JP2008032494A - Measuring method and measuring kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple measuring method having high sensitivity and quantification properties. <P>SOLUTION: The measuring method has an immunoreaction process for successively bringing a mixed liquid, which is prepared by mixing a competitive substance with a specimen solution to be examined containing a target substance, into contact with a competitive capture substance and a judge substance, an enzymatic reaction process for bringing a substrate of enzyme possessed by the competitive substance into contact with at least one of the competitive capture substance and the judge substance to obtain a reaction substance and a calculation process for measuring the amount of the reaction substance to calculate the amount of the target substance. That is, detection sensitivity is enhanced by bonding enzyme in order to quantify the concentration of the target substance in immunochromatography to amplify the concentration corresponding to the target substance. Further, This measuring method can be preferably adapted to the target substance low in molecular weight. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an immunoassay method and a measurement kit capable of measuring a target substance with high sensitivity and simple operation.

  Conventionally, methods utilizing various principles have been developed as methods for measuring trace substances (for example, Patent Documents 1 and 2). Among them, in recent years, methods using an immune reaction, such as immunochromatography and ELISA, have been developed as one of highly sensitive measurement methods. An analysis method using instrumental analysis has also been proposed. In particular, immunochromatography is used in various inspection fields because it is easy to operate and has a short time to detection.

  Immunochromatography is characterized by the simplicity of simply immersing the sample solution in a chromatographic chip or medium prepared as a sample. The reaction solution travels through the membrane by capillary action, binds to the colloidal gold labeled antibody embedded in the membrane, and further binds to the trap antibody at the downstream detection unit to express the presence or absence of the target substance.

  Here, as a feature of immunochromatography, since detection by color development of colloidal gold particles is common, the lower limit of detection is on the order of ppm. However, although it is excellent in convenience, it is mainly qualitative detection by visual observation and its quantitativeness is poor.

  As a feature of the ELISA method, the detection sensitivity is generally good, and it can be quantified by adding a control to the test group. However, there are drawbacks in that the operation is complicated and errors and variations due to the skill level of the measurer are large. In addition, a large amount of reaction reagent and solution are required for working hours as long as several hours.

The characteristic of instrumental analysis is that the highest sensitivity can be expected among the current technologies. On the other hand, expensive equipment is required and engineers with high expertise are also required. Further, since the detection sensitivity is high, the test sample is easily affected by a small amount of impurities, and it is necessary to perform complicated pretreatment on the test sample.
Japanese Patent No. 3005303 JP 2004-138550 A

  The present invention has been completed in view of the above circumstances, and on the convenience of immunochromatography, a measurement method having high sensitivity and quantitative properties, such as an ELISA method and an instrumental analysis method, and a measurement applying the measurement principle. Providing kits for use is a problem to be solved.

Means for Solving the Problems and Effects of the Invention

(1) The measurement method of the present invention that solves the above problems includes an immune reaction step in which a mixed solution obtained by mixing the following competitive substance in a test sample solution containing a target substance is contacted in the order of the following competitive capture substance and the following determination substance. ,
An enzyme reaction step of obtaining a reaction product by contacting at least one of the competitive capture substance and the determination substance with an enzyme substrate provided in the competitive substance;
A calculation step of measuring the amount of the reactant and calculating the amount of the target substance;
It is characterized by having.

  In addition, the measurement kit of the present invention that solves the above-described problems includes the following competitive substance that is brought into contact with the following competitive substance to be mixed with a test sample liquid containing a low molecular weight target substance, and the mixed liquid of the test sample liquid and the competitive substance. A capture substance, a determination substance that contacts the mixed solution after the competitive capture substance, and a substrate of an enzyme included in the competitive substance that contacts at least one of the competitive capture substance and the determination substance And a treatment liquid.

Here, the competitive substance, the competitive capture substance, and the determination substance are as follows.
Competitor: A substance in which an enzyme that promotes a predetermined substrate reaction is bound to one of an antibody that can bind to the target substance and the target substance or a similar substance.
Competitive capture substance: An antibody that can bind to the target substance and a substance that is the other of the target substance or similar substance and is separable from the contacted liquid mixture.
Determination substance: A substance that is an antibody that can bind to the competitive substance and is separable from the mixed solution in contact therewith.

