JP4179419B2 - Test substance detection method, sensitizer and immunochromatography kit - Google Patents

Description

  The present invention labels a test substance and a second antibody that recognizes a site different from the predetermined site of the test substance on a test strip on which a first antibody that recognizes the predetermined site of the test substance is immobilized. The present invention relates to a detection method of a test substance that develops a labeled second antibody bound to the substance, and further relates to a sensitizer and an immunochromatography kit.

  ELISA (enzyme-linked immunosorbent assay) and MEIA (microparticle enzyme-based immunoassay) methods are widely used as a method for detecting a small amount of a test substance in a sample. There are problems such as a long reaction time and complicated measurement operation.

  Therefore, in recent years, an analysis method using an immunochromatography method has attracted attention as an analysis method replacing the ELISA method. The immunochromatography method is excellent in various respects such as storage stability, rapid measurement, ease of determination, and the necessity of a special accessory device. For example, the point of interest for influenza testing, HCV testing, PSA testing, etc. Widely used in the field of care testing.

  Detection of a test substance using an immunochromatography method is performed, for example, through the following steps. First, two types of antibodies for recognizing different parts of the test substance are prepared. One antibody (first antibody) is applied and fixed to a region called a test line of the test strip, and the other antibody (second antibody) is used. The colloidal gold particles are labeled on the antibody. Then, the test solution and the colloidal gold-labeled second antibody are mixed, absorbed at one end of the test strip, and developed. When the test substance is present in the test solution, the test substance-gold colloid-labeled second antibody complex formed in the mixture is captured by the immobilized first antibody on the test line, and the immobilized first An antibody-test substance-gold colloid-labeled first antibody complex is formed. As a result, the red coloration of the colloidal gold is observed on the test line. On the other hand, when the test substance is not present in the test solution, the colloidal gold-labeled second antibody does not accumulate in the test line, so that no color is produced.

  However, the immunochromatography method has a problem in terms of sensitivity, and sufficient sensitivity may not be obtained depending on the measurement object. For example, if the concentration of the test substance in the test solution is very low, the amount of colloidal gold accumulated on the test line becomes insufficient, the color development becomes extremely thin, and there is a possibility that it is erroneously determined as negative.

Therefore, research aimed at improving the detection sensitivity of the immunochromatography method is being promoted in various fields. For example, the secondary antibody is labeled with magnetic fine particles, and the test substance is detected using the change in magnetism on the test strip as an index, or the secondary antibody is labeled with an enzyme such as alkaline phosphatase or peroxidase, and the color-developing agent is added. The method of expanding from is widely known. As a method using an enzyme-labeled secondary antibody, a method of performing enzyme immunoassay in the presence of a base in enzyme immunoassay using peroxidase (see, for example, Patent Document 1) has been proposed.
JP 2001-74740 A

  However, in the method of developing the color former after the development of the magnetic fine particle label or the enzyme-labeled secondary antibody, a special detection device is required, which not only makes the detection operation complicated, but also takes time for detection of magnetic changes, enzyme reaction, etc. There is a problem that it takes.

  Therefore, the present invention has been proposed in view of the above-described circumstances, and provides a method for detecting a test substance capable of quickly and easily detecting a test substance in a test solution with high sensitivity. Objective. Another object of the present invention is to provide a sensitizer and an immunochromatography kit used in the detection method.

  In order to solve the above-described problem, a test substance detection method according to the present invention includes a test substance, a test substance, and a test strip on which a first antibody that recognizes a predetermined site of the test substance is immobilized. After developing a labeled second antibody in which a second antibody that recognizes a site different from the predetermined site of the substance is bound to the labeled material, the predetermined site of the test substance is recognized by a sensitizer that emits a predetermined signal. And developing a sensitizer to which a test substance is bound via an antibody. Further, the sensitizer according to the present invention provides a test strip on which a first antibody that recognizes a predetermined site of the test substance is immobilized, and a test substance and a site different from the predetermined site of the test substance. Used in an immunochromatography method for developing a labeled second antibody in which a second antibody to be recognized is bound to a labeled substance, and via an antibody that recognizes the predetermined site of the test substance to a sensitizer that emits a predetermined signal It is characterized by binding a test substance.

