JP2000074918A - Method and apparatus for immunological measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a specific immunological measuring method for an antigen (or an antibody) which can be quantitatively determined quickly, simply and at a low cost and to provide a specific immunolological measuring apparatus for an antigen (or an antibody) which can be measured quickly, simply, with high accuracy, automatically and at a low cost. SOLUTION: A test piece 2 for measurement, in which an antigen (or an antibody) or an antigen-antibody complex as an object to be measured is immobilized, in a fixed amount, to a solid phase is prepared, An antibody (or an antigen) which can be bonded specifically to the antigen (or the antibody) and to which a marker substance 95 is bonded is brought into contact with the test piece 2 for measurement, inside a reaction container 1. Then, by using an antigen (or an antibody) which is fixed to the test piece 2 for measurement and by using an antigen 92 (or an antibody) in a liquid to be inspected, an antibody 94 (or an antigen) is distributed and bonded according to the concentration of the antigen (or the antibody). The amount of the marker substance 95 which is bonded to the test piece 2 for measurement or the liquid to be inspected is measured. The concentration of the antigen (or the antibody) in the liquid to be detected is calculated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method and apparatus for immunologically measuring the concentration of an antigen or antibody in a solution.

[0002]

2. Description of the Related Art In recent years, so-called allergic symptoms such as allergic rhinitis such as hay fever, allergic bronchial asthma, and atopic dermatitis, which are allergens which are a kind of antigen, are increasing mainly in developed countries. It has been specified that various constituent substances such as mites, pollen, animal hair, and mold are the causative substances. These allergic symptoms can be relatively mild, but they can be as severe as death, such as allergic bronchial asthma. For this reason, as one means to suppress sensitization and onset,
It is necessary to maintain low levels of allergen, a causative agent peculiar to each individual. However, at present, allergen measurement is considered to be a relatively difficult advanced technology,
It requires a professional measurement technician, requires a long measurement time of half a day to a day, and requires expensive measurement equipment.

[0003] It is known that the allergen concentration in the environment generally correlates with the sensitization / onset of allergic disease. Therefore, measurement of the allergen concentration in the environment is an index of how much cleaning and washing should be performed and how often it should be performed. It will be useful in life. It is also generally known that the concentration of IgE antibody in the blood of an allergic disease patient is an indicator of the degree of sensitization of type I allergy in an immediate allergic reaction. It is extremely useful as an index for knowing the degree and thus the degree of allergic disease.

In recent years, infection of microorganisms such as bacteria represented by pathogenic Escherichia coli O-157 and damage such as food poisoning caused by metabolites have become a problem. It is common to identify and quantify types. Therefore, it takes a long time, such as 1 to 7 days, until a test result is obtained, mainly due to the time required for the culturing operation, and requires a specialized measurement technician. In particular, when it becomes necessary to inspect fresh foods and the like, the problem is that the measurement time is long.

[0005] Incidentally, such microorganisms and the above-mentioned antigens are not unrelated. That is, the name of the cells such as pathogenic Escherichia coli O-157 is derived from the name of the antibody, and the pathogenic Escherichia coli O-157 has an antigenic determinant to which the antibody O-157 specifically binds on the cell surface. Expressed pathogenic E. coli ". Each microorganism species has a unique antigenic determinant on the body surface, and the microorganism itself can be regarded as an antigen. For the reasons described above, microorganisms can be identified and quantified as antigens, and are extremely useful for testing fresh foods and the like.

[0006] Against this background, there is a need for a method for specifically, rapidly, conveniently and highly sensitively measuring the concentration of an antigen or an antibody, and an inexpensive measuring apparatus using the method.

Therefore, a conventional method for analyzing the antigen concentration will be described. Note that the measurement of the antigen concentration described below is exactly the same even when the antigen and the antibody are exchanged and the antibody is measured. Therefore, unless otherwise specified, when one is described, the other is also described. Further, the term "antigen (or antibody)" or "antibody (or antigen)" will be explicitly described as necessary.

[0008] Conventional methods for measuring antigens utilizing an antigen-antibody reaction are collectively referred to as immunoassays and are broadly classified into unlabeled immunoassays and labeled immunoassays. The unlabeled immunoassay is a method of measuring the amount of an antigen using a change in a physical quantity or the like due to an antigen-antibody reaction without binding a labeled substance such as an enzyme to an antibody. An unlabeled immunoassay includes an SPR method (surface plasmon resonance method) and a method using a quartz oscillator. The labeled immunoassay is a method for measuring the amount of an antigen by measuring the amount of a labeling substance using a substance obtained by binding a labeling substance such as an enzyme to an antibody. Labeled immunoassays include radioimmunoassay (radioisotope labeling), enzyme immunoassay, and fluorescence immunoassay depending on the type of label (eg, Eiji Ishikawa et al .: “Enzyme immunoassay (third edition)”). , 31-54, Medical Shoin (1987)).

Among these assays, the immunoassay mainly used at present is E-type, a kind of enzyme immunoassay.
This is a method called the LISA method. In particular, a method called a sandwich method (hereinafter, referred to as a "sandwich ELISA method") is generally and widely used mainly because of its high measurement sensitivity and relatively easy operation. For example, an outline of how the sandwich ELISA method is performed will be described with reference to FIG. 10 in the case of measuring mite allergen as an example.

FIG. 10 is a schematic view showing main steps of a conventional sandwich ELISA method. This method has four steps <III-1. Preparation of reaction vessel for measurement>, <III-2.
Reaction step>, <III-3. Measurement process> and <III-
4. Calculation step>. Hereinafter, the four steps will be described.

<III-1. Step of preparing reaction vessel for measurement> This is a step of immobilizing the antibody in the reaction vessel to bring it into a stabilized state. It comprises the following steps:
In FIG. 10, 1 is a reaction vessel, 91 is an antibody, 92 is an antigen, 94 is an enzyme-labeled antibody, and 95 is an enzyme.

(III-1) Step of Immobilizing Antibody 91 that Specifically Binds to Antigen in Reaction Vessel 1 The solution is poured, and the antibody 91 is immobilized by non-specific adsorption on the solid phase of the reaction vessel 1.

(III-1) Step of Washing and Removing Unreacted Antibody 91 The unreacted antibody 91 is removed by washing the reaction vessel 1 three times or more with a buffer or the like.

(III-1) Step of Coating Non-Specific Adsorption Surface of Reaction Vessel 1 with Blocking Agent A blocking agent solution is poured into the reaction container 1, and the blocking agent is applied to the non-specific adsorption surface remaining on the surface of the reaction container 1. Non-specific adsorption is performed so that non-specific adsorption of other organic substances and the like to the reaction vessel 1 is prevented as much as possible.

(III-1) Step of Washing and Removing Unreacted Blocking Agent The unreacted blocking agent is removed by washing the reaction vessel 1 at least three times with a buffer or the like.

(III-1) Step of Performing Processing for Preserving Reaction Container 1 for Measurement The reaction container 1 is kept in a long-term stabilized state by using a freeze-drying method as a preserving means.

<III-2. Reaction Step> This is a step of immobilizing the enzyme-labeled antibody in an amount proportional to the amount of the antigen in the reaction vessel 1. It comprises the following steps:

(III-2) Step of Specificly Immobilizing Antigen 92 in Reaction Vessel 1 A standard antigen solution having a known concentration, a test solution containing antigen 92, and a blank solution are placed in at least three reaction vessels 1. Each of them is poured, and a standard antigen or antigen 92 is specifically bound to the immobilized antibody 91 in the reaction vessel 1 by an antigen-antibody reaction in parallel under the same conditions.

(III-2) Step of Washing and Removing Unreacted Antigen 92 The reaction container 1 is washed three times or more with a buffer solution or the like to remove the unreacted antigen 92.

(III-2) Step of Specific Binding of Enzyme-Labeled Antibody 94 to Reaction Vessel 1 A solution of the enzyme-labeled antibody 94 is poured into the reaction vessel 1, and the enzyme-labeled antibody 94 is separated from the antigen-antibody complex in the reaction vessel 1. Specific binding is achieved by an antigen-antibody reaction via the antigen 92.

(III-2) Unreacted enzyme-labeled antibody 94
Washing and removing step The reaction vessel 1 is washed three times or more with a buffer solution or the like to remove the unreacted enzyme-labeled antibody 94.

<III-3. Measurement step> This is a step of adding a chromogenic substrate to form a color by an enzymatic reaction and measuring the degree of color formation. It comprises the following steps:

(III-3) Step of Coloring Substrate by Enzyme Reaction A substrate solution is added to the reaction vessel 1, and the enzyme 95 of the enzyme-labeled antibody 94 in the reaction vessel 1 generates a dye from the substrate by the enzyme reaction.

(III-3) Step of Stopping Enzyme Reaction The reaction stop solution is added to the reaction vessel 1 to stop the enzyme reaction.

(III-3) Step of Measuring Degree of Color Development at Color Development Wavelength Dye generated at a predetermined wavelength by a spectrophotometer is measured.

<III-4. Calculation Step> This is a step of calculating the antigen concentration from the calibration curve of the standard antigen solution. It consists of the following steps. (III-4) Calculation Step A calibration curve of the standard antigen solution is created from the measurement results, and the calculation of the antigen concentration of the test solution is performed.

[0027]

However, the above-mentioned conventional measuring method will be described below even if the reaction vessel prepared by the <III-1 measuring reaction vessel preparation step> is supplied to the operator. There was such a problem.

(1) A two-step antigen-antibody reaction (the above (III-2) and (III-2)) is required. If all the reactions do not proceed completely, a correct value cannot be obtained.
The problem is that the time required for the measurement is as long as about 24 hours, and especially about 24 hours is required to improve the accuracy.

(2) About 10 kinds of different reagents are required, and a two-step washing operation {(III-2)
), (III-2)｝ must be performed, and the work is complicated and the operability is poor.

(3) In order to perform the test, a dispenser, a thermostat, a refrigerator, and the like are required, and a considerable cost is required.

Accordingly, an object of the present invention is to provide a specific method for measuring an antigen (or an antibody) which can be quantified quickly, simply, and inexpensively.

It is a further object of the present invention to provide an inexpensive and specific antigen (or antibody) measuring device that can automatically and quickly perform a highly accurate measurement.

[0033]

Means for Solving the Problems In order to solve the above-mentioned problems, the immunological assay method of the present invention involves immobilizing a fixed amount of an antigen (or antibody) or an antigen-antibody complex to be measured on a solid phase. A test strip for measurement is prepared, and an antibody (or antigen) that can specifically bind to the antigen (or antibody) and binds to a labeling substance is brought into contact with the test strip for measurement in a reaction vessel. The antigen (or the antibody) immobilized on the test strip and the antigen (or the antibody) in the test solution, the antibody (or the antigen) is distributed and bound in accordance with the respective antigen (or the antibody) concentration, The amount of the labeling substance bound to the test strip or the test solution is measured to calculate the antigen (or antibody) concentration in the test solution.

As a result, a specific immunological assay method for an antigen (or an antibody) which can be quickly, simply, and inexpensively quantified is obtained.

The immunological measuring device of the present invention comprises a measuring test piece in which a predetermined amount of an antigen (or antibody) or an antigen-antibody complex to be measured is immobilized on a solid phase; A reaction vessel containing a test solution containing the antigen (or antibody) to which an antibody (or antigen) to which a labeling substance is bound, which can specifically bind to the test substance, the test piece for measurement, and the inside of the reaction vessel A transfer unit for contacting the test solution with the test solution, and the label of any of the antibodies (or antigens) that have undergone an antigen-antibody reaction with the antigen (or antibody) of the measurement test piece and the test solution, respectively. A measuring unit for measuring the amount of the substance; and a calculating unit for converting the amount of the labeling substance measured by the measuring unit into an antigen (or antibody) concentration in the test solution.

According to the present invention, a rapid, simple, and highly accurate measurement can be automatically performed, and an inexpensive antigen (or antibody) specific measurement device can be obtained.

Further, the measurement test piece of the present invention is a measurement test piece having a fixed amount of an antigen (or antibody) or an antigen-antibody complex to be measured immobilized on a solid phase. ), An antibody (or antigen) that can specifically bind to the labeling substance is brought into contact with the measurement test piece in a reaction vessel, and the antigen (or antibody) immobilized on the measurement test piece is Antigen (or antibody) in test solution
And dividing and binding the antibody (or antigen) in accordance with the concentration of each antigen (or antibody), measuring the amount of the labeling substance bound to the test strip for measurement or the test solution, It is characterized in that the antigen (or antibody) concentration in the test solution is calculated.

As a result, a specific test strip for antigen (or antibody) can be obtained which can be quantified quickly, simply and inexpensively.

[0039]

BEST MODE FOR CARRYING OUT THE INVENTION In the first immunological assay method of the present invention, a test strip for measurement is prepared by immobilizing a fixed amount of an antigen to be measured on a solid phase, and specifically binds to the antigen. An antigen-containing test solution to which an antibody to which a labeling substance has been added can be contacted with the measurement test piece in a reaction vessel, and the antigen immobilized on the measurement test piece and the test solution The antigen is distributed and bound in accordance with the respective antigen concentrations with the antigen in, and the amount of the labeling substance bound to the test strip or the test solution is measured, and the amount of the test substance in the test solution is measured. Since the method for immunological measurement of antigen concentration is characterized by calculating the antigen concentration, the measurement can be simplified and speeded up by using the separately prepared and stored test strip for measurement. Further, since the measurement test piece has only the antigen immobilized thereon, it can be stored for a long time. In addition, since the antigen-antibody reaction is performed only once when the test solution is brought into contact with the test solution, it is not necessary to wash in a short period of time and, if the reaction is performed multiple times, between the reactions. The antibody added to the test solution is distributed at a predetermined ratio according to the respective antigen concentrations by a certain amount of the antigen immobilized on the test piece for measurement and the antigen in the test solution. Since the amount of the distributed antibody and the labeling substance have a predetermined relationship, by measuring the amount of the labeling substance bound to the test strip or the test liquid, the antigen in the test liquid is measured. The density can be calculated.

