bound, and the labeling reagent is bound to the test substance, The present invention can also be applied to immunochromatographic analysis (hereinafter referred to as improved immunochromatographic analysis) in which a labeled test substance is captured and detected by binding a capture substance to the specific reactant.

  In this case, a specific reactive substance that specifically binds to the test substance and the capture substance, respectively, and a site that includes a labeling substance and is different from the binding site of the specific reactive substance of the test substance. The first labeled reagent to be bound is allowed to act, and the specific reaction substance bound to the test substance is bound to the capture substance. As a result, a first labeling reagent-test substance-specific reaction substance complex is formed, and further, this first labeling reagent-test substance-specific reaction substance complex binds to the capture substance, and the first labeling reagent A test substance-specific reaction substance-capture substance complex is formed. The trapping substance is fixed in a striped manner to a strip or the like as a support, and the part to which the trapping substance is fixed serves as a determination unit as in the case of the sandwich method. As the first labeling reagent-test substance-specific reaction substance-capture antibody complex accumulates in the determination unit, the labeling substance is visually recognized, and the presence of the test substance in the sample is detected.

  In the improved immunochromatographic analysis, it is possible to deal with various test substances by selecting specific reaction substances according to the test substances. For example, an analysis using an antibody that recognizes an antigen such as an allergen as a test substance is also possible.

  In the present invention, the test substance to be detected can be applied to any substance as long as it contains an immunoreactive substance, and examples thereof include various antibodies and antigens. Here, as antibodies (IgG, IgM, IgE, IgD, IgA, etc.), autoantibodies and various immunoglobulins typified by anti-DNA antibodies, anti-ENA antibodies, anti-cardioribine antibodies, anti-mitochondrial antibodies, anti-smooth muscle antibodies, etc. For example, the present invention can be applied to analysis of specific immunoglobulins (specific IgE, etc.) that specifically recognize allergens. Examples of the antigen include a glycoprotein, a specific protein composed of a plurality of subunits, and a protein complex such as prostate specific antigen (PSA).

  In the sandwich method, the labeled antibody or the capture substance (capture antibody) of the first labeling reagent may be selected according to the type of the test substance. In the improved immunochromatographic analysis, a specific reaction substance may be selected according to the type of the test substance. For example, when the test substance is the autoantibody (for example, an anti-DNA antibody), the anti-DNA antibody recognizes it. The specific DNA to be used may be used as the specific reactant. When the test substance is an autoantibody whose γ globulin class is IgG, an antigen such as a specific protein or protein complex recognized by the IgG may be used as the specific reaction substance. When the test substance is an immunoglobulin (IgE, IgG, etc.) that specifically recognizes the allergen, the allergen may be used as a specific reaction substance. When the test substance is an antigen such as a glycoprotein, a specific protein, or a protein complex, an antibody that recognizes a predetermined protein portion may be used as the specific reaction substance. As the specific reaction substance, each antigen or antibody itself may be used, or a fragment thereof may be used. By combining these test substances and specific reaction substances, immunochromatographic analysis can be performed on various test substances.

  In the improved immunochromatographic analysis, as the first labeling reagent that binds to and labels the test substance, for example, a binding substance of a labeled antibody that recognizes the test substance as an antigen and a labeling substance can be used. In the improved immunochromatographic analysis, if the labeled antibody that recognizes the test substance as an antigen recognizes a site that is different from the reaction site of the test substance with the specific reaction substance, the test combined with the specific reaction substance It is possible to further bind to the substance. When such a labeling reagent is used, the labeling substance binds to the test substance via the labeling antibody and labels it.

  As the labeling substance, a coloring substance, a luminescent substance or the like can be used as the labeling substance, but it is preferable to use a coloring substance in order to enable quick and simple detection. Here, as the coloring material, metallic fine particles, latex fine particles, organic polymer fine particles, inorganic fine particles, coloring fine particles such as liposomes including a color forming agent, and the like can be used. Examples of the metal fine particles include noble metal fine particles such as gold fine particles, silver fine particles, and platinum fine particles, titanium fine particles, iron fine particles, and nickel fine particles. The metal fine particles may be colloidal metal fine particles having a particle diameter of 1 nm to 100 nm. That is, noble metal colloid particles such as gold colloid particles, silver colloid particles, platinum colloid particles, titanium colloid particles, iron colloid particles, and nickel colloid particles may be used. Latex fine particles include polystyrene, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, vinyl acetate-acrylic. Examples thereof include fine particles containing at least one selected from acid ester copolymers and the like. Examples of the organic polymer fine particles include fine particles containing at least one selected from insoluble agarose, cellulose, insoluble dextran and the like. Examples of the inorganic fine particles include silica and alumina. Among these, it is preferable to use highly versatile noble metal colloid particles, latex fine particles, and the like. In particular, it is preferable to use gold colloid particles because a clear color can be obtained.