  The immunochromatography kit according to the present invention labels a test strip on which a first antibody that recognizes a predetermined site of a test substance is immobilized and a second antibody that recognizes a site different from the predetermined site of the test substance. A labeled second antibody bound to the substance, and a sensitizer obtained by binding the test substance to the sensitizer that emits a predetermined signal via an antibody that recognizes the predetermined site of the test substance. And In addition, the immunochromatography kit according to the present invention includes a test strip on which a first antibody that recognizes a predetermined site of a test substance is immobilized, and a second antibody that recognizes a site different from the predetermined site of the test substance. Characterized in that it comprises: a labeled second antibody in which is bound to a labeled substance; a labeled antibody labeled with an antibody that recognizes the predetermined site of the test substance with a sensitizing substance that emits a predetermined signal; and a test substance To do.

  When detecting the test substance, first, the test solution containing the test substance and the labeled second antibody are mixed and developed. When the test substance is present in the test solution, the test substance-labeled second antibody complex formed in the mixture is captured by the first antibody immobilized on the test strip. As a result, a first antibody-test substance-labeled second antibody complex is formed on the test strip. Up to this point, it is the same as the detection method using the conventional chromatography method. Even at this time, a signal (coloring or luminescence) from the labeling substance accumulated in the determination unit is observed, but when the test substance in the test solution is at a low concentration, the accumulation amount of the labeling substance becomes insufficient and weak. Only a signal may be obtained.

  Therefore, in the present invention, a sensitizer is developed in which a test substance is bound to an sensitizer that emits a predetermined signal via an antibody that recognizes the same site as the first antibody. As a result of the sensitizer binding to the first antibody-test substance-labeled second antibody complex formed on the strip, both the labeling substance and the sensitizing substance are accumulated in the determination section, thereby enhancing the signal. It is done. As described above, by using the sensitizer, a strong signal can be obtained in the determination unit even when the test substance concentration in the test solution is low.

  Since the test substance constituting the sensitizer is already bound to an antibody that recognizes the same site as the first antibody, the complex directly binds to the first antibody immobilized on the test strip. There is nothing. For this reason, when the labeled second antibody is not present in the determination part (when the test substance is not contained in the test solution), signal enhancement does not occur and accurate detection is realized.

  According to the test substance detection method of the present invention, a sufficiently strong signal can be obtained even when the test substance in the test solution has a low concentration, so that the detection sensitivity can be improved. . In addition, according to the detection method of the test substance of the present invention, it is basically possible to detect with the naked eye, and in most cases no special apparatus is required for detection, so that high sensitivity of the test substance in the test solution is obtained. Detection can be performed quickly and easily.

  The immunochromatography kit of the present invention is very useful for applications such as clinical tests (so-called point-of-care tests) performed near a patient. Furthermore, since a sensitizer can be prepared with a material used in a normal immunochromatography method, the material is easily available and inexpensive.

  Hereinafter, a detection method, a sensitizer, and an immunochromatography kit according to the present invention will be described in detail with reference to the drawings.

  In the detection method of the present invention, any substance such as a biological substance and a synthetic substance can be used as a test substance. In addition, as a test solution containing a test substance, for example, a sample solution derived from a living body such as blood, serum, urine, a sample solution containing water or soil collected from the natural environment, a solution obtained by preparing these, etc. Any thing can be used.

  In the detection method of the present invention, as shown in FIG. 1, two types of antibodies, that is, the first antibody 1 and the second antibody 2 are used. These antibodies recognize different sites on the test substance 3 to be detected, respectively, and specifically bind to them. In addition, the test substance detection method of the present invention includes a test strip 4 in which the first antibody 1 is immobilized on a determination unit (test line T), and a labeled second antibody in which the second antibody 2 is labeled with a labeling substance 5. 6 are prepared. Then, after developing the test solution containing the test substance 3 and the labeled second antibody 6 on the test strip 4, an antibody that recognizes the same site as the recognition site of the first antibody 1 is used as a sensitizer that emits a predetermined signal. One of the features is that the sensitizer 7 to which the test substance 3 is bonded is developed. Hereinafter, in the description of FIGS. 1 to 3, the case where the first antibody 1 is used as the antibody constituting the sensitizer 7 and the labeling substance 5 is used as the sensitizer will be described as an example.