  In other words, in order to quantify the concentration of the target substance in immunochromatography, the detection sensitivity could be improved by binding an enzyme capable of chemically amplifying the signal and amplifying the concentration corresponding to the target substance. In addition, the measurement method using a competitive reaction like the method of the present invention can be suitably applied to a low molecular weight target substance. The “low molecular weight” mentioned here means that the molecular weight is such that a sandwich method widely used in immune reactions cannot be applied. In the so-called competitive method employed in the measurement method and measurement kit of the present invention, it was considered that chemical amplification was difficult. In addition, the “competitive reaction (competition method)” here includes a narrowly defined competitive reaction (competition method) in which the competitive substance and the target substance react reversibly, and the competitive substance and the target substance are bound to each other. It is a competing reaction (competitive method) in a broad sense, including an inhibitory reaction (inhibiting method).

  Here, high detection sensitivity can be realized by adjusting the abundance ratio between the competitive substance and the competitive capture substance in consideration of the assumed detection range of the target substance as the measurement target.

  That is, it is desirable that the molar ratio (detection sensitivity of the target substance) :( determination substance) in the immune reaction between the target substance and the determination substance is 1: 1 to 1: 0.01.

In addition, the molar ratio in the immune reaction of the target substance and the competitor (target substance detection sensitivity): (competitor) is 1: 1 to 1: 0.001.
The molar ratio of the target substance and the competitive capture substance in the immune reaction (detection sensitivity of the target substance): (competitive capture substance) is preferably 1: 1 to 1: 0.01. Here, the detection sensitivity of the target substance is the number of moles or the molar concentration of the detection limit that can be measured.

  That is, in the measurement method using a competitive reaction as in the method of the present invention, the target substance and the competitive substance and the competitive substance and the competitive capture substance, or the target substance and the competitive capture substance and the competitive substance and the competitive capture are used. An immune reaction progresses competitively with the substance, and the competitive substance is captured by the competitive capture substance according to the amount of the target substance. At this time, the competing target substance is quantified by directly or indirectly measuring the amount of the competing substance captured by the competitive capture substance. Therefore, a desirable abundance ratio as described above can be set between the target substance, the competitive substance, and the competitive capture substance.

  Here, it is preferable that the competitive capture substance and the determination substance are immobilized on a solid support. The form of the solid carrier is not limited, and includes fine particles and fibers. In particular, the solid support on which the competitive capture substance and the determination substance are immobilized is preferably arranged on the same thin film. The target substance can be easily quantified simply by developing the test sample solution mixed with the competitive substance from the competitive capture substance side on the thin film. The competing substance can also be arranged on the thin film in advance.

(2) The measurement method of the present invention that solves the above-described problem is obtained by mixing a mixture solution obtained by mixing the following competitive substance with a test sample solution containing a low molecular weight target substance, and a plurality of the following competitive captures having different molar concentrations in the immune reaction. An immune reaction step in which each of the substances is contacted independently and then contacted with each of the following determination substances;
A calculation step of calculating the amount of the target substance by measuring the amount of the competitive substance bound to at least one of the set of the competitive capture substance and the determination substance;
It is characterized by having.

  In addition, the measurement kit of the present invention that solves the above-mentioned problems is a substance that allows the following competitive substance to be mixed with a test sample solution containing a low-molecular-weight target substance and the mixed liquid to contact each other independently and in an immune reaction. A plurality of the following competitive capture substances having different molar concentrations, and the following determination substance for bringing the mixed solution brought into contact with the plurality of competitive capture substances into contact with each other independently.

  Here, the competitive substance is one of an antibody that can bind to the target substance and the target substance or a similar substance. The competitive capture substance is an antibody that can bind to the target substance and a substance that is the other of the target substance or a similar substance and is separable from the contacted mixed solution. The determination substance is an antibody that can bind to the competitive substance and is separable from the contacted mixed solution.

  By employing a plurality of competitive capture substances having different concentrations, the dynamic range of the concentration that reacts with the target substance is expanded. In particular, in the method of the present invention using a competitive reaction, when the concentration of the target substance exceeds a predetermined concentration, the competitor substance flows out and binds to the determination substance. Since the predetermined concentration is determined according to the concentration of the competitive capture substance, the concentration of the target substance can be estimated by adopting a plurality of concentrations of the competitive capture substance. In other words, since the amount of competitive substance binding to each judgment substance and competitive capture substance varies greatly from the target substance concentration according to the concentration of these competitive capture substances, the target substance concentration can be easily measured. can do.