In the second immunoassay according to the present invention, a test piece for measurement is prepared by immobilizing a fixed amount of an antigen to be measured on a solid phase, and can specifically bind to the antigen. Contacting the antigen-containing test solution to which the first antibody and the second antibody, which can specifically bind to the first antibody and are bound with a labeling substance, are added, to the test strip for measurement in a reaction vessel; The first antibody and the second antibody are distributed and bound according to the respective antigen concentrations with the antigen immobilized on the measurement test piece and the antigen in the test solution, and the measurement test piece or the The amount of the labeling substance bound to the test solution is measured, and the antigen concentration in the test solution is calculated. Simple and quick measurement by using the test piece for measurement Door can be. Further, since the measurement test piece has only the antigen immobilized thereon, it can be stored for a long time. In addition, since the antigen-antibody reaction is performed only once when the test solution is brought into contact with the test solution, it is not necessary to wash in a short period of time and, if the reaction is performed multiple times, between the reactions. The first antibody and the second antibody added to the test solution can be adjusted to the respective antigen concentrations by a certain amount of the antigen immobilized on the measurement test piece and the antigen in the test solution. Accordingly, since the amount of the second antibody distributed and the labeling substance have a predetermined relationship, the amount of the labeling substance bound to the test strip for measurement or the test solution is measured. This makes it possible to calculate the antigen concentration in the test solution. In addition, since two types of antibodies are used, it is possible to measure a wide variety of antigens by changing the specificity of the first antibody, and widely by changing the labeling substance of the second antibody. Various measurement methods can be used.

According to the third immunological assay method of the present invention, a test strip for measurement in which a predetermined amount of an antibody to be measured is immobilized on a solid phase can be prepared and specifically bound to the antibody. The test solution containing the antibody to which the antigen to which the labeling substance is added is added is brought into contact with the test piece for measurement in a reaction vessel, and the antibody immobilized on the test piece for measurement and the antibody in the test solution are used. The antigen is distributed in accordance with the respective antibody concentration and bound, and the amount of the labeling substance bound to the test strip or the test solution is measured to calculate the antibody concentration in the test solution. Since the method is an immunological method for measuring an antibody concentration, the measurement can be simplified and speeded up by using the separately prepared and stored test piece for measurement. Further, since the measurement test piece has only the antibody immobilized thereon, it can be stored for a long time. In addition, since the antigen-antibody reaction is performed only once when the test solution is brought into contact with the test solution, it is not necessary to wash in a short period of time and, if the reaction is performed multiple times, between the reactions. Can be
The antigen added to the test solution is distributed at a predetermined ratio according to each antibody concentration by the fixed amount of the antibody immobilized on the measurement test piece and the antibody in the test solution. Since there is a predetermined relationship between the amount and the labeling substance, it is possible to calculate the antibody concentration in the test liquid by measuring the amount of the labeling substance bound to the test strip or the test liquid. It becomes possible.

According to the fourth immunoassay of the present invention, an antigen to be measured is immobilized on a solid phase in a fixed amount, and an antibody capable of specifically binding to the antigen and having a labeled substance bound thereto is used as the antigen. To prepare a test piece for measurement in which an antigen-antibody complex of the antigen and the antibody is formed on a solid phase, and contact the test liquid containing the antigen with the test piece for measurement in a reaction vessel. The antibody of the antigen-antibody complex is distributed and bound in accordance with the respective antigen concentration with the immobilized antigen of the antigen-antibody complex and the antigen in the test solution, and the test strip for measurement or the test Measuring the amount of the labeling substance bound to the test solution, and calculating the antigen concentration in the test solution is an immunological measurement method of the antigen concentration, the separately prepared and stored Simple and quick measurement by using test specimens It can be. Furthermore, since the antigen and the antibody are immobilized on the test strip for measurement, the operation of adding the antibody later can be omitted. In addition, since the antigen-antibody reaction is performed only once when the test solution is brought into contact with the test solution, it is not necessary to wash in a short period of time and, if the reaction is performed multiple times, between the reactions. By a fixed amount of antigen immobilized on the test strip for measurement and the antigen in the test solution, the antibody of the antigen-antibody complex formed on the test strip for measurement, the respective antigen concentration Is distributed in a predetermined ratio according to the amount of the distributed antibody and the labeling substance because there is a predetermined relationship, by measuring the amount of the labeling substance bound to the test strip for measurement or the test solution Thus, it is possible to calculate the antigen concentration in the test solution.

According to the fifth immunoassay of the present invention, the antigen to be measured is immobilized on a solid phase in a fixed amount, and the first antibody capable of specifically binding to the antigen and the first antibody A measurement test in which a second antibody capable of specifically binding and binding a labeling substance is brought into contact with the antigen to form an antigen-antibody complex of the antigen, the first antibody and the second antibody on a solid phase. A sample is prepared, and the test solution containing the antigen is brought into contact with the test piece for measurement in a reaction vessel, and the antigen-antibody complex is immobilized on the immobilized antigen of the antigen-antibody complex and the antigen in the test solution. The first antibody and the second antibody of the body are distributed and bound according to the respective antibody concentrations, and the amount of the labeling substance bound to the test strip for measurement or the test solution is measured. Antigen concentration immunology characterized by calculating antigen concentration in test solution Since the measurement method can be simply and speeding the measurement by using the test piece for measuring stored separately fabricated. Furthermore, since the antigen, the first antibody, and the second antibody are immobilized on the test strip for measurement, the operation of adding the first antibody and the second antibody later can be omitted. In addition, since the antigen-antibody reaction is performed only once when the test solution is brought into contact with the test solution, it is not necessary to wash in a short period of time and, if the reaction is performed multiple times, between the reactions. The first antibody and the second antibody of the antigen-antibody complex formed on the test strip for measurement are formed by a fixed amount of antigen immobilized on the test strip for measurement and the antigen in the test solution. Antibodies are distributed at a predetermined ratio according to the respective antigen concentrations, and since the distributed amount of the second antibody and the labeling substance have a predetermined relationship, the antibody bound to the measurement test strip or the test solution is By measuring the amount of the labeling substance, it becomes possible to calculate the antigen concentration in the test solution. Further, since two kinds of antibodies are used, the first type which can specifically bind to the antigen is used.
By changing the antibody, it becomes possible to measure a wide variety of antigens, and by changing the labeling substance of the second antibody that can specifically bind to the first antibody and bind a labeling substance, It is possible to use various measurement methods.

In the sixth immunoassay according to the present invention, an antibody to be measured is immobilized on a solid phase in a fixed amount, and an antigen which can specifically bind to the antibody and has a labeled substance bound thereto is used as an antibody. To prepare a test strip for measurement in which an antigen-antibody complex of the antibody and the antigen is formed on a solid phase, and contact the test solution containing the antibody with the test strip for measurement in a reaction vessel. The antigen of the antigen-antibody complex is distributed and bound according to the respective antibody concentration between the immobilized antibody of the antigen-antibody complex and the antibody in the test solution, and the measurement test piece or the Measuring the amount of the labeling substance bound to the test solution, and calculating the antibody concentration in the test solution, since the method for immunological measurement of the antibody concentration, the separately prepared and stored Simple and quick measurement by using test specimens It can be. Furthermore, since the antibody and the antigen are immobilized on the measurement test piece, the operation of adding the antigen later can be omitted. In addition, since the antigen-antibody reaction is performed only once when the test solution is brought into contact with the test solution, it is not necessary to wash in a short period of time and, if the reaction is performed multiple times, between the reactions. The antigen of the antigen-antibody complex formed on the test strip for measurement by a certain amount of the antibody immobilized on the test strip for test and the antibody in the test solution can be converted into the respective antibody concentrations. Is distributed in a predetermined ratio according to the amount of the distributed antigen and the labeling substance because there is a predetermined relationship, by measuring the amount of the labeling substance bound to the test strip for measurement or the test solution In addition, it is possible to calculate the antibody concentration in the test solution.

In the immunological assay method of the present invention, the labeling substance is preferably an enzyme. By using an enzyme as a labeling substance, an antigen or antibody can be measured quickly, easily, and inexpensively.

In the immunological measurement method of the present invention, the test piece for measurement has a shape in which any one or a plurality of materials selected from bead, film, column, and container are combined. After the partitioning, the separation of the antigen-antibody complex on the solid phase of the test specimen for measurement and the antigen-antibody complex in the liquid phase of the test liquid is performed by using the test specimen for measurement and the test liquid in the reaction container. Is preferably performed by cutting off the contact. Thus, the separation of the antigen-antibody complex on the solid phase of the test specimen from the antigen-antibody complex in the liquid phase of the test liquid can be performed easily in a short time.

In the immunological measurement method of the present invention, it is preferable that the test piece for measurement is simultaneously immersed in a plurality of test solutions. As a result, a plurality of test liquids can be measured at the same time, the measurement time per test liquid is shortened, the measurement operation per test liquid is reduced, and the cost required for measurement per test liquid is reduced. can do.

The first immunological measuring apparatus of the present invention comprises a measuring test piece in which a predetermined amount of an antigen to be measured is immobilized on a solid phase, and a labeling substance capable of specifically binding to the antigen. A reaction container containing the antigen-containing test solution to which the bound antibody has been added, a transfer unit for bringing the measurement test piece into contact with the test solution in the reaction container, and the measurement test piece And a measurement unit for measuring the amount of the labeling substance of any of the antibodies and the antigen-antibody reaction of the test liquid, and the amount of the labeling substance measured by the measurement unit is measured for the test liquid. Because it is an immunological measurement device for antigen concentration, comprising a calculation unit for converting to antigen concentration,
Since only the antigen is immobilized on the measurement test piece, it can be stored for a long period of time, and the measurement can always be performed easily. In addition, since the reaction, measurement, and calculation can be performed by one measuring device, variation in operation and complexity can be reduced, and a device capable of performing highly accurate and simple measurement can be provided.

The second immunological measuring device of the present invention comprises a measuring test piece having a fixed amount of an antigen to be measured immobilized on a solid phase, and a first antibody capable of specifically binding to the antigen. A reaction container containing the antigen-containing test solution to which the second antibody capable of specifically binding to the first antibody and having a label attached thereto is added, the measurement test piece, and the inside of the reaction container. A transfer unit for contacting the test solution with the test solution, and measuring the amount of the labeling substance of any one of the second antibody and the antigen-antibody reaction with each of the antigen of the measurement test piece and the test solution. Since the measurement unit, the immunological measurement device for antigen concentration, comprising a calculation unit that converts the amount of the labeling substance measured by the measurement unit to the antigen concentration of the test solution, Since the test piece for measurement only immobilizes the antigen, Storage can be conveniently carried out measurements at all times. In addition, since the reaction, measurement, and calculation can be performed by one measuring device, variation in operation and complexity can be reduced, and a device capable of performing highly accurate and simple measurement can be provided. Further, by changing the first antibody, a wide variety of antigens can be measured, and by changing the labeling substance of the second antibody, an apparatus can be provided which can use a wide variety of measurement methods. .

The third immunoassay apparatus of the present invention comprises a test strip for measurement in which a predetermined amount of an antibody to be measured is immobilized on a solid phase, and a labeled substance capable of specifically binding to the antigen. A reaction container containing the antibody-containing test solution to which the bound antibody has been added, a transfer unit for bringing the measurement test piece into contact with the test solution in the reaction container, and the measurement test piece And a measuring unit that measures the amount of the labeling substance of each of the antigens that have reacted with the antibody and the antigen of the test liquid, and the amount of the labeling substance measured by the measuring unit is the amount of the test liquid. Since it is an immunological measurement device for antibody concentration, comprising a calculation unit for converting to antibody concentration,
Since only the antibody is immobilized on the test piece for measurement, it can be stored for a long period of time, and the measurement can always be performed easily. In addition, since the reaction, measurement, and calculation can be performed by one measuring device, variation in operation and complexity can be reduced, and a device capable of performing highly accurate and simple measurement can be provided.

The fourth immunoassay apparatus of the present invention is characterized in that an antigen to be measured is immobilized on a solid phase in a fixed amount, and an antibody which can specifically bind to the antigen and has a labeled substance bound thereto is used as an antibody. A measurement test piece formed with an antigen-antibody complex of
A reaction container containing the antigen-containing test solution, a transfer unit for bringing the measurement test piece into contact with the test solution in the reaction container, and each of the measurement test piece and the test solution A measuring unit that measures the amount of the labeling substance of any of the antibodies that have reacted with the antigen and the antigen and an arithmetic unit that converts the amount of the labeling substance measured by the measuring unit into an antigen concentration of the test solution Since it is an immunological measurement device for antigen concentration characterized by comprising, since the test piece for measurement immobilizes the antigen and the antibody, the operation of adding the antibody later can be omitted. it can. In addition, it is possible to provide a device capable of performing reaction, measurement, and calculation with one measuring device, reducing variation and complexity of operation, and performing highly accurate and simple measurement.