  In detecting the test substance 3 in the test solution, first, the test solution and the labeled second antibody 6 are mixed, then absorbed on one end of the membrane 8 constituting the test strip 4 and developed. When the test substance 3 is present in the test solution, the complex of the test substance 3 and the labeled second antibody 6 formed in the mixture moves in the direction indicated by the arrow A as shown in FIG. Then, it is captured by the first antibody 1 (test line T) immobilized on the membrane 8. As a result, a complex composed of the first antibody 1-test substance 3-labeled second antibody 6 is formed. On the other hand, when the test substance 3 is not present in the test solution, the labeled second antibody 6 cannot form a complex and passes through the test line T without being captured by the first antibody 1. Up to this point, it is the same as the detection method using the conventional immunochromatography method.

  In the present invention, next, as shown in FIG. 3, a solution containing the sensitizer 7 is absorbed at one end of the membrane 8 and developed. When the labeled second antibody 6 is captured on the test line T, the first antibody 1 constituting the sensitizer 7 binds to the labeled second antibody 6 in a state where the test substance 3 is sandwiched. Sensitizer 7 is captured by labeled second antibody 6. As a result, a complex composed of the first antibody 1-test substance 3-labeled second antibody 6-sensitizer 7 is formed. As a result of the capture of the sensitizer 7, the accumulation amount of the labeling substance 5 in the test line T is increased, so that sensitization is performed. Therefore, the detection sensitivity of the immunochromatography method can be improved by the present invention.

  Sensitizer 7 and labeled second antibody 6 bind in any of the following states. One is that the portion of the antigen-binding site of the labeled second antibody 6 (second antibody 2) to which the test substance 3 is not bound binds to the test substance 3 constituting the sensitizer 7. The other is that the test substance 3 derived from the test solution bound to the labeled second antibody 6 is bound to the test substance 3 among the antigen binding sites of the first antibody 1 constituting the sensitizer 7. This is due to binding with no site.

  Since the test substance 3 constituting the sensitizer 7 is already bound to the first antibody 1 constituting the sensitizer 7, it does not bind to the first antibody 1 fixed to the membrane 8. For this reason, when the labeled second antibody 6 is not captured on the test line T, that is, when the test substance 3 is not present in the test solution, a signal caused by the sensitizer 7 is generated on the test line T. Never do. Therefore, since the detection method of the present invention enhances the signal intensity at the test line T only when the test substance 3 is present in the test solution, accurate detection is possible.

  The method for preparing the sensitizer used in the detection method as described above is arbitrary. For example, the sensitizer is a sensitizer that emits a predetermined signal, labels the antibody that recognizes the same part of the first antibody and the test substance, mixes the obtained labeled antibody and the test substance, and binds them. Prepared.

  As the antibody constituting the sensitizer, any antibody whose recognition site for the test substance is the same as that of the first antibody can be used. However, since the material is easily available, the first antibody is used as the antibody. It is preferable. For example, when hCG is detected as a test substance and a HαS antibody is immobilized on a test strip, it is preferable to use a HαS antibody as an antibody constituting the sensitizer.

  As the sensitizer constituting the sensitizer, any substance which emits a predetermined signal and is used as a labeling substance in a normal immunochromatography method can be used. However, in order to enable rapid and simple detection, it is preferable to use a coloring substance or a luminescent substance that emits a signal detectable with the naked eye as a sensitizing substance, and it is particularly preferable to use a coloring substance. Specific examples of the coloring substance include colloidal metal having a specific absorption band in the visible region such as gold colloid, liposomes including a coloring agent such as latex fine particles and silver fine particles. Further, when carrying out measurement with higher sensitivity, it is preferable to use a luminescent substance, a fluorescent substance, or the like as a sensitizer, and more specifically, fluorescent fine particles. However, when using a luminescent substance or a fluorescent substance as a sensitizer, a measuring instrument may be required.