  The second labeling reagent also contains the same labeling substance as the first labeling reagent, but unlike the first labeling reagent, an antibody that recognizes a capture substance or a specific reaction substance is used as a labeled antibody. The second labeling reagent binds evenly to the capture substance and the specific reaction substance, and raises the label with the labeling substance contained in the first labeling reagent.

  For example, in the case of the sandwich method, a capture antibody that recognizes a substance to be inspected as an antigen is used as the capture substance fixed to the strip as a support. Alternatively, instead of a capture antibody, a capture antigen that specifically binds to a test substance (antibody) can be immobilized on the strip as a capture substance. In the case of the improved immunochromatographic analysis, an antibody that captures the labeled reagent-test substance-specific reactive substance complex to which the specific reactive substance and the labeling reagent are bound, and recognizes and binds the specific reactive substance as an antigen. Can be used. In this case, the capture antibody may directly recognize a specific reactive substance, or may recognize an antigen immobilized on the surface of the specific reactive substance.

  For example, when the specific reactive substance is an antigen (for example, allergen), the capture antibody may be an antibody that recognizes the allergen as an antigen, or an antibody that recognizes biotin attached to the allergen surface as an antigen. Good. In the latter case, the allergen to which biotin is attached corresponds to the specific reactant. When biotin is attached to the allergen surface and the recognition site on the allergen surface is biotin, a plurality of biotins are attached to the allergen to ensure a fixed portion, and sensitivity can be increased. If the allergen itself is immobilized with a capture antibody, measurement of a test substance (for example, IgE) that recognizes a site close to the recognition site of the capture antibody is inhibited, which may cause a decrease in sensitivity. If biotin is used as the recognition site on the allergen surface, this can be prevented and sensitivity can be increased.

  Examples of the sample solution containing the test substance include a solution derived from a living body such as blood, serum, and urine, a solution containing water or soil collected from the natural environment, a solution obtained by using these, and the like. Any one can be used. Any substance such as a biological substance or a synthetic substance can be used as a test substance.

  To detect the test substance in the sample solution, for example, in the sandwich method, first, the sample solution and the labeled antibody are mixed to form a complex of the test substance and the labeled antibody, and then the capture substance is fixed. Permeate from one end of the strip. In the immunochromatography method, a sample solution is developed utilizing capillary action. The sample solution and the solution containing the labeled antibody may be individually flowed on the strip in this order. When the test substance is present in the sample solution, the capture substance and the labeled antibody fixed on the strip bind to the test substance in a sandwich, and as a result, the amount of labeled antibody corresponding to the test substance is determined. Accumulate in part.

  The analyst can easily determine the presence or absence of the test substance by visually confirming the presence or absence of color development in the determination unit or by detecting the color development using a device such as a densitometer. If the concentration of the test substance is low, sufficient color intensity may not always be obtained. Therefore, in the present invention, after capturing the test substance, the second labeling reagent is flowed to perform so-called sensitization. The second labeling reagent binds to the capture substance fixed on the strip and also accumulates in the determination unit. As a result, color development is enhanced by the resonance plasmon effect of the labeling substance of the first labeling reagent and the labeling substance of the second labeling reagent accumulated in the determination unit, and the detection sensitivity is improved. As described above, since the second labeling reagent binds to the capture substance and is captured at a position close to the first labeling reagent, the resonance plasmon effect is reliably exhibited. In addition, since the amount of the second labeling reagent accumulated is generally determined by the number of capture substances to be immobilized, the color development is raised evenly and variations in sensitization can be suppressed.