The fifth immunoassay apparatus of the present invention comprises a first antibody capable of immobilizing a predetermined amount of an antigen to be measured on a solid phase and capable of specifically binding to the antigen, and a first antibody specific to the first antibody. Secondly bound with a labeled substance
A test piece for measurement in which an antigen-antibody complex with the antigen is formed with an antibody, a reaction container containing a test solution containing the antigen, and the test sample for measurement and the test solution in the reaction container are brought into contact with each other. And a transfer unit for causing the antigen-antibody reaction with the antigen of each of the test piece for measurement and the test solution.
A measuring unit for measuring the amount of the labeling substance of any of the antibodies; and a calculating unit for converting the amount of the labeling substance measured by the measuring unit into an antigen concentration of the test solution. Since it is an immunological measurement device for antigen concentration, since the antigen, the first antibody, and the second antibody are immobilized on the test strip for measurement, the first antibody and the second antibody are The operation of adding can be omitted. In addition, it is possible to provide a device capable of performing reaction, measurement, and calculation with one measuring device, reducing variation and complexity of operation, and performing highly accurate and simple measurement. Further, it is possible to provide an apparatus that can measure a wide variety of antigens by changing the first antibody, and can use a wide variety of measurement methods by changing a labeling substance of the second antibody. it can.

The sixth immunological assay device of the present invention is characterized in that an antibody to be measured is immobilized on a solid phase in a fixed amount, and an antigen which can specifically bind to the antibody and has a labeled substance bound thereto is used as an antigen. A measurement test piece formed with an antigen-antibody complex of
A reaction container containing the antibody-containing test solution, a transfer unit for bringing the measurement test piece into contact with the test solution in the reaction container, and each of the measurement test piece and the test solution. A measuring unit that measures the amount of the labeling substance of any of the antigens that have reacted with the antibody and the antigen-antibody, and a calculating unit that converts the amount of the labeling substance measured by the measuring unit into an antibody concentration of the test liquid Since the immunoassay apparatus for antibody concentration is characterized by comprising: the antibody and the antigen are immobilized on the test strip, so that the operation of adding the antigen later is omitted. Can be. In addition, it is possible to provide a device capable of performing reaction, measurement, and calculation with one measuring device, reducing variation and complexity of operation, and performing highly accurate and simple measurement.

It is preferable that the immunological measurement device of the present invention is provided with a temperature holding device for keeping the temperature of the antigen-antibody reaction and the enzyme reaction constant. Thus, by providing a constant temperature for the antigen-antibody reaction and the enzyme reaction, an appropriate reaction can be performed quickly and stably, and the measurement time can be shortened to provide a device capable of performing highly accurate measurement. be able to.

The immunological measuring device of the present invention preferably has a vibration mechanism or a stirring mechanism. Thus, it is possible to provide a device capable of efficiently progressing the antigen-antibody reaction and the enzyme reaction by vibration or stirring and shortening the measurement time.

The first test strip of the present invention is a test strip in which an antigen to be measured is immobilized on a solid phase in a fixed amount, and is capable of specifically binding to the antigen. The test solution containing the antigen to which the antibody bound is added is brought into contact with the test piece for measurement in a reaction vessel, and the antigen immobilized on the test piece for measurement and the antigen in the test solution are mixed with the antigen. Distributing and binding the antibodies according to their respective antigen concentrations, measuring the amount of the labeling substance bound to the test strip or the test solution, and calculating the antigen concentration in the test solution. The measurement test piece is characterized in that the measurement can be simplified and speeded up by using the separately prepared and stored measurement test piece. Further, since the measurement test piece has only the antigen immobilized thereon, it can be stored for a long time.

The second test strip of the present invention is a test strip in which an antigen to be measured is immobilized on a solid phase in a fixed amount, and the first test strip capable of specifically binding to the antigen is used. Contacting the antigen-containing test solution to which the antibody and the second antibody capable of specifically binding to the first antibody and to which the labeling substance is added, with the test piece for measurement in a reaction vessel; The first antibody and the second antibody are distributed and bound according to the respective antigen concentrations with the antigen immobilized on the test strip and the antigen in the test solution, and the test strip for measurement or the test Measuring the amount of the labeling substance bound to the liquid,
Since the measurement test piece is characterized by calculating the antigen concentration in the test solution, the measurement can be simplified and speeded up by using the separately prepared and stored measurement test piece. Further, since the measurement test piece has only the antigen immobilized thereon, it can be stored for a long time.

The third test strip of the present invention is a test strip in which a predetermined amount of an antibody to be measured is immobilized on a solid phase, and is capable of binding specifically to the antibody. The test solution containing the antibody to which the antigen bound was added was brought into contact with the test piece for measurement in a reaction vessel, and the antibody immobilized on the test piece for measurement and the antibody in the test solution were used for the test. Calculating the antibody concentration in the test liquid by measuring the amount of the labeling substance bound to the test strip or the test liquid by distributing the antigens according to the respective antibody concentrations and binding them. Since the test for measurement is characterized by the above, the measurement can be simplified and speeded up by using the test piece for measurement separately prepared and stored. Further, since the measurement test piece has only the antibody immobilized thereon, it can be stored for a long time.

In the fourth test strip of the present invention, an antigen to be measured is immobilized on a solid phase in a fixed amount, and an antibody which can specifically bind to the antigen and has a labeled substance bound thereto is used as the antigen. A test specimen for measurement in which an antigen-antibody complex of the antigen and the antibody are formed on a solid phase, wherein the test liquid containing the antigen is brought into contact with the test specimen for measurement in a reaction vessel, The immobilized antigen of the antigen-antibody complex and the antigen in the test solution distribute and bind the antibodies of the antigen-antibody complex according to the respective antigen concentrations, and the test strip for measurement or the test solution By measuring the amount of the labeling substance bound to the test solution, the antigen concentration in the test solution is calculated, so that the measurement test piece separately prepared and stored is used. As a result, the measurement can be simplified and speeded up. Furthermore, since the antigen and the antibody are immobilized on the test strip for measurement, the operation of adding the antibody later can be omitted.

The fifth test strip of the present invention comprises a first antibody capable of immobilizing a predetermined amount of an antigen to be measured on a solid phase, a first antibody capable of specifically binding to the antigen, and a first antibody specific for the first antibody. A second antibody capable of binding to a labeling substance and contacting the antigen is brought into contact with the antigen, and the antigen and the first antibody are placed on a solid phase.
A test strip for forming an antigen-antibody complex of an antibody and a second antibody, wherein the test liquid containing the antigen is contacted with the test strip in a reaction vessel to fix the antigen-antibody complex. The first antibody and the second antibody of the antigen-antibody complex are bound and bound according to the respective antibody concentrations with the immobilized antigen and the antigen in the test solution, and the test strip for measurement or the test By measuring the amount of the labeling substance bound to the solution, it is a measuring test piece characterized by calculating the antigen concentration in the test liquid, the separately prepared and stored measuring test piece The use can facilitate and speed up the measurement. Further, since the antigen, the first antibody, and the second antibody are immobilized on the measurement test piece, the first
The operation of adding the antibody and the second antibody later can be omitted.

In the sixth test strip of the present invention, an antibody to be measured is immobilized on a solid phase in a fixed amount, and an antigen which can specifically bind to the antibody and has a labeled substance bound thereto is added to the antibody. Contacting, a test specimen for measurement formed an antigen-antibody complex of the antibody and the antigen on a solid phase, the test liquid containing the antibody is brought into contact with the test specimen for measurement in a reaction vessel, The antigen of the antigen-antibody complex is distributed and bound in accordance with the respective antibody concentration with the immobilized antibody of the antigen-antibody complex and the antibody in the test solution, and the test strip for measurement or the test solution is used. By measuring the amount of the labeling substance bound to the test solution, the antibody concentration in the test solution is calculated, so that the test sample separately prepared and stored is used. As a result, the measurement can be simplified and speeded up. Furthermore, since the antibody and the antigen are immobilized on the measurement test piece, the operation of adding the antigen later can be omitted.

Hereinafter, embodiments of the immunological measurement method and the immunological measurement device of the antigen (or antibody) of the present invention will be described with reference to the drawings.

(Embodiment 1) In Embodiment 1, an immunoassay method and an immunoassay apparatus for measuring the number of cells by using a cell having an antigenic determinant to which an antibody specifically binds as an antigen explain. The measurement of the antigen will be described below. For example, the antigen and the antibody can be reversed, for example, by measuring the amount of antibody in the serum against the bacterial body (antigen). For these reasons, the description of antibody measurement is omitted.

FIG. 1 is a schematic sectional view of the immunological measuring device according to the first and second embodiments of the present invention, and FIG. 2 is a schematic plan view of the immunological measuring device according to the first and second embodiments of the present invention. FIG. 3 is a schematic perspective view showing a reaction vessel according to Embodiments 1 and 2 of the present invention, FIG. 4 is a schematic perspective view showing a test piece for measurement according to Embodiments 1 and 2 of the present invention, and FIG. It is a schematic sectional view showing the state where the reaction container and the test piece for measurement in Embodiments 1 and 2 of the present invention were combined. FIG. 6 is a schematic view showing main steps of the first embodiment of the present invention.

In FIG. 3, 31 to 38 are independent first to third.
An eighth reaction vessel 30 is a reaction vessel connecting frame that integrates the first reaction vessel 31 to the eighth reaction vessel 38. In FIG. 4, reference numerals 41 to 48 denote independent first to eighth measurement test pieces, and reference numeral 40 denotes a measurement test piece connection frame that integrates the first measurement test piece 41 to the eighth measurement test piece 48. is there.

In FIG. 6, 1 is a reaction vessel, 2 is a test strip for measurement, 91 is an antibody, 92 is an antigen, 93 is a labeled antigen, 94 is an enzyme-labeled antibody, and 95 is an enzyme.

The immunological measurement method according to the first embodiment has four steps <I-1. Step of preparing test piece for measurement>, <I-2.
Reaction step>, <I-3. Measurement step> and <I-4.
Calculation step>.

<I-1. Step of preparing test piece 2 for measurement> This is a step of immobilizing the standard antigen 93 on the test piece 2 for measurement to bring it into a stabilized state. It comprises the following steps:

(I-1) Step of Immobilizing Standard Antigen 93 on Test Specimen 2 The standard antigen 93 is an antigen to be measured and is used in the form of a single bacterial suspension.

The reaction vessel 1 is a vessel made of an optically transparent synthetic resin such as polystyrene, and is a collection of independent single or plural vessels, for example, as shown in FIG.
The single or multiple vessels of the reaction vessel 1 preferably have a horizontal cross-sectional area of 10 to 500 mm ² , a depth of about 1 to 30 mm, and an internal volume of 10 μL to 15 mL to secure the liquid volume required for measurement, Becomes easier. More preferably, the content is in the range of 50 to 500 μL. It is preferable to carry out a treatment with a blocking agent in advance so that organic substances do not bind to the reaction vessel 1. For example, a blocking agent solution containing a protein such as bovine serum albumin and milk-derived casein is used as the blocking agent. The solvent of the blocking agent solution is a buffer, preferably a phosphate buffer, a borate buffer, or the like, so that the pH can be kept constant. More preferably, a salt such as sodium chloride, potassium chloride or the like is mixed with the buffer solution. The pH of the buffer is preferably p
By setting H5 to H9, a stable blocking agent can be nonspecifically adsorbed to the reaction vessel 1. The pH is more preferably in the range of 6 to 8. The conditions for the treatment with the blocking agent solution are pH 5 to 9, the treatment time is 10 minutes to 48 hours, and the non-specific adsorption of the blocking agent on the surface of the reaction vessel 1 where non-specific adsorption occurs by immersion treatment at a processing temperature of 4 to 40 ° C. Then, non-specific adsorption of other organic substances is prevented from occurring as much as possible, and more accurate measurement is possible.
More preferably, immersion treatment is performed at a pH of 6 to 8, a treatment time of 30 minutes to 2 hours, and a treatment temperature of 4 to 30 ° C.

The test piece for measurement 2 is a projection such as a bead, a film, a column, a container, or the like made of a synthetic resin such as polystyrene treated so as to have a high binding of an organic substance. Alternatively, it is an aggregate of a plurality of protrusions, for example, in the form shown in FIG. Preferably a horizontal cross-sectional area of 10
~ 500mm ² , depth of about 1-30mm, content 10
By adjusting the volume to μL to 15 mL, the amount of liquid necessary for the measurement is secured, and the operation becomes easy. More preferably, the volume is 50 to
The range is 500 μL.

As a standard antigen 93 solution, a suspension of cells as antigens is used. The standard antigen 93 solution is preferably 10
^{At 4} to 10 ¹² cells / mL, an amount of antigen sufficient for measurement can be brought into contact with the test piece 2 for measurement and immobilized by non-specific adsorption. More preferably, 10 ⁷ to 10 ¹¹ cells / mL
Range. The solvent of the standard antigen 93 solution is a buffer, preferably a phosphate buffer, a borate buffer, or the like, so that the pH can be kept constant. More preferably, a salt such as sodium chloride, potassium chloride or the like is mixed with the buffer solution. By setting the pH of the buffer solution to preferably 5 to 9, stable nonspecific adsorption of bacterial cells can be generated on the test strip 2 for measurement. The pH is more preferably in the range of 6 to 8.