  As the sensitizer (sensitizer), the same material as the label substance constituting the labeled second antibody can be used. In this case, since substantially the same signal is emitted from the sensitizer and the labeling substance, it is possible to reliably obtain the effect of improving the detection sensitivity by enhancing the signal. In addition, it is preferable to use the same material for the sensitizing substance and the labeling substance from the viewpoint of easy availability of the material. When the same kind of material is used for the sensitizing substance and the labeling substance, the respective dimensions (particle diameters) may be the same or different. In the case where the same kind of coloring fine particles are used for the sensitizer and the labeling substance, changes in the coloring wavelength (peak shift) or changes in the coloring wavelength intensity are observed by making the particle diameters different from each other.

  The labeling substance and the sensitizing substance constituting the labeled second antibody can be made of different materials. When the sensitizer and the labeling substance are different materials, examples of the combination of the sensitizer and the labeling substance include gold colloid-fluorescent fine particles, heterogeneous fluorescent fine particles, gold colloid-silver fine particle-containing liposomes, protein color former-silver. Fine particle inclusion liposome etc. are mentioned. In the case of a combination of different kinds of fluorescent fine particles, it is possible to further improve the detection sensitivity by detecting the fluorescence emitted using the principle of FRET (Fluorescence Resonance Energy Transfer).

  When using the FRET principle, beads coated with a fluorescent material, QD (quantum dots), or the like can be used as a labeling substance and a sensitizing substance. Specifically, the beads are coated with a chemical substance (for example, Chromeon 546 etc.) that is excited by light having a wavelength of 546 nm and emits fluorescence at around 561 nm, and this is bound to an antibody to form a labeled second antibody. On the other hand, the beads are coated with a chemical substance (for example, Chromeon 642) that is excited by the fluorescence emitted from the labeled second antibody and emits fluorescence at around 660 nm, and this is bound to the antibody and the test substance is bound. Use as a sensitizer. In this case, when the labeled second antibody and the sensitizer are bound (adjacent), the fluorescence intensity near 561 nm becomes weak and the fluorescence intensity near 660 nm becomes strong. it can.

  The abundance ratio between the antibody and the test substance in the sensitizer is arbitrary, but from the standpoint of ensuring a reliable signal enhancement effect when detecting test solutions of any test substance concentration, It is preferable to have both an antigen binding site to which is bound and an antigen binding site to which the test substance is not bound. A preferable ratio of the antigen binding site to which the test substance is bound to the antigen binding site to which the test substance is not bound is approximately 1: 1.

  For example, if there are too many antigen-binding sites to which the test substance is not bound in the sensitizer, it may be difficult to enhance the signal when testing a test solution having a low test substance concentration. This is because it becomes difficult for the sensitizer to bind to the labeled second antibody as a result of the amount of the test substance presented on the surface of the labeled second antibody being reduced. On the other hand, if the number of antigen binding sites to which the test substance is bound is relatively large, accurate detection may be difficult. This is because the surplus test substance generated when preparing the sensitizer is combined with the labeled second antibody remaining upstream of the test line T, and then developed and captured by the immobilized first antibody. As a result, a signal is emitted on the test line T even though the test substance is not present in the test solution. In addition, signal enhancement may be difficult when the test substance in the test solution is at a high concentration

  In addition, “in the sensitizer, both the antigen binding site to which the test substance is bound and the antigen binding site to which the test substance is not bound” means, for example, an individual sensitizer molecule or an individual antibody. Does not mean that both the antigen-binding site to which the test substance is bound and the antigen-binding site to which the test substance is not bound are present, but when the whole amount of the sensitizer used in the actual test is viewed Represents a state where both an antigen binding site to which the test substance is bound and an antigen binding site to which the test substance is not bound exist.

  When storing the sensitizer, for example, when the sensitizer is dissolved in a buffer solution such as a phosphate buffer, it is preferably stored in an environment of 4 ° C. or lower.