  In the case of the improved immunochromatographic analysis, first, the sample solution, the specific reaction substance, and the labeling reagent are mixed to form a first labeling reagent-test substance-specific reaction substance complex, and then the support on which the capture substance is fixed. (Detection strip). The supplied sample solution (mixed solution) permeates the detection strip and is developed by capillary action. When a test substance exists in the sample solution, the capture substance fixed to the detection strip binds to the specific reaction substance of the complex, and as a result, an amount of labeling substance corresponding to the test substance is determined by the determination unit (capture The material is collected and visualized on the fixed part).

  Also in the improved immunochromatographic analysis, when the concentration of the test substance is low, it is possible to enhance the color intensity by sensitizing with the second labeling reagent. That is, after the first labeling reagent-test substance-specific reaction substance complex is captured by the capturing substance, the second labeling reagent is flowed for sensitization. The second labeling reagent recognizes and binds to the capture substance and the specific reaction substance of the first labeling reagent-test substance-specific reaction substance complex captured by the capture substance, and accumulates them in the determination unit. Therefore, as in the sandwich method, color development is enhanced by the resonance plasmon effect of the labeling substance of the first labeling reagent and the labeling substance of the second labeling reagent accumulated in the determination unit, and the detection sensitivity is improved.

  In the improved immunochromatographic analysis, the sensitivity may be reduced due to an inhibitory effect depending on the sample. For example, when the test substance is a specific immunoglobulin that recognizes a specific allergen, the sample (specimen) includes non-specific immunoglobulin in addition to the specific immunoglobulin. Thus, when the nonspecific immunoglobulin and the specific immunoglobulin to be analyzed are mixed, the first labeling reagent also binds to the nonspecific immunoglobulin, and the detection sensitivity of the specific immunoglobulin is reduced. . In such a case, it is preferable to take measures to avoid the inhibitory effect.

  In order to avoid the inhibitory effect due to the mixture of nonspecific immunoglobulins, a method of avoiding the binding of the first labeling reagent to the nonspecific immunoglobulin (hereinafter referred to as an avoidance method) or capture with a capture substance. Examples include a method of labeling the test substance again with a first labeling reagent (hereinafter referred to as a relabeling method).

  Here, as described above, the avoidance method is a method for avoiding the binding of the first labeling reagent to the nonspecific immunoglobulin, and after immobilizing the test substance bound to the specific reaction substance with the capture substance, In this method, the first labeling reagent is allowed to act. In the avoidance method, a specific reaction substance is allowed to act on a sample, and the specific reaction substance bound to the test substance is captured by the capture substance, and then the test substance is labeled by acting the first labeling reagent. In order to realize this, the first labeling reagent may be flowed with the developing solution after flowing the sample together with the specific reaction material to the detection strip in which the capture material is fixed at a predetermined position.

  In the above avoidance method, the first labeling reagent is allowed to act after the inhibitory substance (for example, non-specific immunoglobulin) has flowed, and the first labeling reagent is selectively bound to the captured test substance. Therefore, the test substance can be detected without receiving the inhibitory effect of the inhibitor and the sensitivity can be improved. At this time, if the second labeling reagent is mixed with the first labeling reagent and allowed to act, it is possible to achieve both avoidance of inhibition of the labeling of the test substance and sensitization by the second labeling reagent.

  On the other hand, the relabeling method is a method in which the test substance that has not been labeled with the first labeling reagent at the time of development is labeled again to enhance color development. In the relabeling method, the specific reaction substance and the first labeling reagent are allowed to act on the sample, the specific reaction substance bound to the test substance is captured by the capture substance, and then the first labeling reagent is allowed to act again. In order to realize this, after flowing the sample together with the specific reaction substance and the first labeling reagent on the detection strip in which the capture substance is fixed at a predetermined position, the first labeling reagent is flowed again with the developing solution. Good.

  In the relabeling method, a test substance that cannot be bound to the first labeling reagent due to the influence of an inhibitor (for example, non-specific immunoglobulin) is also bound to the first labeling reagent by re-labeling, and color development is enhanced. Therefore, it is possible to increase detection sensitivity. Furthermore, when the second labeling reagent is mixed with the first labeling reagent when the labeling is performed again, enhancement of color development by relabeling and sensitization by the second labeling reagent can be realized. Is possible.