  Here, it is preferable that the competitive capture substance and the determination substance are immobilized on a solid support, and the competitive capture substance and the determination substance are arranged side by side at different molar concentrations.

  (3) The measurement kit of the present invention that solves the above-described problems is a competitive substance that is a substance that is developed and brought into contact with the following competitive substance to be mixed with a test sample solution containing a target substance, and the mixed liquid is developed from one side. And the following competitive capture substance immobilized on the thin film in excess of the molar equivalent in the immune reaction against

  Here, the competitive substance is one of an antibody that can bind to the target substance and the target substance or a similar substance, and the competitive capture substance is an antibody that can bind to the target substance, and the target substance. Or it is the substance which is the other of the similar substances, and can be separated from the said mixed liquid contacted.

  In other words, when a mixed solution of a test sample solution and a competitive substance is developed from one of an immobilized amount of a competitive capture substance relative to the competitive substance in a thin film state, the larger the amount of the target substance, the more the side that started the development. Since the competitive substance is bound and captured on the side away from the target, the amount of the target substance can be calculated by measuring the position where the competitive substance is bound and captured.

  The measurement method and measurement kit of the present invention will be described in detail below. The target substance to be measured by the measurement method and measurement kit of the present invention is contained in the test sample solution. The test sample solution is not particularly limited, but it is desirable that the test sample solution be prepared with an appropriate buffer solution. Since the test sample solution may be contained as long as it does not affect the immune reaction and the detection / quantitative reaction, the purification process can be simplified. Although it does not specifically limit as a target substance, It can apply suitably with respect to a low molecular weight compound.

(First embodiment: measurement method and measurement kit)
The measuring method of this embodiment has an immune reaction process, an enzyme reaction process, and a calculation process. The immune reaction step is a step in which a mixed liquid obtained by mixing a competitive substance with a test sample solution is contacted in the order of a competitive capture substance and a judgment substance.

  The competitive substance is an antibody that can bind to the target substance and a substance obtained by binding an enzyme that promotes a predetermined substrate reaction to one of the target substance and a similar substance. Here, as the enzyme to be bound, known enzymes such as alkaline phosphatase and peroxidase can be employed. The competitive substance can be supported on some solid support in addition to the form mixed with the test sample solution. Here, since the enzyme is introduced into the competitor for the purpose of quantifying the amount of the competitor by an enzyme reaction, it is sufficient that the enzyme is bound to the competitor during the enzyme reaction step. In other words, it is possible to bind the enzyme specifically to the competitor as in the avidin-biotin reaction, as well as to allow the enzyme to be bound by covalent bond from the beginning (before the immune reaction step). A process that binds the enzyme after the immune reaction process and before the enzyme reaction process (for example, a reagent that can introduce the enzyme is prepared, the reagent is brought into contact, and can be introduced into the competitor as necessary. This can be realized by removing or deactivating the enzyme that was not present) and then binding.

  A competitive capture substance is the other of an antibody that can bind to a target substance and a target substance or a similar substance. That is, when the competitive substance is an antibody that can bind to the target substance, it is a target substance or a similar substance, and when the competitive substance is a target substance or a similar substance, it is an antibody that can bind to the target substance. This substance is a substance that can be separated from the liquid mixture. For example, it can be made separable by being immobilized on an insoluble solid support. Although it does not specifically limit as a solid support | carrier, A fine powder and fibrous things, such as a silica gel, a cellulose, or those derivatives, can be illustrated.

  The determination substance is an antibody that can bind to the competitive substance. For example, an antibody that specifically binds to an enzyme included in the competitor can be used. The determination substance is also a substance that can be separated from the mixed solution in the same manner as the competitive capture substance. For example, it can be made separable by being immobilized on an insoluble solid support.

  Here, the amount of the competitive substance and the competitive capture substance is not particularly limited, but a suitable range can be set based on the detection sensitivity of the target substance. Specifically, the molar ratio in the immune reaction between the target substance and the competitor (target substance detection sensitivity): (competitor) is preferably 1: 1 to 1: 0.001, preferably 1: 1 to 1: 0.01. Is more preferable. The molar ratio of the target substance and the competitive capture substance in the immune reaction (target substance detection sensitivity): (competitive capture substance) is preferably 1:10 to 1: 0.01, and 1: 1 to 1: 0. .1 is more preferable.