  The sensitizer may be developed on the strip in a state dissolved in, for example, a phosphate buffer containing 1% bovine serum albumin.

  The labeled second antibody is prepared by labeling the second antibody with a labeling substance. The second antibody may be an antibody that recognizes a site different from the first antibody and specifically binds to the test substance. For example, when hCG is detected as a test substance and a HαS antibody is used as the first antibody, an antibody that recognizes the β-subunit of hCG (hCG antibody) is preferably used as the second antibody.

  As a specific labeling substance constituting the labeled second antibody, any substance that emits a predetermined signal and is used as a labeling substance in a normal immunochromatography method can be used. However, in order to enable quick and simple detection, it is preferable to use a coloring substance or a luminescent substance that emits a signal that can be detected with the naked eye as a labeling substance, and it is particularly preferable to use a coloring substance. Specific examples of the color developing substance include colloidal metals such as gold colloid, liposomes including color formers such as latex fine particles and silver fine particles. Further, when carrying out measurement with higher sensitivity, it is preferable to use a luminescent substance, a fluorescent substance, or the like as a labeling substance, and more specifically, fluorescent fine particles or the like. However, when a luminescent substance or a fluorescent substance is used as a labeling substance, a measuring instrument may be required.

  The labeled second antibody may be mixed with a test solution before dropping or absorbing the test strip, or may be held in a state that can be developed upstream of the test line in the test strip.

  As the test strip, any test strip used in this type of immunochromatography method can be used without limitation as long as it has a determination unit (test line) on which the first antibody is immobilized. The membrane constituting the test strip is not particularly limited, and for example, nitrocellulose or the like can be used. Immobilization of the first antibody on the membrane may be performed according to a conventional method, for example, it may be applied. The shape of the test line T is a strip shape substantially orthogonal to the development direction in FIG. 1, but is not limited to this.

  As shown in FIG. 4, the test strip 4 preferably includes a control line C for capturing the labeled second antibody 6 that has not been captured by the test line T on the downstream side of the test line T. The control line C is configured by applying and immobilizing a third antibody 9 that recognizes the labeled second antibody 6. By observing a signal (coloring or luminescence) from the labeling substance in the control line C, the end of the test is indicated.

  As shown in FIG. 4, the test strip 4 is preferably provided with an absorption pad 10 at the downstream end of the membrane 8, and here it is preferable to absorb excess developing solution and the like.

  The immunochromatography kit of the present invention comprises at least a test strip, a labeled second antibody and a sensitizer as described above. By using the kit of the present invention, high-sensitivity detection of a test substance in a test solution can be performed quickly and easily.

  Moreover, the immunochromatography kit of the present invention may include a sensitizer in a raw material state. In this case, the immunochromatography kit comprises at least a test strip, a labeled second antibody, a labeled antibody obtained by labeling an antibody that recognizes the same site of the first antibody and the test substance with a sensitizer, and the test substance. Composed. In testing, a sensitizer may be prepared by mixing a labeled antibody and a test substance. For example, when the sensitizing substance is gold colloid or fluorescent fine particles, the kit can be stored at room temperature by freeze-drying the test substance and the labeled antibody. Even when the test substance and the labeled antibody are freeze-dried, it is preferably stored in a low-temperature environment such as 4 ° C. for long-term storage.

  When a substance that is difficult to detect with the naked eye (for example, a fluorescent substance, a luminescent substance, etc.) is used as a sensitizer and a labeling substance, the immunochromatography kit may further include a measuring instrument that can detect a signal from the substance. preferable.

Examples of the present invention will be described below with reference to experimental results.
In this example, a model experiment was performed using HCG (human gonadotropin), which is a pregnancy test marker, as a test substance. As the first antibody, a monoclonal antibody (anti-HαS antibody) that recognizes the α-subunit of HCG was used. As the second antibody, a monoclonal antibody (anti-HCG antibody) that recognizes the β-subunit of HCG was used.

(Experiment 1)
In Experiment 1, the effect of the sensitizer was examined.
First, an anti-HαS antibody was applied to the region corresponding to the membrane test line, and an anti-mouse IgG antibody was applied to the region corresponding to the control line. Thereafter, blocking and washing were performed to obtain a test strip.