  The immunochromatographic analysis method described above can be easily carried out by using an immunochromatographic analysis kit composed of a pad containing the first labeling reagent and a specific reactive substance, a detection strip on which a capture substance is fixed, and a developing solution. Is possible. The first labeling reagent and the second labeling reagent may be separately attached. In this case, the immunochromatography analysis kit includes a detection strip in which a capture substance is fixed at a predetermined position, and a test in a sample containing the labeling substance. A first labeling reagent that recognizes and binds to a substance and a second labeling reagent that includes a labeling substance and an antibody that recognizes the same recognition site as the capturing substance or the capturing substance itself may be provided. The first labeling reagent and the second labeling reagent can be mixed with a developing solution and used as a sensitizing solution.

  The immunochromatographic analysis kit for performing the improved immunochromatographic analysis includes a detection strip in which a capture substance is fixed at a predetermined position, and a specific reaction substance that specifically binds to the test substance and the capture substance. A first pad, a second pad holding a labeling reagent that contains a first labeling substance and binds to a site different from the binding site of the specific reactant of the test substance, and further a sensitizing solution containing a second labeling reagent It only has to have. For example, if a first pad, a second pad, and a detection strip are arranged in order and combined to form an immunochromatography analysis kit, the sample solution is simply dropped onto the first pad. It can be done quickly.

  When the sample solution is dropped on the first pad of the immunochromatography analysis kit having the above-described configuration, the sample solution penetrates into the first pad, and the test substance contained in the sample solution reacts with the specific reactant contained in the first pad. Further, the sample solution penetrates into the second pad, and the test substance to which the specific reaction substance is bound reacts with the first labeling reagent contained in the second pad. Thereby, the first labeling reagent-test substance-specific substance complex is formed. The solution containing this complex penetrates into the detection strip and is developed. In the detection strip, the complex is captured by the immobilized capture substance, and the labeling substance is accumulated to be visualized. If the sensitizing solution is flowed after capturing the complex, sensitization with the second labeling reagent is performed, and the color development intensity is enhanced.

  In the immunochromatography analysis kit for performing the avoidance method, it is necessary to arrange the second pad, the first pad, and the detection strip in this order. In many cases, the immunochromatography analysis kit is provided in a state where the first pad, the second pad, and the detection strip are accommodated in a container made of plastic or the like. In this case, the container includes the first pad and the second pad. Solution supply holes are respectively formed at positions corresponding to the two pads. In the analysis, after dropping the sample solution from the solution supply hole corresponding to the first pad, the developing solution is dropped from the solution supply hole corresponding to the second pad. If the sample solution is dropped from the solution supply hole corresponding to the first pad, only the specific reactant reacts with the sample, and the specific reactant bound to the test substance is captured by the capture antibody. Next, when a developing solution is dropped from the solution supply hole corresponding to the second pad, the captured test substance and the labeling reagent come into contact with each other and are labeled. Thereafter, if a sensitizing solution containing the second labeling reagent is poured, sensitization with the second labeling reagent is performed.

  In the immunochromatography analysis kit for performing the relabeling method, the first pad, the second pad, and the detection strip may be arranged in this order, unlike the case of the avoidance method. The first pad, the second pad, and the detection strip are accommodated in a container, and a solution supply hole is formed at a position corresponding to the first pad of the container. A developing solution containing the first labeling reagent and the second labeling reagent is attached. In the analysis, first, a sample solution is dropped from the solution supply hole and developed as usual, and after fixing the first labeled reagent-test substance-specific substance complex with the capture antibody, the first labeled reagent and A developing solution containing the second labeling reagent is dropped. Thereby, the labeling of the complex is enhanced by the first labeling reagent, and the color development is raised by the second labeling reagent.

  Next, the immunochromatographic analysis method and the immunochromatographic analysis kit of the present invention will be described more specifically by taking as an example an improved immunochromatographic analysis (relabeling method) using specific IgE that recognizes allergen as a test substance.

  FIG. 1 is a schematic diagram for explaining an immunochromatographic analysis method and an immunochromatographic analysis kit of the present embodiment. In this embodiment, the test substance is specific IgE that recognizes the allergen, and therefore the allergen (here, allergen to which biotin is attached) corresponds to the specific reaction substance. The labeling reagent is a gold colloid labeled anti-IgE antibody. Biotin is attached to the surface of the allergen, and an anti-biotin antibody that recognizes biotin is used as a capture substance (capture antibody).