  It is desirable that the competitive trapping substance and determination substance are immobilized on a solid support and arranged on a thin film, in a capillary, in a column, or the like. And a liquid mixture can be made to contact in order of a competitive capture substance and a determination substance by expand | deploying a liquid mixture from the side which arrange | positioned the competitive capture substance. When such a configuration is adopted, it is desirable that the competing substance is also carried on the part before reaching the competitive capturing substance.

  When the mixed solution of the test sample solution and the competitive substance comes into contact with the competitive capture substance, the competitive substance contained therein is bound to and captured by the competitive capture substance according to the amount of the target substance contained in the test sample liquid. The That is, the competitive substance is bound and captured by the competitive capture substance in the absence of the target substance, and the competitive substance flows to the determination substance without binding to the competitive capture substance as the amount of the target substance increases. In the determination substance, a competitive substance that is not bound or captured by the competitive capture substance is bound or captured.

  The enzyme reaction step is a step of obtaining a reaction product by bringing a substrate (corresponding to an enzyme included in the competitive substance) into contact with at least one of the competitive capture substance and the determination substance.

  The calculation step is a step of calculating the amount of the target substance by measuring the amount of the generated reactant. It does not specifically limit as a method of measuring the quantity of a reaction material. For example, measurement of absorbance or fluorescence change can be mentioned. As a method for measuring absorbance or fluorescence, a method based on image analysis is exemplified. The amount of the target substance is calculated from the result of quantifying the amount of the reactant. For example, the target substance can be calculated using a calibration curve prepared in advance.

(Second Embodiment: Measurement Method and Measurement Kit)
The measurement method of this embodiment is substantially the same as the measurement method described in the first embodiment, but differs in the following points. That is, a competitive capture substance and a determination substance have a plurality of sets in parallel with varying concentrations of the competitive capture substance, that the enzyme is not necessarily bound as a competitive substance, and The three points are that the enzyme reaction step is not essential.

  In other words, the concentration at which the competitive capture substance is saturated is changed by arranging the competitive capture substances having different concentrations in parallel and reacting the mixed solution sequentially. As a result, depending on the concentration of the competitive capture substance, the amount of the target substance contained in the test sample solution when the competitive substance moves from the competitive capture substance to the determination substance will differ, and the amount of the target substance will be determined. Becomes easier.

  More specific description will be given below. In the measuring method of the present invention, the target substance and the competitive capture substance (or the competitive substance and the target substance) are competitively bound to the competitive substance (or competitive capture substance), and the competitive capture substance is bound. The amount of the target substance is indirectly measured by measuring the amount of the competitive substance that could not be bound. Therefore, if the concentration of the competitive capture substance is decreased, the amount (concentration) of the competitive substance and the amount (concentration) of the target substance do not change. Therefore, even if the amount of the target substance is relatively small, many competitor substances are judged substances. Will be combined and captured. On the other hand, when the concentration of the competitive capture substance is increased, the amount of the target substance is relatively reduced, so that the amount of the competitive substance flowing to the determination substance is reduced. In other words, if the concentration of the competitive capture substance is increased, it can be handled in the same way as when the detection limit of the target substance is increased, so the concentration of the competitive capture substance can be detected at an appropriate stage. By changing stepwise, the amount of the target substance can be determined without quantifying the amount of the competitive substance in the determination substance.

  In the present embodiment, the presence of the enzyme is not essential for the competing substance. Therefore, when the enzyme is contained and the enzyme amplification is not performed, a conventional configuration such as a gold colloid can be employed as it is. A desirable form is a form in which an enzyme is bound to a competitor and an enzyme reaction step is provided, as in the first embodiment.

(Third embodiment: measurement method and measurement kit)
The measurement kit of this embodiment has a competitive substance and a competitive capture substance. The competitive substance can adopt the same configuration as that described in the first embodiment or the second embodiment.

  The compound to be contained as the competitive capture substance is the same as that described in the first embodiment and the second embodiment, but the arrangement form and the arrangement quantity are different. That is, the amount of the competitive capture substance is set to an excess amount than the amount of the competitive substance. Here, the determination of whether the amount is excessive is determined based on the molar ratio in the immune reaction. Excess amounts include, for example, more than 1 time, more than 1.5 times, more than 2 times, more than 2.5 times, more than 3 times based on the molar ratio in the immune reaction. This molar ratio can also be calculated experimentally.