The standard antigen 93 solution was poured from the first reaction vessel 31 of the reaction vessel 1 to the eighth reaction vessel 38 so as to have the same volume and the same concentration. The first to eighth measurement test pieces 41 of the test piece for measurement 2 are inserted into the solution 38.

The reaction conditions of the reaction container 1 and the test piece 2 for measurement with the standard antigen 93 solution are pH 5 to 9, the treatment time is 10 minutes to 48 hours, and the test temperature is 4 to 40 ° C. It becomes possible to non-specifically adsorb the cells as the standard antigen 93 to the piece 2 at a high density. More preferably, immersion treatment is performed at a pH of 6 to 8, a treatment time of 30 minutes to 2 hours, and a treatment temperature of 4 to 30 ° C.

(I-1) Step of Washing and Removing Unreacted Standard Antigen 93 The test piece 2 for measurement and the reaction vessel 1 are preferably washed with a buffer solution.
By washing 10 to 10 times, the unreacted standard antigen 93 can be efficiently removed, and the accuracy of the subsequent measurement can be improved. More preferably, it is in the range of 2 to 5 times.

(I-1) Reaction vessel 1 with blocking agent
Step of coating non-specific adsorption surface of (1) The blocking agent solution is poured into the reaction vessel 1 and the measurement test piece 2 is inserted. Non-specific adsorption remaining on the test piece 2 for measurement by immersion at a treatment temperature of 4 to 40 ° C. at a treatment condition of pH 5 to 9, a treatment time of 10 to 48 hours, and a treatment temperature of 4 to 40 ° C. The non-specific adsorption of the blocking agent on the surface causing the non-specific adsorption of non-specific substances is prevented as much as possible, and a more accurate measurement is possible. More preferably, immersion treatment is performed at a pH of 6 to 8, a treatment time of 30 minutes to 2 hours, and a treatment temperature of 4 to 30 ° C.

(I-1) Step of Washing and Removing Unreacted Blocking Agent The test specimen 2 for measurement and the reaction vessel 1 were washed with a buffer solution containing a surfactant.
Is preferably washed 1 to 10 times, whereby the unreacted blocking agent can be efficiently removed, and the accuracy of the subsequent measurement can be improved. More preferably, 2 to 5
Range of times.

As the solvent for the surfactant-containing buffer, a buffer containing a polyoxyethylene (20) sorbitan monolaurate, a polyoxyethylene (80) sorbitan monolaurate, or the like as a surfactant is used. By setting the concentration of the surfactant to preferably 0.001 to 1 w / v%, unreacted proteins and the like can be efficiently removed, and the accuracy of the subsequent measurement can be improved. More preferably, it is in the range of 0.01 to 0.2 w / v%.

(I-1) Step of Preserving the Test Specimen 2 for Measurement The freeze-drying method, air-drying, or the like is used as a preserving means to make the test specimen 2 for measurement long-term stable.

Next, <I-1. <I-2. The immunological measurement method of the present invention in which the number of cells is measured using the measurement test piece 2 prepared in the measurement test piece preparation step>. Reaction Step>, <I
-3. Measurement step>, <I-4. Arithmetic Step> and an immunological measurement apparatus for performing the immunological measurement method will be described.

The subsequent steps and subsequent steps are schematic sectional views of the immunological measuring device according to the first and second embodiments of the present invention shown in FIG. 1 and the immunological measuring device according to the first and second embodiments of the present invention shown in FIG. A description will be given along a schematic plan view.

1 and 2, reference numeral 1 denotes a reaction container for holding a standard solution, a test solution, and the like. Reference numeral 2 denotes a test piece for measurement. Reference numeral 3 denotes a reaction container driving motor that supplies a driving force for sequentially moving the reaction container 1 in the horizontal direction. Reference numeral 4 denotes a reaction container driving belt for transmitting the driving force of the reaction container driving motor 3 to the reaction container 1. Reference numeral 5 denotes a motor for moving the test specimen up and down, which supplies a driving force for vertically moving the test specimen 2 for measurement. Reference numeral 6 denotes a measurement test piece vertical movement support for fixing the measurement test piece vertical movement motor 5 and the measurement test piece 2. The motor 3 for driving the reaction container, the belt 4 for driving the reaction container, the motor 5 for vertically moving the test piece for measurement, and the support 6 for vertically moving the test piece for measurement constitute a transfer section of the immunological measurement apparatus of the first embodiment. I have.

Reference numeral 7 denotes a substrate solution tank lid for preventing the evaporation of the substrate solution in the substrate solution tank 8. Reference numeral 8 denotes a substrate solution tank for storing the substrate solution. Reference numeral 9 denotes a substrate solution pump for injecting the substrate solution in the substrate solution tank 8 into the reaction vessel 1. 10 is a substrate solution pump 9
A substrate solution injection nozzle for guiding the substrate solution discharged from the reaction vessel 1 to the reaction vessel 1.

Reference numeral 11 denotes a reaction stopping liquid tank lid for preventing the reaction stopping liquid in the reaction stopping liquid tank 12 from evaporating. Reference numeral 12 denotes a reaction stopping liquid tank for storing a reaction stopping liquid. Reference numeral 13 denotes a reaction stopping liquid pump for injecting the reaction stopping liquid in the reaction stopping liquid tank 12 into the reaction vessel 1. Reference numeral 14 denotes a reaction stop liquid injection nozzle for guiding the reaction stop liquid discharged from the reaction stop liquid pump 13 to the reaction vessel 1.

Reference numeral 15 denotes a temperature sensor that uses a thermocouple or the like to measure the temperature during the reaction. Also, 16
Reference numeral denotes a temperature adjustment unit that controls the temperature adjustment heater 17 so as to reach a set temperature in response to a temperature measurement result from the temperature sensor 15. Reference numeral 17 denotes a temperature control heater using a nichrome wire or the like to supply heat for temperature control.

Reference numeral 18 denotes a light emitting section using a light emitting diode, a halogen lamp, or the like for irradiating visible light. Reference numeral 19 denotes a light receiving unit using a filter, a lens, a photomultiplier, or the like as necessary to detect light transmitted through the reaction vessel 1. Reference numeral 20 denotes a calculation unit for calculating the cell concentration using a microcomputer or the like.

Reference numeral 21 denotes a display unit using a liquid crystal or the like. Reference numeral 22 denotes an input unit. Reference numeral 23 denotes a printing unit using a thermal head printer or the like. Also,
Reference numeral 24 denotes an input / output interface unit for inputting and outputting external information. Reference numeral 25 denotes a power supply unit, reference numeral 26 denotes a control unit, and reference numeral 27 denotes a reaction container insertion port into which the reaction container 1 is inserted. Reference numeral 28 denotes a reaction vessel outlet for taking out the reaction vessel 1. Reference numeral 29 denotes a housing.

Next, how to carry out an immunological measurement method using this apparatus will be described.

<I-2. Reaction Step> This is a step of distributing the enzyme-labeled antibody 94 in an amount proportional to the amount of antigen to the reaction container 1 and the test strip 2 for measurement. It consists of the following steps.

(I-2) Step of Distributing Enzyme-Labeled Antibody 94 in an Amount Proportionate to the Amount of Antigen into the Reaction Vessel 1 and the Test Specimen 2 It is fixed to the moving support 6. Subsequently, the standard antigen suspension was used as a stock solution, which was sequentially diluted with a surfactant-containing buffer solution to prepare six 1.5- to 20-fold, preferably 2- to 5-fold, standard dilution series solutions. A certain amount is poured from the second reaction vessel 32 of the reaction vessel 1 to the seventh reaction vessel 37, respectively. A buffer solution containing a surfactant is poured as a blank solution into the first reaction vessel 31 of the new reaction vessel 1 in the same amount as the standard dilution series solution. The test solution is poured into the eighth reaction vessel 38 of the new reaction vessel 1 in the same amount as the standard dilution series solution. Further, a fixed amount of the antibody solution is added to each of the first to eighth reaction containers 31 to 38 of the new reaction container 1 into which the test solution, the standard dilution series solution, and the blank solution are poured. The blank solution and the standard dilution series solution are used for preparing a calibration curve.

An antibody solution is prepared by binding a labeling substance to a monoclonal (or polyclonal) antibody capable of specifically binding to the cells as antigen 92, or binding specifically to cells as antigen 92. A solution containing a combination of a monoclonal (or polyclonal) antibody 91 having an ability and an antibody 94 obtained by binding a labeling substance to a monoclonal (or polyclonal) antibody having a specific binding ability to the antibody 91 can be used. . The labeling substance is enzyme 95, preferably peroxidase, β-galactosidase, alkaline phosphatase or the like. FIG. 6 shows a case where a solution containing a combination of an antibody 91 and an antibody 94 having a specific binding ability to an antibody having a specific binding ability to the antibody 91 is used.

The reaction container 1 is inserted through the reaction container insertion port 27 provided in the housing 29. Further, the correspondence between the cell concentration of the solution in the standard dilution column and each reaction container of the reaction container 1 is input to the input unit 2.
2 to the arithmetic unit 20. A plurality of components constituting the reaction container 1 are formed by moving the measurement test piece vertical movement support 6 and thus the measurement test piece 2 up and down by forward and reverse rotations of the measurement test piece vertical movement motor 5 by operation from the input unit 22. A plurality of protrusions constituting the test piece for measurement 2 are immersed in the test solution, the solution in the standard dilution line, and the blank solution stored in the container, and are brought into contact with each other, followed by stirring. The treatment conditions of the test specimen 2 with the test liquid, the standard dilution rate solution, and the blank liquid are as follows: the treatment time is 10 minutes to 3 hours, and the test specimen 2 is immersed at a treatment temperature of 4 to 40 ° C. First, an enzyme-labeled antibody 94 or a complex of the antibody 91 and the enzyme-labeled antibody 94 is dissolved in a standard antigen 93 immobilized on a measurement test piece 2 which is a solid phase, and in a liquid phase in the reaction vessel 1. And the antigen 92 that is present. More preferably, the immersion treatment is performed at a treatment temperature of 4 to 30 ° C. for a treatment time of 30 minutes to 2 hours. The reaction vessel 1 and the measurement test piece 2 are separated by moving the measurement test piece vertical movement support 6 and thus the measurement test piece 2 upward by the measurement test piece vertical movement motor 5,
The contact between the test piece for measurement 2 and the test liquid is cut off.

<I-3. Measurement step> This is a step of adding a chromogenic substrate to form a color by an enzymatic reaction and measuring the degree of color formation. It comprises the following steps:

(I-3) Step of Coloring Substrate by Enzyme Reaction A substrate solution is prepared in advance, and this substrate solution is filled in the substrate solution tank 7. The substrate solution contains a chromogenic substrate corresponding to the labeling substance as a solute, and in the case of peroxidase, o-phenylenediamine dihydrochloride,
', 5,5'-Tetramethylbenzidine, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 5-aminosalicylic acid and the like can be used. The substrate solution contains hydrogen peroxide as a substrate, uses a buffer solution as a solvent, and has a pH of 5.0 ± 0.5.
One 0.1 mol / L citrate buffer or the like can be used.

The reaction container driving belt 3 and the reaction container 1 are sequentially moved in the horizontal direction by the reaction container driving motor 3 so that the substrate solution tank 7
The substrate solution therein is added via a substrate solution injection nozzle 10 by a substrate solution injection pump 9 to advance the reaction. The reaction conditions are
When the reaction is performed at a reaction temperature of 10 to 40 ° C. for a reaction time of 1 to 60 minutes, the labeling substance remaining in the reaction container 1 can efficiently generate a dye from the chromogenic substrate by an enzyme reaction. More preferably, the reaction time is 10 to 10.
The reaction is carried out at a reaction temperature of 15 to 25 ° C. for 20 minutes.

(I-3) Step of Stopping Enzyme Reaction A reaction stop solution is prepared in advance and filled in the reaction stop solution tank 12. The reaction terminating solution contains a reaction terminator corresponding to the labeling substance as a solute, and sulfuric acid, hydrochloric acid, nitric acid,
Phosphoric acid, sodium hydroxide and the like can be used.

The reaction container driving belt 3 and the reaction container 1 are sequentially moved in the horizontal direction by the reaction container driving motor 3, and the reaction stop liquid in the reaction stop liquid tank 12 is stopped in each reaction container of the reaction container 1. The enzyme reaction is stopped by the liquid injection pump 13 through the reaction stop liquid injection nozzle 14.

(I-3) Step of Measuring Color Degree at Coloring Wavelength Reaction vessel driving belt 4 by reaction vessel driving motor 3
Eventually, the reaction vessel 1 is sequentially moved in the horizontal direction, and the light emitting portion 18 is used as a light source, and the wavelength 490 at which the dye specifically absorbs.
The light of nm is detected by the light receiving unit 19 and output to the calculation unit 20.
As a result, calibration-related data indicating the relationship between the absorbance corresponding to the cell concentration of the standard dilution series solution is obtained.

<I-4. Calculation Step> This is a step of calculating the antigen concentration from the calibration curve of the standard antigen solution. It consists of the following steps.

<I-4> Calculation Step The calculation section 20 calculates the absorbance from the detection value of the light receiving section 19, subtracts the absorbance of the blank solution from the absorbance of each standard light source solution, and calculates the regression line or curve of the calibration curve. Determined in part 20. If the absorbance of any test solution is measured from the regression line or regression curve of the calibration curve, the concentration of the test solution can be calculated, and the result can be output to the display unit 21 and displayed. Further, the printing unit 23 can print out the result or output the result to an external arithmetic unit or the like via the input / output interface 24.