  The immunochromatographic analysis kit comprises a strip-shaped detection strip 1 and a first pad 2 and a second pad 3 coupled to the end of the detection strip 1, and further a sensitizing solution is attached. Yes. The arrangement order of the first pad 2 and the second pad 3 is the order of the first pad 2, the second pad 3, and the detection strip 1 from the upstream side. An absorption pad that absorbs the developed sample solution may be provided on the downstream side of the detection strip 1.

  The first pad 2 and the second pad 3 are made of, for example, glass wool or the like laminated with filter paper having excellent water absorption on the upper surface, and the first pad 2 has a specific reactive substance (biotin attached thereto). Allergen 4) is retained. The second pad 3 holds a colloidal gold labeled anti-IgE antibody 5 as a first labeling reagent. The colloidal gold-labeled anti-IgE antibody 5 is obtained by immobilizing the anti-IgE antibody 5b on the surface of a gold colloid 5a that is a coloring substance. Therefore, this colloidal gold-labeled anti-IgE antibody 5 recognizes IgE as an antigen and binds to it.

  The detection strip 1 can be used without limitation as long as it is a membrane used in this type of immunochromatography, and for example, nitrocellulose or the like can be used. An anti-biotin antibody 6 that is a capture antibody is fixed in the middle of the detection strip 1 and constitutes a determination unit. The determination part is formed as a linear pattern in a direction orthogonal to the development direction in the detection strip 1.

  When specific IgE (test substance) is detected using the immunochromatography analysis kit having the above-described configuration, as shown in FIG. 1 (a), a sample solution 7 obtained by diluting serum as a specimen with a developing solution is used as a first solution. Drip onto the pad 2. The sample solution 7 contains non-specific IgE9 that does not recognize allergens in addition to specific IgE8 that is a test substance.

  When the sample solution 7 is dropped onto the first pad 2, the sample solution 7 penetrates into the first pad 2, the second pad 3, and further the detection strip 1. In this process, allergen 4 and colloidal gold-labeled anti-IgE antibody 5 are eluted from each pad, and bind to specific IgE8 in sample solution 7 by antigen-antibody reaction. That is, as shown in FIG. 1B, a complex 10 in which allergen 4 or colloidal gold-labeled anti-IgE antibody 5 is bound to specific IgE8 is formed. The colloidal gold-labeled anti-IgE antibody 5 also binds to nonspecific IgE9. Therefore, a part of specific IgE8 bound to allergen 4 is developed as it is without binding to gold colloid-labeled anti-IgE antibody 5.

  After the reaction, the sample solution 7 is developed in the detection strip 1 from the left to the right in the figure by capillary action, and as shown in FIG. 1C, the allergen 4 and the anti-biotin antibody 6 are detected in the determination unit. The complex 10 is captured by the binding. At this time, not only the complex 10 but also specific IgE8 bound only to the allergen 4 that is not labeled with the colloidal gold-labeled anti-IgE antibody 5 and excess allergen 4 are captured.

  As described above, the colloidal gold-labeled anti-IgE antibody 5 is bound to the determination portion of the detection strip 1 depending on the concentration of specific IgE8, and the colloidal gold 5a is accumulated. As a result of the accumulation of the gold colloid 5a, the determination part (test line) develops a linear color, and the presence or absence of allergy can be determined visually.

  However, when immunochromatography analysis is performed according to the procedure shown in FIGS. 1 (a) to 1 (c), as shown in FIG. 1 (c), the determination part is not specifically labeled with the colloidal gold-labeled anti-IgE antibody 5. IgE8 is also captured and the detection sensitivity is reduced. In particular, when a large amount of non-specific IgE9 is present in the sample, the colloidal gold-labeled anti-IgE antibody 5 and the specific IgE8, which are labeling reagents, are formed when the complex 10 is formed after application of the sample solution or the developing solution. Prevent binding competitively. As a result, the ratio of specific IgE8 that is not labeled out of specific IgE8 trapped in the determination unit increases, and the color development of the test line is suppressed.