  An excessive amount of competitive capture substance is immobilized in a thin film. Here, it is desirable that the competitive trapping substance is immobilized at a uniform concentration on the thin film.

  Since the measurement method and the measurement kit of the present invention have the above-described configuration, the position where the competitive substance is bound and captured changes as the amount of the target substance increases, and the position is measured. Thus, the amount of the target substance can be easily quantified.

(Detection of PCB as a target substance)
Preparation of antibody-enzyme complex (competitive substance) 50 μL of 0.1 mol / L β-mercaptoethylamine was added to 2 mg / PBS of coplana PCB antibody, and the mixture was allowed to stand at 37 ° C. for 90 minutes. Thereafter, the antibody reductant was isolated by gel filtration using a phosphate buffer (pH 6, 5 mM EDTA).

  30 μL of Sulfo-SMCC was added to 1 mg of alkaline phosphatase and allowed to stand for 1 hour. Thereafter, the remaining Sulfo-SMCC was removed with a desalting column to obtain maleimide-labeled alkaline phosphatase.

  By mixing equal amounts of the antibody reductant and maleimide-labeled alkaline phosphatase, the two reacted to obtain an alkaline phosphatase-labeled coplana PCB antibody (AP-labeled coplana PCB antibody).

-Immobilization of competitive capture substance and determination substance on thin film 0.1 μg of dichlorobenzene-streptavidin as a competitive capture substance was immobilized on the thin film by a drop drying method (FIG. 1: T2). Then, 1 μg of anti-mouse antibody as a determination substance was arranged and fixed on the thin film at a predetermined interval by a drop drying method (T1) to obtain an immunochromatographic device (part of a measurement kit). In FIG. 1, a cover is formed on the thin film, but the thin film itself is rectangular. A test sample solution or the like is dropped from the window 10 formed on the cover.

-Measurement A test sample solution was prepared by diluting PCB (KC-500) having a known concentration to various concentrations with DMSO. AP-labeled coplana PCB antibody was diluted with a dilution buffer (10 mM Phosphate, 150 mM NaCl, 0.1% casein), and 1/10 amount of the test sample solution was added. After allowing the reaction to proceed for 30 minutes at room temperature, 75 μL was dropped on the window 10 formed on the competitive capture substance side of the immunochromatographic device and allowed to stand for 20 minutes as the development time (immune reaction step). Thereafter, BCIP / NBT (manufactured by Nacalai Tesque) as a substrate diluted 1/100 with the attached dilution buffer was dropped into the window 10 as an aftertreatment solution in the same manner as the test sample solution (enzyme). Reaction step).

  The degree of color development in the determination substance was measured with an immuno server (model number: AIS-GP100) manufactured by Aisin Seiki Co., Ltd., and a calibration curve for PCB was prepared. The color development was measured by using two green LEDs irradiated through a filter having a wavelength of 500 nm to 600 nm from a diagonal direction of 45 ° as a light source, and detecting with a photodiode disposed above the sample.

・ Comparative example (see Patent Document 2)
Instead of binding enzyme, colloidal gold particles (average particle size 40 nm, BB International) were bound. The binding method was carried out according to the protocol of BB International. A calibration curve for PCB was prepared in the same manner as in Example 1 by employing the obtained colloidal gold particle labeled coplanar PCB antibody instead of the competitor.

  The results are shown in FIG. As is apparent from FIG. 2, a sensitivity difference of about two digits was recognized between Example 1 and the comparative example.

(Detection of biotin as a target substance)
-Immobilization of competitive capture substance and determination substance on thin film Three types of biotin-BSA as a competitive capture substance: 3 x ^10-5 mol, 3 x ^10-6 mol, and 3 x ^10-7 mol The amount was immobilized on a nitrocellulose thin film. Then, an anti-Rabbit antibody as a determination substance was immobilized at a concentration of 1 mg / mL on a thin film on which the biotin-BSA antibody was immobilized at a predetermined interval to obtain an immunochromatography device (part of a measurement kit). . The immobilization of the competitive capture substance and the determination substance was performed by dropping each on a thin film and drying at 50 ° C. for 1 hour.