As described above, according to the present embodiment, the reacted test piece 2 is prepared as much as possible on the supplier side, and the test piece 2 and the test liquid are reacted in the reaction vessel 1. The labeled antibody (or antigen) is distributed to the reaction container 1 or the measurement test piece 2 in accordance with the antigen 92 concentration of the test solution, and the concentration of the liquid in the reaction container 1 or the label substance on the measurement test piece 2 is caused. It is possible to measure the antigen (or antibody) concentration in the test solution by quantifying the When comparing the conventional measurement method and measurement apparatus, for example, the sandwich ELISA method currently most commonly used with the measurement method of the present invention,
Whereas the sandwich ELISA method requires two steps of the antigen-antibody reaction step and two steps of the washing operation, the measurement method of the present invention requires only one step of the antigen-antibody reaction step, thus eliminating the need for the washing operation. This is a specific immunological measurement method that allows simple, rapid, and inexpensive quantification of an antigen (or antibody).

Further, by practicing the measurement method of the present invention, it is also possible to provide a specific immunological apparatus capable of simply, rapidly, sensitively and inexpensively quantifying an antigen (or antibody).

(Embodiment 2) In Embodiment 2, the allergen concentration is measured using an allergen having an antigenic determinant to which an antibody specifically binds as an antigen. Hereinafter, the measurement of the antigen will be described. For example, it is also possible to reverse the antigen and the antibody, for example, by measuring the amount of an antibody in the serum against the allergen (antigen). For these reasons, the description of antibody measurement is omitted.

FIG. 7 is a schematic diagram showing main steps of the second embodiment of the present invention. In FIG. 7, reference numeral 1 denotes a reaction vessel;
Is a test piece for measurement, 92 is an antigen, 93 is a standard antigen,
94 is an enzyme-labeled antibody, and 95 is an enzyme.

The immunological assay method according to the second embodiment has four steps <II-1. Step of preparing test piece for measurement>, <II-2.
Reaction step>, <II-3. Measurement step> and <II-4.
Calculation step>.

<II-1. Step of preparing test piece 2 for measurement> This is a step of immobilizing the standard antigen 93 and the enzyme-labeled antibody 94 on the test piece 2 for measurement to bring them into a stabilized state. It comprises the following steps:

(II-1) Step of Immobilizing Standard Antigen 93 on Test Piece for Measurement 2 The standard antigen 93 is an antigen to be measured, and a single allergen solution is used.

The reaction vessel 1 is a vessel made of an optically transparent synthetic resin such as polystyrene or the like, and is a collection of independent single or plural vessels, for example, as shown in FIG. Preferably has a horizontal cross-sectional area of 10 to 500 mm ² and a depth of about 1 to 1
By setting the content to 30 μm and the content to 10 μL to 15 mL, the amount of liquid necessary for the measurement is secured, and the operation becomes easy. More preferably, the content is in the range of 50 to 500 μL. It is preferable to carry out a treatment with a blocking agent in advance so that organic matter is not bound to the reaction vessel 1. For example, a blocking agent solution containing a protein such as bovine serum albumin and milk-derived casein is used as the blocking agent. The solvent for the blocking agent solution is a buffer, preferably a phosphate buffer, a borate buffer, or the like to adjust the pH.
Can be kept constant. More preferably, a salt such as sodium chloride, potassium chloride or the like is mixed with the buffer solution. By setting the pH of the buffer solution to preferably 5 to 9, stable nonspecific adsorption of the blocking agent can be generated in the reaction vessel 1. The pH is more preferably in the range of 6 to 8. The conditions for the treatment with the blocking agent solution are pH 5 to 9, the treatment time is 10 minutes to 48 hours, and the non-specific adsorption of the blocking agent on the surface of the reaction vessel 1 where non-specific adsorption occurs by immersion treatment at a processing temperature of 4 to 40 ° C Then, non-specific adsorption of other organic substances is prevented from occurring as much as possible, and more accurate measurement is possible. More preferably, immersion treatment is performed at a pH of 6 to 8, a treatment time of 30 minutes to 2 hours, and a treatment temperature of 4 to 30 ° C.

The test piece for measurement 2 is a protrusion such as a bead, a film, a column, a container, etc. made of a synthetic resin such as polystyrene treated so that an organic substance is highly bonded. Alternatively, it is an aggregate of a plurality of protrusions (for example, in the form of a test piece 2 for measurement shown in FIG. 4), and preferably has a horizontal sectional area of 10 to 500 mm ² and a depth of about 1 to 30 mm.
By adjusting the volume to 10 μL to 15 mL, the amount of liquid necessary for the measurement is secured, and the operation becomes easy. More preferably, the volume is in the range of 50 to 500 μL.

As the standard antigen 93 solution, a solution of an allergen as an antigen is used. The standard antigen 93 solution is preferably 0.0001 to 100 μg / mL, and a sufficient amount of antigen for measurement can be brought into contact with the measurement test piece 2 and immobilized by non-specific adsorption. More preferably 0.001
-10 μg / mL. The solvent of the standard antigen 93 is a buffer, preferably a phosphate buffer, a borate buffer, or the like, so that the pH can be kept constant. More preferably, a salt such as sodium chloride, potassium chloride or the like is mixed with the buffer solution. By setting the pH of the buffer solution to preferably 5 to 9, stable non-specific adsorption of allergen can be generated on the test strip 2 for measurement. The pH is more preferably in the range of 6 to 8.

The standard antigen 93 solution was poured from the first reaction vessel 31 of the reaction vessel 1 to the eighth reaction vessel 38 so as to have the same volume and the same concentration. The first to eighth measurement test pieces 41 of the test piece for measurement 2 are inserted into the solution 38.

The treatment conditions of the reaction vessel 1 and the test piece 2 for measurement with the standard antigen 93 solution are pH 5 to 9, treatment time is 10 minutes to 48 hours, and immersion treatment is performed at a treatment temperature of 4 to 40 ° C. Allergen as an antigen can be non-specifically adsorbed on the piece 2 at high density. More preferably, the pH is 6 to 8, the treatment time is 30 minutes to 2 hours, and the treatment temperature is 4
This is an immersion treatment in the range of 30 ° C.

(II-1) Step of Washing and Removing Unreacted Standard Antigen 93 The test piece 2 for measurement and the reaction vessel 1 are preferably washed with a buffer solution.
By washing 10 to 10 times, the unreacted standard antigen 93 can be efficiently removed, and the accuracy of the subsequent measurement can be improved. More preferably, it is in the range of 2 to 5 times.

(II-1) Reaction vessel 1 with blocking agent
Step of coating non-specific adsorption surface of (1) The blocking agent solution is poured into the reaction vessel 1 and the measurement test piece 2 is inserted. Non-specific adsorption remaining on the test piece 2 for measurement by immersion at a treatment temperature of 4 to 40 ° C. at a treatment condition of pH 5 to 9, a treatment time of 10 to 48 hours, and a treatment temperature of 4 to 40 ° C. The non-specific adsorption of the blocking agent on the surface causing the non-specific adsorption of non-specific substances such as other organic substances is prevented as much as possible, so that more accurate measurement can be performed. More preferably, immersion treatment is performed at a pH of 6 to 8, a treatment time of 30 minutes to 2 hours, and a treatment temperature of 4 to 30 ° C.

(II-1) Step of Washing and Removing Unreacted Blocking Agent The test piece 2 for measurement and the reaction vessel 1 were washed with a buffer solution containing a surfactant.
Is preferably washed 1 to 10 times, whereby the unreacted blocking agent can be efficiently removed, and the accuracy of the subsequent measurement can be improved. More preferably, 2 to 5
Range of times.

The solvent of the surfactant-containing buffer solution is such that the buffer solution contains polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (80) sorbitan monolaurate or the like as a surfactant. .
The concentration of the surfactant is preferably 0.001-1 w / v%
By doing so, unreacted proteins and the like can be efficiently removed, and the accuracy of subsequent measurements can be improved. More preferably, it is in the range of 0.01 to 0.2 w / v%.

(II-1) Step of Specific Adsorption of Enzyme-Labeled Antibody 94 to Standard Antigen 93 on Test Specimen 2 As an enzyme-labeled antibody 94 solution, an antibody that has specific binding ability to an antigen allergen is used. An antibody 94 in which a labeling substance is bound to a monoclonal (or polyclonal) antibody, or a monoclonal (or polyclonal) antibody having a specific binding ability to an antigen allergen and a monoclonal having a binding ability specific to this antibody A solution containing a combination of a nal (or polyclonal) antibody and an antibody having a labeled substance bound thereto can be used. The labeling substance is enzyme 95, preferably peroxidase, β
-Galactosidase, alkaline phosphatase and the like. FIG. 7 shows a case where a solution containing an antibody 94 in which a labeling substance is bound to an antibody capable of specifically binding to an antigen is used.

The solution of the enzyme-labeled antibody 94 was placed in the first
The same volume from the reaction vessel 31 to the eighth reaction vessel 38
Pour the mixture so as to have the same concentration.
The first to eighth measurement test pieces 41 of the measurement test piece 2 are inserted into the solutions of the first to eighth reaction vessels 38, respectively. By treating the test piece 2 for measurement with the enzyme-labeled antibody 94 solution at a pH of 5 to 9, a treatment time of 10 minutes to 48 hours, and a immersion treatment at a treatment temperature of 4 to 40 ° C.
The enzyme-labeled antibody 94 can specifically bind to the immobilized standard antigen 93 on the measurement test piece 2 by the antigen-antibody reaction. More preferably, the pH is 6 to 8, and the treatment time is 30 minutes to 2 hours.
It is an immersion treatment at a treatment temperature of 4 to 30 ° C. for a time.

(II-1) Step of Washing and Removing Unreacted Enzyme-Labeled Antibody 94 The test specimen 2 for measurement and the reaction vessel 1 were washed with a buffer containing a surfactant.
Is preferably washed 1 to 10 times, whereby the unreacted enzyme-labeled antibody 94 can be efficiently removed, and the accuracy of the subsequent measurement can be improved. More preferably 2-
The range is five times.

(II-1) Step of Performing Processing for Preserving Measurement Test Piece 2 The measurement test piece 2 is kept in a long-term stabilized state by using a freeze-drying method, air drying or the like as a storage means.

Next, <II-1. Measurement Test Piece Preparation Step> The immunological measurement method of the present invention for measuring the allergen concentration using the measurement test piece 2 prepared in <II-2. Reaction step>, <II-3. Measurement step>, <II-4. Calculation process>,
An immunological measurement device for performing the immunological measurement method will be described. Hereinafter, description will be given according to the immunological measurement apparatus shown in FIGS.

1 and 2, reference numeral 1 denotes a reaction vessel;
Is a test piece for measurement, 3 is a motor for driving the reaction vessel, 4 is a belt for driving the reaction vessel, 5 is a motor for vertically moving the test piece for measurement, 6 is a support for vertically moving the test piece for measurement, and 7 is a substrate. Reference numeral 8 denotes a substrate solution tank, 9 denotes a substrate solution pump, and 10 denotes a substrate solution injection nozzle. Reference numeral 11 denotes a reaction stop liquid tank lid, 12 denotes a reaction stop liquid tank, 13 denotes a reaction stop liquid pump, and 14 denotes a reaction stop liquid injection nozzle. 15 is a temperature sensor, 16 is a temperature adjustment unit,
17 is a temperature control heater, 18 is a light emitting unit, 19 is a light receiving unit, 20 is a calculation unit, 21 is a display unit, 22 is an input unit, 23 is a printing unit, and 24 is an input / output interface unit. , 25 are a power supply unit, 26 is a control unit, 27 is a reaction vessel insertion port, 28 is a reaction vessel discharge port, and 29 is a housing.

The components having the same reference numerals as those described in the first embodiment are the same as those in the first embodiment, and the detailed description will be omitted.

Next, how to carry out an immunoassay using this apparatus will be described.

<II-2. Reaction Step> This is a step of distributing the enzyme-labeled antibody 94 in an amount proportional to the amount of antigen to the reaction container 1 and the test strip 2 for measurement. It consists of the following steps.

(II-2) Step of Distributing Enzyme-Labeled Antibody 94 in an Amount Proportionate to the Amount of Antigen to the Reaction Vessel 1 and the Test Specimen 2 It is fixed to the moving support 6. Subsequently, the standard antigen solution was used as a stock solution, and was sequentially diluted with a surfactant-containing buffer solution to prepare six kinds of standard dilution series solutions of 1.5 to 20 times, preferably 2 to 5 times, and a new reaction vessel was prepared. 1 second reaction vessel 3
A certain amount is poured into each of the second to seventh reaction vessels 37. A buffer solution containing a surfactant is poured as a blank solution into the first reaction vessel 31 of the new reaction vessel 1 in the same amount as the standard dilution series solution. The test solution is poured into the eighth reaction vessel 38 of the new reaction vessel 1 in the same amount as the standard dilution series solution. Further, a fixed amount of the antibody solution is added to each of the first to eighth reaction containers 31 to 38 of the new reaction container 1 into which the test solution, the standard dilution series solution, and the blank solution are poured. The blank solution and the standard dilution series solution are used for preparing a calibration curve.