  Further, an anti-HCG antibody and a colloidal gold solution (Tanaka Kikinzoku, particle size 40 nm) were mixed to prepare a colloidal gold-labeled anti-HCG antibody as a labeled second antibody. O.D. of solution containing colloidal gold labeled anti-HCG antibody D. The value (580 nm) was adjusted to 6.0.

  The sensitizer was prepared as follows. First, gold colloids with different particle sizes (40 nm and 80 nm) were labeled on the anti-HαS antibody, respectively. O. of the solution. The D value (580 nm) was adjusted to 6.0. The gold colloid-labeled anti-HαS antibody-containing solution thus obtained was mixed with 40 μl of an HCG solution adjusted to a concentration of 1 ng / ml to obtain a sensitizer.

(Comparative example)
HCG was detected using the test strip, gold colloid-labeled anti-HCG antibody and sensitizer. First, 4 μl of the gold colloid-labeled anti-HCG antibody was added to and mixed with 40 μl of the antigen solution (HCG concentrations: 0.1 ng / ml, 0.05 ng / ml, 0.025 ng / ml). The mixed solution was absorbed into the test strip, and the coloration due to the accumulation of colloidal gold in the test line was observed. The results are shown in FIG. In FIG. 5, a line indicated by an arrow corresponds to a test line.

(Example)
In the examples, a sensitizer was developed on the test strip after the operation of the comparative example was performed. Specifically, a sensitizer was prepared by mixing 40 μl of an HCG solution adjusted to a concentration of 1 ng / ml and 4 μl of the gold colloid-labeled anti-HαS antibody-containing solution, and added to the test strip after the operation of the comparative example. The solution containing the sensitizer was absorbed and developed. Thereafter, coloration due to accumulation of colloidal gold in the test line was observed. The results are shown in FIG.

(result)
As is clear from FIG. 5, in the comparative example in which the mixture of the test solution and the colloidal gold-labeled anti-HCG antibody was developed, red coloration on the test line was hardly confirmed, and detection of HCG was difficult. It was.

  In contrast, as shown in FIG. 6, by developing a sensitizer containing gold colloid with a particle size of 40 nm, the color development on the test line is enhanced and detection of HCG at a concentration of 0.05 ng / ml is realized. It was done. In addition, when a sensitizer containing a gold colloid with a particle size of 80 nm is developed, it is possible to detect a lower concentration of HCG (0.025 ng / ml). The enhancement of color development on these test lines is because the sensitizer (gold colloid-labeled first antibody) is further bound to the colloidal gold-labeled anti-HCG antibody captured on the test line, and the amount of colloidal gold accumulated on the test line is reduced. Presumed to be due to an increase.

  As shown by the above experimental results, it was possible to detect a low concentration of antigen (HCG) that was sensitized by developing a sensitizer and could not be detected by ordinary immunochromatography.

(Experiment 2)
In Experiment 2, the binding between the sensitizer and the colloidal gold-labeled anti-HCG antibody was analyzed using an analyzer manufactured by BIACORE.

First, an antibody (anti-HαS antibody) having the same concentration as the first antibody immobilized on the membrane by a normal immunochromatography method was physically adsorbed on the Au substrate of the sensor chip. As a sample, colloidal gold (particle size 40 nm) labeled anti-HCG antibody and HCG having a concentration of 0.02 nm / ml were mixed and allowed to flow. Thereafter, a sensitizer (gold colloid-labeled first antibody) using a gold colloid having a particle size of 40 nm was flowed. The mass change caused by the binding and dissociation of two molecules on the surface of the sensor chip at this time was measured using surface plasmon resonance. The results are shown in FIG. In FIG. 7, the vertical axis represents mass change on the surface of the sensor chip, and the horizontal axis represents time. The unit of the vertical axis is represented by Resonance unit (RU), and 1RU corresponds to 1 pg / mm ² .