Measurement / Biotin (target substance) at a known concentration was diluted to various concentrations with PBS buffer to prepare a test sample solution. Alkaline phosphatase-labeled anti-biotin antibody (Rabbit-derived: AP-labeled anti-biotin antibody) as a competitor is diluted with the aforementioned dilution buffer so that it becomes a predetermined amount (10 ⁻¹¹ mol to 10 ⁻⁵ mol), 1/10 amount of the test sample solution was added and reacted at room temperature for 30 minutes.

  Thereafter, 75 μL was dropped on the competitive capture substance side of the immunochromatographic device and allowed to stand for 20 minutes as the development time (immune reaction step). Then, 75 μL of BCIP / NBT (manufactured by Nacalai Tesque) as a substrate diluted 1/100 with the attached dilution buffer was dropped at the same place as the test sample solution (enzyme reaction step).

  The degree of color development in the determination substance was measured by the same method as in Example 1 using the above-mentioned immunoserver manufactured by Aisin Seiki Co., Ltd.

The results are shown in FIG. As is clear from FIG. 3, it was found that the lower the concentration of biotin-BSA as a competitive capture substance within the range of 3 × 10 ^{−5 to} 3 × 10 ⁻⁷ mol, the better. Then, it was found that the concentration of the AP-labeled anti-biotin antibody as a competitor is preferably about 10 ⁻¹⁰ to 10 ⁻⁷ mol, and more preferably about 10 ⁻¹⁰ to 10 ⁻⁸ mol.

  Further, instead of immobilizing biotin-BSA as a competitive capture substance on a thin film, an anti-BSA antibody may be immobilized, and biotin, biotin-BSA, and anti-biotin antibody (AP label) may be mixed as a competitive reaction. .

  An immunodevice as shown in FIG. 4 was prepared in substantially the same manner as in Example 1. Specifically, the amount of competitive capture substance was changed and immobilized at the position of T2 in Example 1 (T2 '). Then, the determination substances whose amount was changed were immobilized on the downstream side of the competitive capture substance whose concentration was changed (T1 '). Each of the three types of competitive capture substances and determination substance sets was separated and separated so that the test sample solution was developed independently.

Measurement In a microtube, mix 100 μL of AP-labeled PCB antibody (competitive capture substance: 1 ng / mL) / PBS and 10 μL of DMSO solution (test sample solution) containing 0.5 ppm, 5 ppm and 50 ppm of PCB. And left to stand for 10 minutes. Then, the whole amount was dripped at the window part 11 shown in FIG. 4, and left to stand for 10 minutes.

  Subsequently, 100 μl of BCIP / NBT solution was dropped from the L window 11 and left for 30 minutes. As a result, the portion where the AP-labeled PCB antibody was bound and captured developed color. The results are shown in FIGS. 4 (a) to 4 (c). As is clear from FIGS. 4A to 4C, the portion where the amount of the immobilized competitive capture substance or the like is small reacts with a lower concentration of the target substance, and the T1 ′ portion develops color. Sequentially, as the concentration of the target substance increased, the portion of T2 'corresponding to the portion where the concentration of the competitive capture substance was high developed. Therefore, by setting the concentration of competitive capture substances etc. so that color development proceeds within the target range and at the target concentration, it is possible to quickly test visually without measuring the degree of color development with an instrument. It was found that the amount of target substance contained in the sample solution can be measured.

It is the outline of the immuno device in the kit for a measurement used in Examples 1 and 2. It is a graph which shows the calibration curve of Example 1 and a comparative example. 6 is a graph showing detection limits measured in Example 2. 6 is a schematic diagram of an immuno device of a measurement kit used in Example 3. FIG.

Claims (13)