The reaction vessel 1 is inserted through the reaction vessel insertion port 27 provided in the housing 29. Further, the correspondence between the allergen concentration of the standard dilution series solution and each reaction container of the reaction container 1 is input to the calculation unit 20 via the input unit 22. The rotation of the motor 5 for the vertical movement of the test piece for measurement
The reaction vessel 1 is moved up and down by the reverse rotation of the support 6 for vertically moving the test piece for measurement and thus the test piece 2 for measurement.
A plurality of projections constituting the test piece for measurement 2 are immersed in the test solution, the solution in the standard dilution row, and the blank solution stored in the plurality of containers constituting the above, and are brought into contact with each other, followed by stirring. The processing conditions for the test piece 2 for measurement using the test solution, the solution in the standard dilution line, and the blank solution are as follows: a processing time of 10 minutes to 3 hours.
By immersion treatment at a treatment temperature of 4 to 40 ° C., the enzyme-labeled antibody 94 or a complex of the antibody 91 and the enzyme-labeled antibody 94 is immobilized on the measurement test piece 2 which is a solid phase. It becomes possible to partition the standard antigen 93 and the antigen 92 dissolved in the liquid phase in the reaction vessel 1 by a chemical equilibrium reaction. More preferably, the immersion treatment is performed at a treatment temperature of 4 to 30 ° C. for a treatment time of 30 minutes to 2 hours.
The reaction container 1 and the measurement test piece 2 are separated by moving the measurement test piece vertical movement support 6 and, consequently, the measurement test piece 2 upward by the measurement test piece vertical movement motor 5. Cut off the contact with the test solution.

<II-3. Measurement step> This is a step of adding a chromogenic substrate to form a color by an enzymatic reaction and measuring the degree of color formation. It comprises the following steps:

(II-3) Step of Coloring Substrate by Enzymatic Reaction A substrate solution is prepared in advance, and this substrate solution is filled in the substrate solution tank 7. The substrate solution contains a chromogenic substrate corresponding to the labeling substance as a solute, and in the case of peroxidase, o-phenylenediamine dihydrochloride,
', 5,5'-Tetramethylbenzidine, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 5-aminosalicylic acid and the like can be used. The substrate solution contains hydrogen peroxide as a substrate, uses a buffer solution as a solvent, and has a pH of 5.0 ± 0.5.
One 0.1 mol / L citrate buffer or the like can be used.
Reaction vessel driving belt 4 by reaction vessel driving motor 3
As a result, the reaction vessel 1 is sequentially moved in the horizontal direction, and the substrate solution in the substrate solution tank 7 is supplied to the plurality of reaction vessels of the reaction vessel 1 by the substrate solution injection pump 9.
And the reaction proceeds. The reaction conditions include a reaction time of 1 minute to 60 minutes and a reaction temperature of 10 to 40 ° C., whereby the labeling substance remaining in the reaction vessel 1 efficiently generates a dye from a chromogenic substrate by an enzymatic reaction. Becomes possible. More preferably, the reaction is carried out at a reaction temperature of 15 to 25 ° C. for a reaction time of 10 to 20 minutes.

(II-3) Step of Stopping Enzyme Reaction A reaction stop solution is prepared in advance and filled in the reaction stop solution tank 12. The reaction terminating solution contains a reaction terminator corresponding to the labeling substance as a solute, and sulfuric acid, hydrochloric acid, nitric acid,
Phosphoric acid, sodium hydroxide and the like can be used.

The reaction vessel driving belt 4 and, consequently, the reaction vessel 1 are sequentially moved in the horizontal direction by the reaction vessel driving motor 3, and the reaction stopping liquid in the reaction stopping liquid tank 12 is stopped in each reaction vessel of the reaction vessel 1. The enzyme reaction is stopped by the liquid injection pump 13 through the reaction stop liquid injection nozzle 14.

(II-3) Step of Measuring Color Degree at Coloring Wavelength The reaction container driving belt 4 is driven by the reaction container driving motor 3.
Eventually, the reaction vessel 1 is sequentially moved in the horizontal direction, and the light emitting portion 18 is used as a light source, and the wavelength 490 at which the dye specifically absorbs.
The light of nm is detected by the light receiving unit 19 and output to the calculation unit 20.

<II-4. Calculation Step> This is a step of calculating the antigen concentration from the calibration curve of the standard antigen solution. It consists of the following steps.

(II-4) Calculation Step The calculation section 20 calculates the absorbance from the detected value of the light receiving section 19, subtracts the absorbance of the blank solution from the absorbance of each standard light source solution, and calculates the regression line or curve of the calibration curve. Determined in part 20. If the absorbance of any test liquid is measured from the regression line or regression curve of the calibration curve, the concentration of the test liquid can be calculated, and the result can be output to the display unit 21 and displayed. Further, the printing unit 23 prints out the result, and outputs the result to an external arithmetic unit or the like via the input / output interface 24.

As described above, according to the present embodiment, a reacted measurement test piece 2 is prepared as much as possible on the supplier side, and the measurement test piece 2 and the test solution are reacted in the reaction vessel 1 to obtain a test piece. The labeled antibody (or antigen) is distributed to the reaction vessel 1 or the measurement test piece 2 according to the antigen 92 concentration of the test solution, and the concentration of the liquid in the reaction vessel 1 or the labeling substance on the measurement test piece 2 is determined. By quantifying, it is possible to measure the antigen (or antibody) concentration in the test solution. When comparing the conventional measurement method and measurement apparatus, for example, the sandwich ELISA method currently most commonly used with the measurement method of the present invention,
The sandwich ELISA method has two immunological reaction steps,
In contrast to the need for a washing step, the assay method of the present invention requires only a single antigen-antibody reaction step, eliminating the need for a washing step, allowing simple, rapid, and inexpensive quantification of antigen (or antibody). This is a specific immunological measurement method that can be performed.

Further, by practicing the measurement method of the present invention, it is also possible to provide a specific immunological apparatus capable of quantifying an antigen (or antibody) simply, quickly, with high sensitivity and at low cost.

[0137]

EXAMPLES The present invention will be described below by way of examples. In the following examples, unless otherwise specified, "%" is used.
Means "% by weight". In Examples 1 and 2, the apparatus shown in FIGS. 1 and 2 was used as an immunological measurement apparatus.

Example 1 In Example 1, Legionella pneumophila serogroup 1 was used.
The number of cells of a serogroup I) was measured. FIG. 8 shows Legionella pneumophila serogroup 1 (Legio) prepared using the measuring apparatus of the present invention.
nella pneumophilaserosgrou
It is a calibration curve of pI).

The immunological measurement method of the present invention comprises four steps <I-1. Step of preparing test piece for measurement>, <I-2.
Reaction step>, <I-3. Measurement step> and <I-4.
Calculation step>.

<I-1. Step of preparing test piece 2 for measurement> This is a step of immobilizing the standard antigen 93 on the test piece 2 for measurement to bring it into a stabilized state. It comprises the following steps:

(I-1) Step of Immobilizing Standard Antigen 93 on Test Specimen 2 Legionella pneumophila serogroup 1 (Le) treated with 70% ethanol for 24 hours as standard antigen 93
gionella pneumophilaselog
loop I, hereinafter referred to as "Lp"). Dulbecco's phosphate buffered saline (hereinafter referred to as “PBS”) having a pH of 7.4 ± 0.2 was used for the dilution.

The reaction vessel 1 is made of polystyrene, has a horizontal sectional area of about 24 mm ² (inner diameter of 5.5 mm) and a depth of about 11.5 m.
This is a cylindrical container having a length of m, and has a structure in which eight units from a first reaction container 31 to an eighth reaction container 38 whose container bottoms are optically transparent and uniform are formed in a row by a reaction container connection frame 30.
A pH 7.4 ± 0.2 PBS solution (hereinafter, “PBS-B”) containing 1 w / v% bovine serum albumin (hereinafter, referred to as “BSA”) as a blocking agent so that organic substances do not bind to the reaction vessel 1 in advance. For 1 hour,
The one immersed under the condition of 25 ° C. was used.

The test piece for measurement 2 is made of polystyrene treated so as to have a high binding of organic substances. The protrusion has a horizontal sectional area of about 1.8 mm ² (outer diameter of 3.5 mm) and a depth of about 11.5 mm.
An eight-mm cylindrical test piece 41 to an eighth test piece 48 having a structure in which eight test strip connecting frames 40 were formed in a line were used. The first reaction container 31 to the eighth reaction container 38 of the reaction container 1 are fixed at the joints between the first measurement test piece 41 to the eighth measurement test piece 48 and the measurement test piece connection frame 40. Each of which was provided with a reaction container insertion guide 49 so as to fit in the column.

A suspension of Lp, which is an antigen, in PBS at 10 ¹⁰ cells / mL was added to the reaction vessel 1 and 100 μL of the suspension was added to each solution in the reaction vessel 38 from the first reaction vessel 31 in the reaction vessel 1. Test piece 2 in which antigen Lp is solid-phased by inserting first test piece 41 to eighth test piece 48 of test piece 2 and immersing for 2 hours at 25 ° C. Was immobilized non-specifically on the protrusions.

(I-1) Step of Washing and Removing Unreacted Standard Antigen 93 The first to eighth measurement test pieces 41 to 48 of the measurement test piece 2 and the reaction vessel 1 are measured using PBS as a buffer solution. The insides of the first to eighth reaction vessels 31 to 38 were washed three times to remove unreacted Lp.

(I-1) Reaction vessel 1 with blocking agent
Step 25: covering the non-specific adsorption surface of the reaction vessel 1 from the first reaction vessel 31 to the eighth reaction vessel 38 with 25 parts of PBS-B.
0 μL was added, and the first measurement test piece 41 of the measurement test piece 2 was added.
From the first measurement test piece 41 to the eighth measurement test piece 48 of the measurement test piece 2, and then immersion treatment is performed at 25 ° C. for 1 hour. BSA was non-specifically adsorbed on the specific adsorption surface so that non-specific adsorption of other organic substances was prevented as much as possible.

(I-1) Step of Washing and Removing Unreacted Blocking Agent 0.05% w / v polyoxyethylene (20) sorbitan monolaurate is contained in PBS as a buffer solution.
7.4 ± 0.2 solution (hereinafter referred to as “PBS-T”)
It was used. Each of the first to eighth measurement test pieces 48 to 48 of the measurement test piece 2 and the first to eighth measurement test pieces 38 to 38 in the reaction container 1 is performed three times with PBS-T. Unreacted BSA was removed by washing.

<I-1> Step of Performing Processing for Preserving Test Specimen 2 for Measurement The test specimen 2 for measurement was kept in a long-term stabilized state by using a freeze-drying method as a preserving means.

<I-2. Reaction Step> This is a step of distributing the enzyme-labeled antibody 94 in an amount proportional to the amount of antigen to the reaction container 1 and the test strip 2 for measurement. It consists of the following steps.

(I-2) Step of Distributing Enzyme-Labeled Antibody 94 in an Amount Proportionate to the Amount of Antigen to the Reaction Vessel 1 and the Test Specimen 2 It was fixed to the moving support 6. Subsequently, the standard antigens were sequentially diluted with PBS-T 10-fold from 5 × 10 ⁹ cells / mL to prepare seven standard dilution series of solutions. 100 μL of T was added to each of the first to eighth reaction vessels 31 to 38 of the new reaction vessel 1.

Further, 20.0 μg / m 2 of the antibody solution was used.
L anti-Lp mouse-derived monoclonal IgG antibody (hereinafter referred to as “ALp”) and 10.0 μg / mL horseradish-derived peroxidase-labeled anti-mouse IgG
(Γ) Goat-derived monoclonal antibody (hereinafter “AMGP
O) was poured into each of the first to eighth reaction vessels 31 to 38 of the reaction vessel 1.

The test piece 2 for measurement is immersed in the reaction vessel 1 at 25 ° C. for 2 hours, and ALp is immobilized on the test piece 2 for measurement according to the concentration of the immobilized antigen Lp and the antigen Lp in the solution. And A
The composite of MGPO and the solid phase of the test piece 2 for measurement and the reaction vessel 1
In each liquid phase.

<I-3. Measurement step> This is a step of adding a chromogenic substrate to form a color by an enzymatic reaction and measuring the degree of color formation. It comprises the following steps:

(I-3) Step of Coloring Substrate by Enzymatic Reaction 10 mL of 0.1 mol of pH 5.0 ± 0.1 in advance.
4 mg of o-phenylenediamine dihydrochloride (hereinafter referred to as “OPD”) was dissolved in 1 / L citrate buffer (hereinafter referred to as “citrate buffer”), and 10 μL of 30% hydrogen peroxide was added. Solution (hereinafter referred to as "substrate solution")
Was prepared. The substrate solution was filled in the substrate solution tank 7.

In each of the reaction vessels 31 to 38, 1
Add 00 μL, react at 25 ° C. for 15 minutes, and add AMGP
A dye was generated from peroxidase, which is a labeling substance (enzyme) of O, from the chromogenic substrate OPD by an enzymatic reaction.

(I-3) Step of Stopping Enzyme Reaction 5 mL of a 1 mol / L sulfuric acid solution (hereinafter referred to as “reaction stop solution”) was charged into the reaction stop solution tank 12 in advance.

In each of the reaction vessels 31 to 38, 5
0 μL was added and the reaction was allowed to proceed at 25 ° C. for 15 minutes to stop the enzyme reaction.

(I-3) Step of Measuring Coloring Degree at Coloring Wavelength Light having a wavelength of 490 nm using light emitting section 18 as a light source is light receiving section 1.
9 and output to the calculation unit 20.