  Therefore, in this embodiment, specific IgE8 that could not bind to the colloidal gold-labeled anti-IgE antibody 5 due to the influence of the inhibitor (non-specific IgE9) is labeled again, and the colloidal gold-labeled anti-biotin that is the second labeling reagent. Color development is enhanced by the action of antibodies.

  That is, after trapping the complex 10, as shown in FIG. 1 (d), a sensitizing solution 12 containing a colloidal gold-labeled anti-IgE antibody 5 and a colloidal gold-labeled anti-biotin antibody 11 is dropped onto the first pad 2, Perform the deployment again. As the sensitizing solution, a developing solution containing a gold colloid-labeled anti-IgE antibody 5 as a first labeling reagent and a gold colloid-labeled anti-biotin antibody 11 as a second labeling reagent is used. The colloidal gold-labeled anti-biotin antibody 11 as the second labeling reagent is one in which the anti-biotin antibody 11b is immobilized on the surface of the colloidal gold 11a, and recognizes and binds biotin on the allergen surface in the same manner as the capture antibody.

  At this time, nothing is held in the first pad 2 or the second pad 3, and as shown in FIG. 1 (e), the gold colloid-labeled anti-IgE antibody 5 and the gold colloid-labeled anti-biotin antibody are obtained by the development. Only 11 will flow. As shown in FIG. 1 (f), the colloidal gold labeled anti-IgE antibody 5 that has flowed to the determination unit binds to specific IgE8 that is not bound to the colloidal gold labeled anti-IgE antibody 5, and labels it. The colloidal gold-labeled anti-biotin antibody 11 recognizes and binds to biotin present on the surface of the allergen 4 contained in the complex 10 captured by the anti-biotin antibody 6 that is a capture antibody.

  As described above, by developing the sensitizing solution 12 containing the colloidal gold-labeled anti-IgE antibody 5 and the colloidal gold-labeled anti-biotin antibody 11 after the development of the sample solution 7 is completed, the sample is not labeled due to the influence of nonspecific IgE9. The colloidal gold-labeled anti-IgE antibody 5 binds to the specific IgE8, color development is enhanced, and detection sensitivity is improved. Further, the colloidal gold-labeled anti-biotin antibody 11 is bound to the surface of the allergen 4 to raise the color development, and the detection sensitivity is further improved. At this time, the colloidal gold-labeled anti-biotin antibody 11 accumulates in proportion to the number of allergens 4 captured (that is, the number of immobilized anti-biotin antibodies 6), and uniform color development is achieved. In addition, since the binding position of the colloidal gold-labeled anti-biotin antibody 11 is the allergen 4 surface and close to the binding position of the colloidal gold-labeled anti-IgE antibody 5, the resonance plasmon effect is reliably expressed, and the color development is surely raised. Made.

  Furthermore, as shown in FIG. 1 (g), the colloidal gold-labeled anti-IgE antibody 5 in the sensitizing solution 12 is non-binding to the colloidal gold-labeled anti-IgE antibody 5 that has already been immobilized through the specific IgE8. It also binds to specific IgE9 and has an effect of increasing the degree of accumulation of colloidal gold 5a on the test line, which also contributes to an effect of increasing detection sensitivity.

  Hereinafter, specific examples of the present invention will be described based on experimental results.

(Detection of cedar pollen-specific IgE)
Using specific IgE that recognizes cedar pollen as an allergen as a test substance, immunochromatographic analysis was performed by the relabeling method shown in FIG. However, the case where the immunochromatography analysis is performed by the operation up to FIG. 1C is a comparative example, and the case where the immunochromatography analysis is performed by the operation up to FIG.

  As a sample, 20 μL of a positive serum sample or 20 μL of a negative serum sample was used, which was diluted with 20 μL of a diluted solution (1% BSA phosphate buffer) to obtain a sample solution. As the developing solution, 30 μL of a phosphate buffer solution containing 0.2% of a surfactant (Tween) was used. On the detection strip, an anti-biotin antibody was fixed to form a linear determination part, and the allergen modified with biotin was carried on the first pad. The second pad was loaded with colloidal gold-labeled anti-IgE antibody. In the examples, 30 μL of gold colloid-labeled anti-biotin antibody (OD520 = 0.006) was added to 30 μL of PBS containing 10% gold colloid-labeled anti-IgE antibody / 0.2% Tween20 to obtain a sensitizing solution.