An immune reaction step in which a mixed liquid obtained by mixing the following competitive substance in a test sample solution containing a target substance is contacted in the order of the following competitive capture substance and the following determination substance;
An enzyme reaction step of obtaining a reaction product by contacting at least one of the competitive capture substance and the determination substance with an enzyme substrate provided in the competitive substance;
A calculation step of measuring the amount of the reactant and calculating the amount of the target substance;
A method for measuring a target substance, comprising:
Competitor: A substance in which an enzyme that promotes a predetermined substrate reaction is bound to one of an antibody that can bind to the target substance and the target substance or a similar substance.
Competitive capture substance: An antibody that can bind to the target substance and a substance that is the other of the target substance or similar substance and is separable from the contacted liquid mixture.
Determination substance: A substance that is an antibody that can bind to the competitive substance and is separable from the mixed solution in contact therewith.   The measurement method according to claim 1, wherein a molar ratio (detection sensitivity of the target substance) :( determination substance) in the immune reaction between the target substance and the determination substance is 1: 1 to 1: 0.01. The molar ratio in the immune reaction of the target substance and the competitor (target substance detection sensitivity): (competitor) is 1: 1 to 1: 0.001.
The molar ratio in the immune reaction between the target substance and the competitive capture substance (target substance detection sensitivity): (competitive capture substance) is 1: 1 to 1: 0.01. Measuring method.   The measurement method according to claim 1, wherein the competitive capture substance and the determination substance are immobilized on a solid support. The following competitor substance to be mixed with the test sample solution containing the target substance:
The following competitive capture substance in contact with the test sample solution and the mixture of the competitive substance,
The following determination substance for contacting the mixed solution after the competitive capture substance,
A post-treatment liquid containing an enzyme substrate of the competitive substance that is brought into contact with at least one of the competitive capture substance and the determination substance;
A measurement kit comprising:
Competitor: A substance in which an enzyme that promotes a predetermined substrate reaction is bound to one of an antibody that can bind to the target substance and the target substance or a similar substance.
Competitive capture substance: An antibody that can bind to the target substance and a substance that is the other of the target substance or similar substance and is separable from the contacted liquid mixture.
Determination substance: A substance that is an antibody that can bind to the competitive substance and is separable from the mixed solution in contact therewith.   The measurement kit according to claim 5, wherein a molar ratio (detection sensitivity of the target substance): (determination substance) in the immune reaction between the target substance and the determination substance is 1: 1 to 1: 0.01. The molar ratio in the immune reaction of the target substance and the competitor (target substance detection sensitivity): (competitor) is 1: 1 to 1: 0.001.
The molar ratio in the immune reaction between the target substance and the competitive capture substance (target substance detection sensitivity): (competitive capture substance) is 1: 1 to 1: 0.01. Measurement kit.   The measurement kit according to claim 5, wherein the competitive capture substance and the determination substance are immobilized on a solid support.   The measurement kit according to claim 8, wherein the solid support on which the competitive capture substance and the determination substance are immobilized is arranged on the same thin film. A mixture obtained by mixing the following competitive substance with a test sample solution containing the target substance is contacted independently with a plurality of competitive capture substances having different molar concentrations in the immune reaction, and then contacted with the following determination substances respectively. An immune reaction process;
A calculation step of calculating the amount of the target substance by measuring the amount of the competitive substance bound to at least one of the set of the competitive capture substance and the determination substance;
A method for measuring a target substance, comprising:
Competing substance: One of an antibody capable of binding to the target substance and the target substance or a similar substance.
Competitive capture substance: An antibody that can bind to the target substance and a substance that is the other of the target substance or similar substance and is separable from the contacted liquid mixture.
Determination substance: A substance that is an antibody that can bind to the competitive substance and is separable from the mixed solution in contact therewith. The following competitor substance to be mixed with the test sample solution containing the target substance:
A plurality of the following competitive capture substances that are the substances that contact the mixed solution independently and have different molar concentrations in the immune reaction;
The following determination substances that contact each of the mixed solutions contacted with the plurality of competitive capture substances independently,
A measurement kit comprising:
Competing substance: One of an antibody capable of binding to the target substance and the target substance or a similar substance.
Competitive capture substance: An antibody that can bind to the target substance and a substance that is the other of the target substance or similar substance and is separable from the contacted liquid mixture.
Determination substance: A substance that is an antibody that can bind to the competitive substance and is separable from the mixed solution in contact therewith. The competitive capture substance and the determination substance are immobilized on a solid support,
The measurement kit according to claim 11, wherein the competitive capture substance and the determination substance are arranged side by side at different molar concentrations. The following competitor substance to be mixed with the test sample solution containing the target substance:
The following competitive capture substance, which is a substance that is developed and brought into contact with the mixed solution from one side, and is immobilized in a thin film in an excess amount than the molar equivalent in the immune reaction to the competitive substance to be mixed;
A measurement kit comprising:
Competing substance: One of an antibody capable of binding to the target substance and the target substance or a similar substance.
Competitive capture substance: An antibody that can bind to the target substance and a substance that is the other of the target substance or similar substance and is separable from the contacted liquid mixture.

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