<I-4. Calculation Step> This is a step of calculating the antigen concentration from the calibration curve of the standard antigen solution. It consists of the following steps.

(I-4) Calculation Step The calculation section 20 calculates the absorbance from the detection value of the light receiving section 19, and subtracts the absorbance of the blank PBS-T solution from the absorbance of each standard antigen solution to create a calibration curve. Then, FIG. 8 was obtained. When the regression line was obtained from the straight line portion in FIG. 8 by the least square method, the following equation (1) was obtained. [Absorbance (-)] = 0.255 x ln [Lp concentration (piece (n) / mL)] -2.615 (1)

FIG. 8 shows that in this embodiment, 1 × 10 ⁵ to 1 × 1
0 ⁷ cells / mL of Legionella pneumophila serogroup 1 is understood to be measurable. Then, if the absorbance of any test liquid was measured, the concentration of the test liquid could be calculated from equation (1), and the result could be output to the display unit 21 and displayed. Further, the result can be output to an external arithmetic unit or the like through the print output of the result by the printing unit 23 or the input / output interface 24.

Example 2 In Example 2, r, the allergen of Dermatophagoides farinae, was used.
The concentration of Der f II was measured.

FIG. 9 is a calibration curve of the allergen rDer f II of Dermatophagoides farinae prepared using the measuring apparatus of the present invention.

The immunological measurement method of the present invention comprises four steps <II-1. Preparation of test piece for measurement>, <II-
2. Reaction step>, <II-3. Measurement process> and <II-
4. Calculation step>.

<II-1. Step of preparing test piece 2 for measurement> This is a step of immobilizing the standard antigen 93 and the enzyme-labeled antibody 94 on the test piece 2 for measurement to bring them into a stabilized state. It comprises the following steps:

(II-1) Step of Immobilizing Standard Antigen 93 on Measurement Test Piece 2 As standard antigen 93, allergen derived from Dermatophagoides farinae was produced and purified by genetic manipulation of Escherichia coli.
er f II (hereinafter referred to as “rDer f II”) was used. Dulbecco's phosphate buffered saline (hereinafter referred to as “PBS”) having a pH of 7.4 ± 0.2 was used for the dilution.

The reaction vessel 1 is made of polystyrene, has a horizontal sectional area of about 24 mm ² (inner diameter of 5.5 mm) and a depth of about 11.5 m.
This is a cylindrical container having a length of m, and has a structure in which eight units from a first reaction container 31 to an eighth reaction container 38 whose container bottoms are optically transparent and uniform are formed in a row by a reaction container connection frame 30.
A pH 7.4 ± 0.2 PBS solution (hereinafter, “PBS-B”) containing 1 w / v% bovine serum albumin (hereinafter, referred to as “BSA”) as a blocking agent so that organic substances do not bind to the reaction vessel 1 in advance. For 1 hour,
The one immersed under the condition of 25 ° C. was used.

The test piece for measurement 2 is made of polystyrene treated so as to have a high binding of organic substances. The protrusion has a horizontal sectional area of about 1.8 mm ² (outer diameter of 3.5 mm) and a depth of about 11.5 mm.
An eight-mm cylindrical test piece 41 to an eighth test piece 48 having a structure in which eight test strip connecting frames 40 were formed in a line were used. The first reaction container 31 to the eighth reaction container 38 of the reaction container 1 are fixed at the joints between the first measurement test piece 41 to the eighth measurement test piece 48 and the measurement test piece connection frame 40. Each of which was provided with a reaction container insertion guide 49 so as to fit in the column.

In the reaction container 1, the standard antigen 93, rDer
fII PBS solution was adjusted to 0.5 μg / mL, and 100 μL of each solution was poured into the first reaction vessel 31 in the reaction vessel 1.
By inserting the first test piece 41 to the eighth test piece 48 of the test piece 2 of the test piece 2 into each solution in the reaction vessel 38, and immersing for 2 hours at 25 ° C. From the first measurement test piece 41 to the eighth measurement test piece 48 of the measurement test piece 2 in which the antigen rDer f II is a solid phase
Was immobilized non-specifically.

(II-1) Step of Washing and Removing Unreacted Standard Antigen 93 The first to eighth measurement test pieces 41 to 48 of the measurement test piece 2 and the reaction vessel 1 were measured with PBS as a buffer solution. The insides of the first to eighth reaction vessels 31 to 38 were washed three times to remove unreacted rDer f II in the rDer f II solution.

(II-1) Reaction vessel 1 with blocking agent
Of PBS-B is added to each of the first to eighth reaction vessels 31 to 38 of the reaction vessel 1, and 250 μL of PBS-B is added to each of the first to fourth reaction vessels. Non-specific adsorption surface where the test piece 48 for measurement is inserted and immersed for 1 hour at 25 ° C. to leave the test piece 48 for measurement from the first test piece 41 to the eighth test piece 48 of the test piece 2 for measurement. Was non-specifically adsorbed to prevent non-specific adsorption of other organic substances.

(II-1) Step of Washing and Removing Unreacted Blocking Agent PBS containing 0.05% (w / v) polyoxyethylene (20) sorbitan monolaurate as a buffer,
7.4 ± 0.2 solution (hereinafter referred to as “PBS-T”)
It was used. Each of the first to eighth measurement test pieces 48 to 48 of the measurement test piece 2 and the first to eighth measurement test pieces 38 to 38 in the reaction container 1 is performed three times with PBS-T. Unreacted BSA was removed by washing.

(II-1) Step of Specific Adsorption of Enzyme-Labeled Antibody 94 to Antigen on Test Specimen 2 As an enzyme standard antibody 94, an anti-rDer f II mouse monoclonal IgG antibody is labeled with horseradish peroxidase. 13A4PO (hereinafter "13A4PO")
) Was used.

From the first reaction vessel 31 of the reaction vessel 1 to the eighth reaction vessel 38, a 0.5 μg / mL 13A4PO solution 25 was added.
0 μL is poured, and the first measurement test piece 41 of the measurement test piece 2
To the eighth measurement test piece 48, and immersion treatment at 25 ° C. for 2 hours, and the surface of the first measurement test piece 41 to the eighth measurement test piece 48 of the measurement test piece 2 The above immobilized antigen rDer f II was specifically bound by an antigen-antibody reaction.

(II-1) Step of Washing and Removing Unreacted Enzyme-Labeled Antibody 94 The first to eighth measurement test pieces 41 to 48 of the measurement test piece 2 and the reaction vessel 1 were washed with PBS-T. The first measurement test piece 31 to the eighth measurement test piece 38 were each washed three times to remove unreacted 13A4PO.

(II-1) Step of Performing Processing for Preserving the Test Specimen for Measurement 2 The freeze-drying method was used as a preserving means to make the test specimen for measurement 2 stable for a long time.

<II-2. Reaction Step> This is a step of distributing the enzyme-labeled antibody 94 in an amount proportional to the amount of antigen to the reaction container 1 and the test strip 2 for measurement. It consists of the following steps.

(II-2) Step of Distributing Enzyme-Labeled Antibody 94 in an Amount Proportionate to the Amount of Antigen to the Reaction Vessel 1 and the Test Specimen 2 It was fixed to the moving support 6. Subsequently, the standard antigen was diluted three times with PBS-T three times sequentially from 0.1 μg / mL to prepare seven kinds of standard dilution series of solutions, and PBS-T was used as a blank of the seven standard dilution series solutions. New reaction vessel 1
From the first reaction vessel 31 to the eighth reaction vessel
00 μL was added.

The test piece 2 for measurement is immersed in the reaction vessel 1 for 2 hours at 25 ° C., and the immobilized antigen rDer f II of the test piece 2 and the antigen rDer f in the antigen in the solution are measured.
According to the concentration of II, 13 specifically adsorbed to the test piece 2 for measurement.
The test piece 2 for A4PO solid phase and the reaction vessel 1 for liquid phase were distributed.

<II-3. Measurement step> This is a step of adding a chromogenic substrate to form a color by an enzymatic reaction, and measuring the degree of color development. It comprises the following steps:

(II-3) Step of Coloring Substrate by Enzyme Reaction: 10 mL of 0.1 mol of pH 5.0 ± 0.1 in advance.
4 mg of o-phenylenediamine dihydrochloride (hereinafter referred to as “OPD”) was dissolved in 1 / L citrate buffer (hereinafter referred to as “citrate buffer”), and 10 μL of 30% hydrogen peroxide was added. Solution (hereinafter referred to as "substrate solution")
Was prepared. The substrate solution was filled in the substrate solution tank 7.

In each of the reaction vessels 31 to 38, 1
Add 00 μL, react at 25 ° C. for 15 minutes, and add 13AP
A dye was generated from peroxidase, which is a labeling substance (enzyme) of O, from the chromogenic substrate OPD by an enzymatic reaction.

(II-3) Step of Stopping Enzyme Reaction 5 mL of a 1 mol / L sulfuric acid solution (hereinafter referred to as “reaction stop solution”) was charged into the reaction stop solution tank 12 in advance. 50 μL of the substrate solution is added to each of the reaction vessels 31 to 38,
The reaction was allowed to proceed at 25 ° C. for 15 minutes to stop the enzyme reaction.

(II-3) Step of Measuring Color Degree at Coloring Wavelength Light having a wavelength of 490 nm using light emitting section 18 as a light source is light receiving section 1.
9 and output to the calculation unit 20.

<II-4. Calculation Step> This is a step of calculating the antigen concentration from the calibration curve of the standard antigen solution. It consists of the following steps.

(II-4) Calculation Step The calculation section 20 calculates the absorbance from the detection value of the light receiving section 19, and subtracts the absorbance of the blank PBS-T solution from the absorbance of each standard antigen solution to create a calibration curve. Then, FIG. 9 was obtained. When the regression line is obtained from the straight line portion in FIG. 9 by the least square method, the following equation (2) is obtained. [Absorbance (-)] = 0.292 × ln [rDer f II concentration (μg / mL)] + 2.224 (2)

As shown in FIG. 9, in this embodiment, 0.002 to 0.0
It can be seen that 3 μg / mL rDer f II can be measured. Then, if the absorbance was measured for any test liquid, the concentration of the test liquid could be calculated from equation (2), and the result could be output to the display unit 21 and displayed.
Further, the result can be output to an external arithmetic unit or the like through the print output of the result by the printing unit 23 or the input / output interface 24.

Various apparatuses for measuring an antigen (or an antibody) other than the above Examples 1 and 2 can be considered. For example, more automated equipment, the detection method is electrochemical,
A method using a detection of a radioisotope, a method using a column for an antigen-antibody reaction, and the like can be considered.

[0189]

As described above, according to the present invention, by reacting a test strip for measurement with a test solution, the labeled antibody (or antigen) depending on the test solution concentration can be converted into a liquid phase or a solid phase. An antigen (or antibody) that can be quantified quickly, simply, and inexpensively by measuring the concentration of the antigen (or antibody) in the test liquid by allowing the partitioning and quantifying the concentration of the label substance in the liquid phase or the solid phase (or Antibody) can be provided.

Furthermore, by practicing the method for measuring a solution component according to the present invention, a rapid, simple and highly sensitive measurement can be automatically performed, and an inexpensive antigen (or antibody) specific measurement device is provided. You can also.

By utilizing the antigen (or antibody) measuring method and measuring apparatus of the present invention, the concentration of the antigen (or antibody) component can be obtained in a specific, simple, rapid and inexpensive manner. It is possible to provide useful information not only in clinical tests, confirmation of cleaning degree of cleaning companies and cleaning companies, but also in personal allergen management at home. Further, the research speed and the research accuracy can be improved in basic research and application / development research on each component.

[0192]

[Brief description of the drawings]

FIG. 1 is a schematic sectional view of an immunological measurement device according to Embodiments 1 and 2 of the present invention.

FIG. 2 is a schematic plan view of the immunological measurement device according to Embodiments 1 and 2 of the present invention.

FIG. 3 is a schematic perspective view showing a reaction vessel according to Embodiments 1 and 2 of the present invention.

FIG. 4 is a schematic perspective view showing a test piece for measurement in Embodiments 1 and 2 of the present invention.

FIG. 5 is a schematic cross-sectional view showing a state where a reaction container and a test piece for measurement are combined in Embodiments 1 and 2 of the present invention.

FIG. 6 is a schematic view showing main steps of the first embodiment of the present invention.

FIG. 7 is a schematic view showing main steps of a second embodiment of the present invention.

FIG. 8 shows Embodiment 1 of the present invention in Embodiment 1;
Legionella pneumophila cello group 1 (Legionellap) prepared using the measuring device in
It is a graph which shows the calibration curve of neuophila serogroup 1).

FIG. 9 shows Embodiment 2 of the present invention;
4 is a graph showing a calibration curve of the allergen rDer f II of Dermatophagoides farinae prepared using the measurement device in FIG.