  As shown in FIG. 7, first, the binding between the colloidal gold-labeled anti-HCG antibody and the first antibody (anti-HαS antibody) was observed. Next, it was confirmed that a sensitizer (gold colloid-labeled first antibody) was further bound to the complex of the gold colloid-labeled anti-HCG antibody and the first antibody (anti-HαS antibody). The amount of gold colloid-labeled anti-HCG antibody bound is represented by the difference between the 2 and 3 lines in FIG. The binding amount of the sensitizer is represented by the difference between the 1 line and the 2 line in FIG.

It is an example of the detection method of the test substance of this invention, and is a principal part expansion perspective view which shows the test line vicinity before test solution expansion | deployment. It is an example of the detection method of the to-be-tested substance of this invention, and is a principal part expansion perspective view which shows the test line vicinity after developing a test solution and a labeled 2nd antibody. It is an example of the detection method of the test substance of this invention, and is a principal part expansion perspective view which shows the test line vicinity after developing a sensitizer. It is a schematic perspective view which shows an example of the test strip used for this invention. It is a photograph which shows the test result of a comparative example. It is a photograph which shows the test result of an Example. It is a characteristic view which shows the result of having analyzed the coupling | bonding of a sensitizer etc.

Explanation of symbols

1 First antibody, 2nd antibody, 3 test substance, 4 test strip, 5 labeled substance, 6 labeled 2nd antibody, 7 sensitizer, 8 membrane

Claims (12)

For a test strip in which a first antibody that recognizes a predetermined site of a test substance is immobilized, a test substance and a second antibody that recognizes a site different from the predetermined site of the test substance are bound to a labeling substance. After developing the labeled second antibody,
A method for detecting a test substance, comprising: developing a sensitizer obtained by binding a test substance to an sensitizer that emits a predetermined signal via an antibody that recognizes the predetermined site of the test substance.   The method for detecting a test substance according to claim 1, wherein the first antibody is used as an antibody constituting the sensitizer.   The method for detecting a test substance according to claim 1 or 2, wherein at least one selected from a coloring substance and a luminescent substance is used as the sensitizing substance.   The method for detecting a test substance according to claim 1, wherein the sensitizing substance and the labeling substance are made of the same kind of material.   The method for detecting a test substance according to any one of claims 1 to 3, wherein the sensitizing substance and the labeling substance are made of different materials.   6. The method for detecting a test substance according to claim 5, wherein the combination of the sensitizing substance and the labeling substance is a gold colloid-silver fine particle-containing liposome.   6. The method for detecting a test substance according to claim 5, wherein different fluorescent fine particles are used for the sensitizing substance and the labeling substance, and fluorescence emitted using the principle of fluorescence resonance energy transfer is detected.   8. The sensitizer according to any one of claims 1 to 7, wherein both an antigen binding site to which a test substance is bound and an antigen binding site to which the test substance is not bound exist. A method for detecting a test substance.   9. The test substance according to claim 8, wherein the ratio of the antigen binding site bound to the test substance in the sensitizer and the antigen binding site not bound to the test substance is approximately 1: 1. Detection method. For a test strip in which a first antibody that recognizes a predetermined site of the test substance is immobilized, a test substance and a second antibody that recognizes a site different from the predetermined site of the test substance are bound to a labeling substance. Used in an immunochromatographic method for developing a labeled second antibody,
A sensitizer, wherein a test substance is bound to a sensitizer that emits a predetermined signal via an antibody that recognizes the predetermined site of the test substance. A test strip in which a first antibody that recognizes a predetermined site of a test substance is immobilized;
A labeled second antibody in which a second antibody that recognizes a site different from the predetermined site of the test substance is bound to a labeling substance;
An immunochromatography kit comprising: a sensitizer that binds a test substance to an sensitizer that emits a predetermined signal via an antibody that recognizes the predetermined site of the test substance. A test strip on which a first antibody that recognizes a predetermined site of a test substance is immobilized;
A labeled second antibody in which a second antibody that recognizes a site different from the predetermined site of the test substance is bound to a labeling substance;
A labeled antibody obtained by labeling an antibody that recognizes the predetermined site of the test substance with a sensitizer that emits a predetermined signal;
A kit for immunochromatography comprising a test substance.

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