  The sample solution (positive control serum sample and negative control serum sample) was developed for 15 minutes, and then the difference in color intensity (color development intensity) of the judgment part depending on the presence or absence of sensitization by the sensitizer was measured with a densitometer. The results are shown in FIG.

  As is clear from FIG. 2, in the example in which sensitization was performed, the difference in color intensity between the positive and negative cases was larger than that in the comparative example in which sensitization was not performed, and the color development was discriminated. It turns out that it is easy.

It is a schematic diagram explaining the basic principle of the immunochromatography analysis method in embodiment (relabeling method) to which this invention is applied, (a) is dripping of a sample solution, (b) is a development process, (c) is based on a capture antibody. Capture, (d) shows the dropping of the sensitizing solution onto the first pad, (e) shows the development process of the sensitizing solution, and (f) and (g) show the relabeling and sensitization states, respectively. In immunochromatographic analysis of positive control serum and negative control serum, it is a characteristic figure which shows the difference of the coloring intensity in an Example and a comparative example.

Explanation of symbols

1 detection strip, 2 first pad, 3 second pad, 4 allergen, 5 gold colloid labeled anti-IgE antibody, 5a gold colloid, 5b anti-IgE antibody, 6 anti-biotin antibody, 7 sample solution, 8 specific IgE, 9 Non-specific IgE, 10 complex, 11 gold colloid labeled anti-biotin antibody, 11a gold colloid, 11b anti-biotin antibody, 12 sensitizing solution

Claims (12)

The test substance in the sample is labeled with a first labeling reagent that contains a labeling substance and recognizes and binds to the test substance, and a specific reaction substance that specifically binds to the test substance and the capture substance is allowed to act. An immunochromatographic analysis method characterized in that it is captured by an immobilized capture substance and sensitized by a second labeling reagent containing an antibody containing a labeling substance that recognizes the specific reaction substance or the capture substance itself . The specific reaction substance and the first labeling reagent are allowed to act on the sample, the specific reaction substance bound to the test substance is captured by the capture substance, and then the first labeling reagent and the second labeling reagent are allowed to act. The immunochromatographic analysis method according to claim 1, wherein A sample is flowed together with a specific reaction substance and a first labeling reagent to a detection strip in which a capture substance is fixed at a predetermined position, and then a sensitizing solution containing a first labeling reagent and a second labeling reagent is allowed to flow. The immunochromatographic analysis method according to claim 2 . The immunochromatographic analysis method according to any one of claims 1 to 3 , wherein the specific reaction substance contains an antigen, and the test substance is a specific immunoglobulin that reacts with the antigen. The immunochromatographic analysis method according to claim 4 , wherein the sample contains a specific immunoglobulin that reacts with the antigen and a non-specific immunoglobulin that does not react with the antigen. 6. The immunochromatographic analysis method according to claim 5 , wherein the specific immunoglobulin is specific IgE, and the non-specific immunoglobulin is non-specific IgE. The immunochromatographic analysis method according to any one of claims 4 to 6 , wherein biotin is immobilized on the surface of the antigen, and the capture substance binds to biotin on the antigen surface. The immunochromatographic analysis method according to claim 7 , wherein the second labeling reagent recognizes and binds to biotin on the antigen surface. The immunochromatographic analysis method according to any one of claims 1 to 8 , wherein the first labeling reagent and the second reagent contain gold colloid as a labeling substance. A detection strip in which a capture substance is fixed at a predetermined position, a first labeling reagent that recognizes and binds a test substance in a sample containing a labeling substance, and a specification that specifically binds to the test substance and the capture substance An immunochromatography analysis kit comprising: a reaction substance ; and a second labeling reagent containing an antibody containing a labeling substance that recognizes the specific reaction substance or the capture substance itself. The immunochromatographic analysis kit according to claim 10, further comprising a sensitizing solution in which the first labeling reagent and the second labeling reagent are mixed. A first pad that holds a specific reaction substance that specifically binds to a test substance and a capture substance and a second pad that holds a first labeling reagent are bound to the detection strip. The immunochromatographic analysis kit according to claim 10 or 11 .

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