FIG. 10 is a schematic view showing main steps of a conventional sandwich ELISA method.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 reaction vessel 2 test piece for measurement 3 motor for driving reaction vessel 4 belt for driving reaction vessel 5 motor for vertically moving test piece for measurement 6 support for vertically moving test piece for measurement 7 substrate solution tank lid 8 substrate solution tank 9 substrate solution pump 10 Substrate solution injection nozzle 11 Reaction stop liquid tank lid 12 Reaction stop liquid tank 13 Reaction stop liquid pump 14 Reaction stop liquid injection nozzle 15 Temperature sensor 16 Temperature adjustment unit 17 Heater 18 Light emitting unit 19 Light receiving unit 20 Operation unit 21 Display unit 22 Input unit Reference Signs List 23 printing section 24 input / output interface section 25 power supply section 26 control section 27 reaction vessel insertion port 28 reaction vessel discharge port 29 housing 30 reaction vessel connecting frame 31 first reaction vessel 32 second reaction vessel 33 third reaction vessel 34 fourth reaction Vessel 35 Fifth reaction vessel 36 Sixth reaction vessel 37 Seventh reaction vessel 38 Eighth reaction vessel 40 Test specimen connecting frame 41 First measurement test piece 42 Second measurement test piece 43 Third measurement test piece 44 Fourth measurement test piece 45 Fifth measurement test piece 46 Sixth measurement test piece 47 No. 7 Test piece for measurement 48 Test piece for 8th test 49 Guide for insertion of reaction vessel 50 Standard solution or test solution 91 Antibody 92 Antigen 93 Standard antigen 94 Enzyme-labeled antibody 95 Enzyme

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Takashi Inoue 1-1-21 Midori, Moriya-cho, Kitasoma-gun, Ibaraki Prefecture Asahi Breweries, Ltd. Basic Research Laboratory (72) Inventor Yasushi Okumura Midori-cho, Moriya-cho, Kitasoma-gun, Ibaraki 1-chome, 21 Asahi Breweries, Ltd.

Claims (23)

[Claims] 1. A test piece for measurement in which a predetermined amount of an antigen to be measured is immobilized on a solid phase, and an antibody capable of specifically binding to the antigen and having a labeled substance added thereto is added. An antigen-containing test solution is brought into contact with the test piece for measurement in a reaction vessel, and the antibody immobilized on the test piece for measurement and the antigen in the test solution are prepared according to the respective antigen concentrations. Distributing and binding, measuring the amount of the labeling substance bound to the test strip or the test solution, and calculating the antigen concentration in the test solution; Measurement method. 2. A test piece having a fixed amount of an antigen to be measured immobilized on a solid phase is prepared, and a first antibody capable of specifically binding to the antigen and a first antibody specific to the first antibody are prepared. The test solution containing the antigen, to which the second antibody to which the labeling substance has been attached and which can be bound to the test sample in a reaction vessel, is brought into contact with the test sample for measurement, and the antigen immobilized on the test sample for measurement is contacted with the antigen. The first antibody and the second antibody are distributed and bound to the antigen in the test solution according to the respective antigen concentrations, and the amount of the labeling substance bound to the test strip for measurement or the test solution. And measuring the antigen concentration in the test solution. 3. A test piece for measurement, in which a predetermined amount of an antibody to be measured is immobilized on a solid phase, is prepared, and the antigen to which a labeling substance-bound antigen that can specifically bind to the antibody is added. An antibody-containing test solution is brought into contact with the test piece for measurement in a reaction container, and the antigen immobilized on the test piece for measurement and the antibody in the test solution are subjected to the antigen in accordance with the respective antibody concentrations. Distributing and binding, measuring the amount of the labeling substance bound to the test strip or the test solution, and calculating the antibody concentration in the test solution; Measurement method. 4. An antigen to be measured is immobilized on a solid phase in a fixed amount, and an antibody capable of specifically binding to the antigen and binding to a labeling substance is brought into contact with the antigen. Prepare a test strip for measurement in which an antigen-antibody complex of the antibody is formed, contact the test solution containing the antigen with the test strip for measurement in a reaction vessel, and fix the antigen-immobilized antigen of the antigen-antibody complex. With the antigen in the test solution, the antibodies of the antigen-antibody complex are distributed and bound according to the respective antigen concentrations,
A method for immunologically measuring an antigen concentration, comprising measuring an amount of the labeling substance bound to the test strip or the test solution to calculate an antigen concentration in the test solution. 5. An antigen to be measured is immobilized on a solid phase in a fixed amount, and a first antibody capable of specifically binding to the antigen and a labeling substance capable of specifically binding to the first antibody are used. The bound second antibody is brought into contact with the antigen, and a test strip for measurement in which an antigen-antibody complex of the antigen, the first antibody and the second antibody is formed on a solid phase is prepared. The test solution is brought into contact with the test piece for measurement in the reaction vessel,
The first antibody and the second antibody of the antigen-antibody complex are composed of the immobilized antigen of the antigen-antibody complex and the antigen in the test solution.
Distribute and bind the antibodies according to their respective antibody concentrations,
A method for immunologically measuring an antigen concentration, comprising measuring an amount of the labeling substance bound to the test strip or the test solution to calculate an antigen concentration in the test solution. 6. An antibody to be measured is immobilized on a solid phase in a fixed amount, and an antigen capable of specifically binding to the antibody and having a label attached thereto is brought into contact with the antibody. Prepare a test piece for measurement in which an antigen-antibody complex of the antigen is formed, contact the test solution containing the antibody with the test piece in a reaction vessel, and immobilize the antigen-antibody complex immobilized antibody. And the antigen in the antigen-antibody complex with the antibody in the test solution is distributed and bound according to the respective antibody concentration,
A method for immunologically measuring an antibody concentration, comprising measuring an amount of the labeling substance bound to the test strip or the test solution to calculate an antibody concentration in the test solution. 7. The immunological assay method according to claim 1, wherein the labeling substance is an enzyme. 8. The test piece for measurement has a shape in which any one or more selected from a bead, a film, a column, and a container are combined, and is used for measurement after distributive bonding. Separation of the antigen-antibody complex on the solid phase of the test piece from the antigen-antibody complex in the liquid phase of the test solution by disconnecting the test piece for measurement from the test solution in the reaction vessel The immunological measurement method according to any one of claims 1 to 7, characterized in that: 9. The method according to claim 1, wherein the test piece for measurement is immersed in a plurality of test solutions at the same time. 10. A test strip for measurement in which a predetermined amount of an antigen to be measured is immobilized on a solid phase, and the antigen-containing test piece to which an antibody capable of specifically binding to the antigen and having a labeled substance added thereto is added. A reaction container containing a test solution, a transfer unit for bringing the measurement test piece into contact with the test solution in the reaction container, and the respective antigens of the measurement test piece and the test solution. A measurement unit for measuring the amount of the labeling substance of any of the antibodies that have undergone antigen-antibody reaction, and a calculation unit for converting the amount of the labeling substance measured by the measurement unit into an antigen concentration of the test solution. An immunoassay device for measuring an antigen concentration. 11. A test strip for measurement in which a predetermined amount of an antigen to be measured is immobilized on a solid phase, a first antibody capable of specifically binding to the antigen, and a specific binding to the first antibody. A reaction container containing the antigen-containing test solution to which the second antibody bound to the labeling substance is added, and a transfer for bringing the measurement test piece into contact with the test solution in the reaction container. And a measuring unit for measuring the amount of the labeling substance of any of the second antibodies that have undergone an antigen-antibody reaction with the antigen of each of the measurement test strip and the test solution, and wherein the measuring unit measures A device for converting the amount of the labeling substance into an antigen concentration of the test solution. 12. A test strip for measurement in which a predetermined amount of an antibody to be measured is immobilized on a solid phase, and a test piece containing the antibody to which an antigen capable of specifically binding to the antibody and having a labeled substance added thereto is added. A reaction container containing a test solution, a transfer unit for bringing the measurement test piece into contact with the test solution in the reaction container, and the antibody of any of the measurement test piece and the test solution A measuring unit for measuring the amount of the labeling substance of each of the antigens reacted with the antigen-antibody, and a calculating unit for converting the amount of the labeling substance measured by the measuring unit into an antibody concentration of the test solution. An immunoassay device for measuring an antibody concentration. 13. An assay for immobilizing a predetermined amount of an antigen to be measured on a solid phase and forming an antigen-antibody complex with the antigen using an antibody that can specifically bind to the antigen and is bound with a labeling substance. A test piece, a reaction container containing the antigen-containing test solution, a transfer unit for bringing the measurement test piece into contact with the test solution in the reaction container, the measurement test piece and the test sample A measurement unit for measuring the amount of the labeling substance of any of the antibodies that have reacted with the antigen and the antigen in the test solution, and the amount of the labeling substance measured by the measurement unit is set to the antigen concentration of the test liquid. An immunological measurement device for measuring an antigen concentration, comprising a calculation unit for conversion. 14. An antigen to be measured is immobilized on a solid phase in a fixed amount, and a first antibody capable of specifically binding to the antigen and a labeling substance capable of specifically binding to the first antibody are bound thereto. A measurement test piece in which an antigen-antibody complex with the antigen is formed with the second antibody, a reaction container containing a test solution containing the antigen, and a test container in the measurement container and a test container in the reaction container. A transfer unit for contacting a liquid, and a measurement unit for measuring the amount of the labeling substance of the second antibody that has reacted with the antigen and the antigen-antibody of each of the measurement test piece and the test solution. An arithmetic unit for converting the amount of the labeling substance measured by the measuring unit into an antigen concentration of the test solution. 15. An assay for immobilizing a predetermined amount of an antibody to be measured on a solid phase and forming an antigen-antibody complex with the antibody using an antigen which can specifically bind to the antibody and has a labeled substance bound thereto. A test piece, a reaction container containing the antibody-containing test solution, a transfer section for bringing the measurement test piece into contact with the test solution in the reaction container, the measurement test piece and the test sample. A measurement unit for measuring the amount of the labeling substance of any of the antigens that have reacted with the respective antibodies and antigens in the test solution, and the amount of the labeling substance measured by the measurement unit to the antibody concentration of the test liquid An immunological measurement device for antibody concentration, comprising: a calculation unit for conversion. 16. The immunological measurement device according to claim 10, further comprising a temperature holding device for keeping the temperature of the antigen-antibody reaction and the enzyme reaction constant. 17. The immunological measurement device according to claim 10, further comprising a vibration mechanism or a stirring mechanism. 18. A test strip for measurement in which a predetermined amount of an antigen to be measured is immobilized on a solid phase, wherein the antigen can be specifically bound to the antigen and an antibody to which a label is bound is added. The test solution containing the solution is brought into contact with the test piece for measurement in a reaction vessel, and the antibody is distributed according to the respective antigen concentration between the antigen immobilized on the test piece for measurement and the antigen in the test solution. And measuring the amount of the labeling substance bound to the test sample or the test solution to calculate the antigen concentration in the test solution. 19. A test strip comprising a fixed amount of an antigen to be measured immobilized on a solid phase, a first antibody capable of specifically binding to the antigen, and a first antibody specifically binding to the first antibody. The test solution containing the antigen to which the second antibody to which the labeling substance has been bound can be bound is contacted with the test piece for measurement in a reaction vessel, and the antigen immobilized on the test piece for measurement and the antigen The first antibody and the second antibody are distributed and bound to the antigen in the test solution according to the respective antigen concentrations, and the amount of the labeling substance bound to the test strip for measurement or the test solution is determined. A test piece for measurement, which measures and calculates the antigen concentration in the test solution. 20. A test strip comprising a fixed amount of an antibody to be measured immobilized on a solid phase, wherein the antibody to which an antigen capable of specifically binding to the antibody and having a labeled substance added thereto is added. The test solution containing the sample is brought into contact with the test piece for measurement in a reaction vessel, and the antigen is distributed according to the respective antibody concentration between the antibody immobilized on the test piece for measurement and the antibody in the test solution. And measuring the amount of the labeling substance bound to the test specimen or the test liquid to calculate the antibody concentration in the test liquid. 21. An antigen to be measured is immobilized on a solid phase in a fixed amount, and an antibody capable of specifically binding to the antigen and having a label attached thereto is brought into contact with the antigen. A measurement test piece formed with an antigen-antibody complex of the antibody, the antigen-containing test solution is contacted with the measurement test piece in a reaction vessel, and the antigen-antibody complex immobilized antigen and With the antigen in the test solution, the antibodies of the antigen-antibody complex are bound and bound according to the respective antigen concentrations,
A test strip for measuring, wherein the amount of the labeling substance bound to the test strip or the test solution is measured to calculate the antigen concentration in the test solution. 22. A fixed amount of an antigen to be measured is immobilized on a solid phase, and a first antigen capable of specifically binding to the antigen is provided.
An antibody and a second antibody capable of specifically binding to the first antibody and binding a labeling substance are brought into contact with the antigen, and an antigen-antibody complex of the antigen, the first antibody, and the second antibody on a solid phase Is a measurement test piece formed, the antigen-containing test solution is brought into contact with the measurement test piece in a reaction vessel,
The first antibody and the second antibody of the antigen-antibody complex are composed of the immobilized antigen of the antigen-antibody complex and the antigen in the test solution.
Distribute and bind the antibodies according to their respective antibody concentrations,
A test strip for measuring, wherein the amount of the labeling substance bound to the test strip or the test solution is measured to calculate the antigen concentration in the test solution. 23. An antibody to be measured is immobilized on a solid phase in a fixed amount, and an antigen capable of specifically binding to the antibody and having a labeled substance bound thereto is brought into contact with the antibody. A test strip for forming an antigen-antibody complex of the antigen, the antibody-containing test solution is brought into contact with the test strip for measurement in a reaction vessel, and the antigen-antibody complex immobilized antibody. With the antibody in the test solution, the antigen of the antigen-antibody complex is distributed and bound according to the respective antibody concentration,
A test strip for measuring the amount of the labeling substance bound to the test strip or the test solution to calculate the antibody concentration in the test solution.